PRODUCTS AND PROCESSES FOR MODULATING PEPTIDE-PEPTIDE BINDING DOMAIN INTERACTIONS

BACKGROUND OF THE INVENTION

The invention relates to compounds (e.g., peptidomimetics and non-peptides) that inhibit a cellular proliferative disorder and methods of treating such disorders. The invention also provides three-dimensional structures of a Polo-like kinase and methods for designing or selecting small molecule inhibitors using these structures. Desirably, these compounds have certain structural, physical, and spatial characteristics that enable the compounds to interact with specific amino acid residues.

Cyclin-dependent kinases (Cdks) have long been considered the master regulators of the cell-cycle, but an increasing number of diverse protein kinases are now emerging as critical components of cell-cycle progression. Among these are members of the Polo-like kinase family (Plks) that play key roles during all stages of mitosis and in the cell cycle checkpoint response to genotoxic stress. Many protein kinases involved in cell-cycle control function, in part, by generating phosphoserine/threonine-containing sequence motifs in their substrates that are subsequently recognized by phosphoserine/threonine-binding proteins. These include the WW and proline isomerase domain of Pin1 that regulates mitotic progression, 14-3-3 proteins that control the G2/M transition in response to DNA damage, and the WD40 repeat of Cdc4p which regulates S-phase entry.

In several instances, a phosphopeptide-binding domain and a kinase domain are combined within a single molecule, best exemplified by the SH2 domain-containing Src kinases and the Rad53p/Chk2-family of FHA domain-containing kinases. In these proteins the phosphopeptide-binding domain targets the kinase to pre-phosphorylated (primed) sites, mediates processive phosphorylation at multiple sites within a single substrate, or facilitates kinase activation. Polo-like kinases are distinguished by the presence of a conserved Ser/Thr kinase domain and a non-catalytic C-terminal region composed of two homologous ˜70-80 residue segments termed Polo-boxes.

Humans, mice and frogs each have three Plk homologues denoted Plk1, Plk2/Snk, and Plk3/Fnk/Prk, while budding yeast, fission yeast, and flies contain only a single Plk family member denoted Cdc5p, Plo1, and Polo, respectively. In addition, humans and mice have a serine/threonine kinase, Sak, that is an extremely divergent member of the Plk family, containing only a single Polo-box and lacking a canonical PBD.

The most extensively studied Polo-like kinases, Plk1 and Cdc5p, have been implicated in numerous mitotic processes including activation of Cdc25C and Cdc2-cyclinB at the G2-M transition, centrosome maturation and spindle assembly, cohesin release/cleavage during sister chromatid separation, anaphase promoting complex (APC) activation during mitotic exit, and septin regulation during cytokinesis. In contrast human Plk2 and Plk3 appear to serve different functions. Plk2 shows peak expression and activity in early G1, while Plk3 is activated by several stress response pathways, including DNA damage and spindle disruption. In fact, Plk3 plays some roles that may directly antagonize Plk1 function. For example, DNA damage directly inhibits Plk1, but activates Plk3 in an Ataxia-Telangiectasia-Mutated (ATM)-dependent manner. Consistent with these results, Plk1 overexpression causes oncogenic transformation in NIH 3T3 cells, while overexpression of Plk3 induces apoptosis.

SUMMARY OF THE INVENTION

We have developed a proteomic approach for identifying targets downstream of kinases in signaling pathways. Our strategy involves using an immobilized library of partially degenerate phosphopeptides, biased toward a kinase phosphorylation motif, to isolate interacting effector proteins targeted by substrates of that kinase. Utilizing this approach for cyclin-dependent kinases, we discovered that the carboxy-terminal region of the cell cycle regulating kinase, Plk-1, encodes a phosphopeptide recognition domain that consists of the non-kinase region of this protein (amino acids 326-603). This phosphopeptide recognition domain, termed the Polo-box domain (PBD), binds phosphoserine and phosphothreonine residues in a sequence-specific context. Specifically, this PBD recognizes and binds to the core phosphopeptide sequence serine-phosphoserine or serine-phosphothreonine.

We performed oriented peptide library screening on the PBDs from all three human Plk homologues, as well as on the Plk1 orthologues Plx1 from Xenopus and Cdc5p from budding yeast. Despite differences in cellular function, we found that all PBDs show strong conserved selection for the core sequence S-[pSer/pThr]-P/X.

To determine the structural basis of PBD activity, the crystal structure of the human Plk1 PBD in complex with its optimal phosphothreonine-containing peptide was determined. We identified a mode of phosphopeptide binding that is unique among structurally characterized phosphodependent binding protein/modules and that is crucial for PBD targeting to substrates both in vitro and in vivo. The architecture of the Plk1 PBD differs significantly from that recently observed for homodimers of the single Polo-box from murine Sak, which lacks a formal PBD (Leung et al., Nat. Struct. Biol. 9:719-724, 2002). The Plk1 PBD represents a new protein fold. Site-directed mutagenesis based on the structural identification of critical phosphothreonine-binding residues has enabled us to demonstrate that phosphodependent substrate recognition by the PBD is necessary for proper mitotic progression. Furthermore, binding of the optimal Plk1 phosphopeptide to the PBD in full-length Plk1 enhances the in vitro activity of the kinase domain, leading to a model for Plk regulation in which intramolecular inhibition of the kinase by the PBD is relieved by PBD-ligand binding. We conclude that phosphoserine/threonine-dependent binding is a general feature of PBD activity across the Plk family and critically important for the function of this domain in Polo-like kinase targeting and regulation. These studies have identified sites that may be targeted in designing therapeutics useful in treating diseases or disorders characterized by inappropriate cell cycle regulation or inappropriate cell death.

We applied the same proteomic approach to identify phosphopeptide-binding modules mediating signal transduction events in the DNA damage response pathway. Using a library of partially degenerate phosphopeptides biased to resemble the phosphorylation motif of the phosphoinositide-like kinases ATM and ATR, we identified tandem BRCT domains in PTIP and BRCA 1 as phosphoserine (pSer)- or phosphothreonone (pThr)-specific binding modules that recognize a subset of ATM (ataxia telangiectasia—mutated) and ATR (ataxia telangiectasia—and RAD3-related)-phosphorylated substrates following γ-irradiation. PTIP tandem BRCT domains are responsible for phosphorylation-dependent protein localization into 53BP1- and phospho-H2AX (_-H2AX)—containing nuclear foci, a marker of DNA damage. These findings provide a new molecular rationale for BRCT domain function in the signaling response to DNA damage and may help to explain why the BRCA1 BRCT domain mutation Met1775 3 Arg, which fails to bind phosphopeptides, predisposes women to breast and ovarian cancer.

In one aspect, the invention generally features computer containing a processor in communication with a memory; the memory having stored therein (i) at least one atomic coordinate, or surrogates thereof, from Table 5 for each of the following residues: His-538, Lys-540, Trp-414, or Leu-491 of a Polo-box domain or atomic coordinates that have a root mean square deviation of the coordinates of less than 3 Å; and (ii) a program for generating a three-dimensional model of the coordinates. In one embodiment, the coordinate is for a heteroatom. In another embodiment, the coordinate is for a side-chain atom. In another embodiment, the coordinate is for a side-chain and a heteroatom.

In another aspect, the invention generally features a computer containing a processor in electrical communication with a memory; the memory having stored therein (i) atomic coordinates, or surrogates thereof, as shown in Table 5 for atoms of residues His-538, Lys-540, Trp-414, or Leu-491 of a Plk1 Polo-box domain or atomic coordinates that have a root mean square deviation from the coordinates of the residues of less than 1, 2, 3, 4, or 5 Å; and (ii) a program for displaying a three-dimensional model of the Polo-box domain.

In another aspect, the invention provides a computer containing a processor in communication with a memory; the memory having stored therein (i) x-ray diffraction data for at least one of the non-hydrogen atoms of residues His-538, Lys-540, Trp-414, or Leu-491 of a Polo-box domain or x-ray diffraction data for amino acids that have a root mean square deviation from the backbone atoms of the residues of less than 1, 2, 3, 4, or 5 Å; and (ii) a program for generating a three-dimensional model of the Polo-box domain.

In another aspect, the invention provides a computer containing a processor in communication with a memory; the memory having stored therein a pharmacophore model of a phosphopeptide that binds a Polo-box domain and a program for displaying the model, the model containing at least one of the following: a phosphate group on threonine that participates in at least 1 hydrogen-bonding interaction; and a serine at the pThr-1 position, where the Ser-1 side chain is directed towards the Plk1 surface. In one embodiment, the serine engages in at least two of the following (i) a hydrogen bonding interaction with Trp-414 main-chain atoms of PBD; (ii) a hydrogen bonding interaction with Leu-491 main-chain carbonyl of PBD; and (iii) a van der Waals interaction with CM from the Trp-414 indole side chain of PBD. In one embodiment, the model further comprises a Proline at the pThr+1 position, where the proline introduces a kink that allows a pThr+2 main chain amino group to contact PBD.

In another aspect, the invention provides a method of selecting or designing a candidate ligand for a Polo-box domain, the method involves the steps of: (a) generating a three-dimensional structure of a Polo-box domain having at least one atomic coordinate, or surrogate thereof, from Table 5 for each of the following residues: His-538, Lys-540, Trp-414, or Leu-491 or atomic coordinates that have a root mean square deviation from the coordinates of less than 1, 2, 3, 4, or 5 Å; and (b) selecting or designing a candidate ligand having sufficient surface complementary to the structure to bind a Polo-box domain in an aqueous solution. In another aspect, the invention provides a method for manufacturing a Polo-box domain ligand, the method involves the steps of: (a) obtaining the atomic coordinates of at least one residue of a Polo-box domain with a ligand; (b) determining one or more moieties in the ligand to be modified; where the modified ligand maintains the ability to bind the Polo-box domain; and (c) modifying the ligand based on the determination. In one embodiment, the method further involves crystallizing a Polo-box domain with a ligand. In another embodiment, the ligand specifically binds the Polo-box domain. In another embodiment, the modification increases the affinity of the ligand for the Polo-box domain. In another embodiment, the modification increases the solubility of the ligand. In another embodiment, the modification increases the half-life of the ligand in vivo.

In another aspect, the invention provides a method for manufacturing a Polo-box domain ligand, the method involves manufacturing a ligand that binds a Polo-box domain; where the ligand is designed or selected based on information obtained using a model of the atomic coordinates of at least a portion of the Polo-box domain.

In another aspect, the invention provides a method of evaluating the ability of a candidate ligand to bind a Polo-box domain, the method involves the steps of: (a) generating a three-dimensional structure of a Polo-box domain having at least one atomic coordinate, or surrogate thereof, from Table 5 for each of the following residues: His-538, Lys-540, Trp-414, or Leu-491 or atomic coordinates that have a root mean square deviation from the coordinates of less than 1, 2, 3, 4, or 5 Å; and (b) employing a means to measure the interaction between the candidate ligand and the Polo-box domain.

In another aspect, the invention provides a method of identifying a candidate ligand for a Polo-box domain, the method involves the steps of: (a) generating a three-dimensional pharmacophore model of Polo-box domain ligands using a computer of a previous aspect; and (b) selecting a candidate ligand satisfying the criteria of the pharmacophore model. In various embodiments, of any previous aspect, the method further involves determining the ability of the candidate ligand to bind the Polo-box domain in vitro or in vivo. In other embodiments, the method further involves determining the ability of the candidate ligand to alter the enzymatic activity of the Polo-box domain in vitro or in vivo. In other embodiments, the three-dimensional structure further comprises the hydrogen atoms of residues His-538, Lys-540, Trp-414, or Leu-491.

In various embodiments of the above aspects, the coordinate is for a heteroatom, or a side-chain atom, or a side-chain and a heteroatom. In other embodiments, the memory stores at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 coordinates or surrogates thereof for His-538; at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 coordinates or surrogates thereof for Lys-540, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 coordinates or surrogates thereof for Trp-414; or at least 1, 1, 2, 3, 4, 5, 6, 7, or 8 coordinates or surrogates thereof for Leu-491. In other embodiments, the coordinate is any one or all of the atomic coordinates in Table 5. In other embodiments of the previous aspect, the coordinates are for any residue required for the biological activity of a Polo box domain, or for binding a phosphopeptide or peptide mimetic. In other embodiments of any of the above aspects, root mean square deviation of the coordinates of less than 1, 2, 3, 4, 5, 6, or 7 Å.

In another aspect, the invention features a crystal of a Polo-like kinase complex containing a Polo-box domain bound to a phosphopeptide. In one embodiment, the Polo-like kinase is Plk-1 (SEQ ID NO: 1). In another embodiment, the Plk-1 comprises at least amino acids 1-603 of SEQ ID NO:1. In another embodiment, the Plk-1 comprises at least amino acids 95-603. In another embodiment, the Plk-1 comprises at least amino acids 326-603. In another embodiment, the Plk-1 comprises at least amino acids 367-603. In another embodiment, the phosphopeptide comprises the amino acid sequence [Pro/Phe]-[φ/Pro]-[φAla_Cdc5p/Gln_Plk2]-[Thr/Gln/His/Met]-Ser-[pThr/pSer]-[Pro/X] (SEQ ID NO: 2), where φ represents hydrophobic amino acids. In another embodiment, the phosphopeptide comprises the amino acid sequence MAGPMQ-S-pT-P-LNGAKK (SEQ ID NO: 3). In another embodiment, the Polo-like kinase is Plk-2 (SEQ ID NO: 4). In another embodiment, the Polo-like kinase is Plk-3 (SEQ ID NO: 5).

In another aspect, the invention provides a method of obtaining a structural model of a Polo-box domain of interest, the method involves homology modeling using at least a portion of the atomic coordinates in Table 5 and at least a portion of the amino acid sequence of the Polo-box domain of interest, thereby generating a model of the Polo-box domain of interest.

In another aspect, the invention provides a method of determining the three-dimensional structure of a Polo-box domain/phosphopeptide complex of interest, the method involves the steps of: (a) crystallizing the Polo-box domain/phosphopeptide complex of interest; (b) generating an X-ray diffraction pattern from the crystallized Polo-box domain of interest; and (c) applying at least a portion of the atomic coordinates in Table 5 to the diffraction pattern to generate a three-dimensional electron density map of at least a portion of the Polo-box domain/phosphopeptide complex of interest.

In another aspect, the invention features an isolated, less than full-length fragment of Polo-box domain containing residues 367-603 of human Plk-1 Polo-box domain) in complex with a phosphopeptide containing S-[pS/pT]-P/X, where X is any amino acid.

In another aspect, the invention features an isolated, less than full-length fragment of Polo-box domain containing residues residues 500-685 of human Plk-2 Polo-box domain in complex with a phosphopeptide containing S-[pS/pT]-P/X, where X is any amino acid.

In another aspect, the invention features an isolated, less than full-length fragment of Polo-box domain containing residues residues 421-607 of human Plk-3 Polo-box domain in complex with a phosphopeptide containing S-[pS/pT]-P/X, where X is any amino acid.

In another aspect, the invention features an isolated Polo-box domain protein or fragment thereof containing a mutation, where the mutation is (a) a mutation that enhances the ability of Polo-box domain to crystallize; (b) a mutation of a residue that is otherwise post-translationally modified in an organism used for recombinant expression; (c) a mutation of the NH₂— or COOH-terminal residue of Polo-box domain; (d) a mutation that increases or decreases the affinity of a Polo-box domain for a phosphopeptide; or (e) a mutation that alters the folding of Polo-box domain. In one embodiment, the PBD further comprises a mutation at His-538, Lys-540, Trp-414, or Leu-491. In other embodiments, the nucleic acid encodes a protein of any previous aspect.

In another aspect, the invention features a phosphopeptide containing the amino acid sequence [Pro/Phe]-[φ/Pro]-[φ/Ala_Cdc5p/Gln_Plk2]-[Thr/Gln/His/Met]-Ser-[pThr/pSer]-[Pro/X] (SEQ ID NO: 2), where φ represents hydrophobic amino acids. In one embodiment, the phosphopeptide comprises Pro-Met-Gln-Ser-pThr-Pro-Leu (SEQ ID NO: 6), where the phosphopeptide binds human Plk-1.

In another aspect, the invention features a phosphopeptide containing the amino acid sequence,

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where pSer and pThr are phosphorylated serine and phosphorylated threonine, and where the amino acids designated in P-3, P-2, or P1 may be natural or unnatural amino acids. In one embodiment, the phosphopeptide of the previous aspect further contains the amino acid sequence,

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where X₁aa and X₂aa are any amino acids and where pSer and pThr are phosphorylated serine and phosphorylated threonine. In another embodiment, the X₁aa is proline and where X₂aa is any amino acid. In another embodiment, the X₁aa is any amino acid and where X₂aa is alanine, leucine, valine, isoleucine, phenylalanine, tyrosine, and tryptophan. In another embodiment, the X₂aa is leucine. In another embodiment, the amino acid at position P-3 is methionine. In another embodiment, the amino acid at position P-2 is glutamine. In another embodiment, the amino acid at position P-1 is serine. In another embodiment, the amino acid at position P0 is phosphorylated serine. In another embodiment, the amino acid at position P0 is phosphorylated threonine. In another embodiment, the amino acid at position P+1 is proline. In another embodiment, the amino acid sequence is Met-Gln-Ser-pThr-Pro-Leu or Met-Gln-Ser-pSer-Pro-Leu (SEQ ID NO: 9), where X₁aa is any amino acid and pThr is phosphorylated threonine and pSer is phosphorylated serine. In another embodiment, the phosphopeptide does not exceed 25 amino acids residues. In another embodiment, the phosphopeptide does not exceed 15 amino acids residues. In another embodiment, the phosphopeptide does not exceed 10 amino acids residues.

In another aspect, the invention features a pharmaceutical composition containing a therapeutic effective dose of any of the phosphopeptides of the previous aspects and a pharmaceutically acceptable excipient, where the pharmaceutical composition is useful for the treatment of a disorder characterized by inappropriate cell cycle regulation. In one embodiment, the cellular proliferative disorder is a neoplasm. In another embodiment, the composition further comprises a second chemotherapeutic agent. In another embodiment, the second chemotherapeutic agent is selected from the group consisting of paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, mechlorethamine, melphalen, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, pentostatin, procarbazine, alemtuzumab, rituximab, streptozocin, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, trastuzumab, vincristine, vindesine, rofecoxib, celecoxib, etodolac and vinorelbine.

In another aspect, the invention features a method for treating or inhibiting a cellular proliferative disorder in a patient, the method involves administering a pharmaceutical composition of the phosphopeptide of a previous aspect, where the phosphopeptide is in an amount sufficient to treat or inhibit the cellular proliferative disorder in the patient. In one embodiment, method includes administering a second chemotherapeutic agent, the phosphopeptide and the chemotherapeutic agent are in amounts sufficient to treat or inhibit the cellular proliferative disorder in the patient, and where the chemotherapeutic agent is administered simultaneously or within 1, 2, 3, 5, 7, 10, 14, or 28 days of administering the phosphopeptide. In another embodiment, the second chemotherapeutic agent is selected from the group consisting of paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, mechlorethamine, melphalen, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, pentostatin, procarbazine, alemtuzumab, rituximab, streptozocin, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, trastuzumab, vincristine, vindesine, rofecoxib, celecoxib, etodolac and vinorelbine, or any other chemotherapeutic known in the art. In other embodiments, the cellular proliferative disorder is a neoplasm.

In another aspect, the invention features a method for identifying a peptidomimetic compound that modulates Polo-like kinase biological activity, the method involves the steps of: a) contacting the phosphopeptide of a previous aspect and a Polo-box domain (PBD) polypeptide to form a complex between the phosphopeptide and the PBD; b) contacting the complex with a candidate compound; and c) measuring the displacement of the phosphopeptide from the PBD, where the displacement of the phosphopeptide from the PBD indicates that the candidate compound is a peptidomimetic compound that modulates Polo-like kinase biological activity.

In another aspect, the invention provides a method for identifying a peptidomimetic compound that modulates Polo-like kinase biological activity, the method involves the steps of: a) contacting the phosphopeptide of a previous aspect and a PBD in the presence of a candidate compound; and b) measuring binding of the phosphopeptide and the PBD, where a reduction in the amount of binding relative to the amount of binding of the phosphopeptide and the polypeptide in the absence of the candidate compound indicates that the candidate compound is a peptidomimetic compound that modulates Polo-like kinase biological activity. In one embodiment, the phosphopeptide or the PBD is detectably labeled. In another embodiment, the phosphopeptide and the PBD are differentially labeled. In another embodiment, the PBD is selected from a group consisting of the PBDs of CdcS, Plo-1, Polo, Plx-1, Plx-2, Plx-3, Plk-1, Prk/Fnk, Snk, and Cnk. In another embodiment, the PBD is Plk-1 PBD. In another embodiment, the Plk-1 PBD is human Plk-1 PBD.

In another aspect, the invention provides a method for identifying a binding pair consisting of a peptide and a peptide-binding domain, the method involes the steps of: a) providing a biased peptide library containing a collection of peptides fixed to a solid support, each peptide having at least two known amino acid residues whose position is invariant; b) providing a pooled cDNA library, where the cDNA library is positioned for protein expression; c) expressing the pooled cDNA library in the presence of a detectable label; d) contacting the peptide library and the expressed cDNA library; and e) detecting a peptide and peptide-binding domain interaction, where an interaction identifies a peptide and peptide-binding domain binding pair. In one embodiment, the biased peptide library is covalently bound to a solid support. In another embodiment, the biased peptide library is noncovalently bound to a solid support. In another embodiment, the peptide is a phosphopeptide and the peptide binding domain is a phosphopeptide binding domain.

In another aspect, the invention provides a method for identifying a binding pair containing a phosphopeptide and a phosphopeptide binding domain, the method involves the steps of: a) providing a biased phosphopeptide library, containing a collection of peptides fixed to a solid support, each peptide having at least two known amino acid residues whose position is invariant; where each phosphopeptide is covalently linked to a biotin group at the amino terminus; b) providing a pooled cDNA library, where the pooled cDNA library is positioned for protein expression; c) expressing the pooled cDNA library in the presence of a detectable label; d) contacting the phosphopeptide library and the expressed cDNA library; and e) detecting a phosphopeptide and the phosphopeptide binding domain interaction, where the presence of an interaction identifies a phosphopeptide and phosphopeptide binding domain. In one embodiment, method further comprises the steps of f) providing a non-phosphorylated peptide of step a), and g) detecting a peptide and phosphopeptide-binding domain interaction, where the absence of an interaction indicates the phosphopeptide and phosphopeptide binding domain interaction is authentic.

In another aspect, the invention provides a method for identifying a binding pair consisting of a peptide and a peptide-binding domain; the method involves the steps of: a) providing a biased peptide library containing a collection of peptides fixed to a solid support, each peptide having at least two known amino acid residues whose position is invariant; b) contacting the biased peptide library with a detectably labeled peptide library; and c) detecting a biased peptide and detectably labeled peptide interaction, where an interaction identifies a peptide and peptide-binding domain binding pair.

In another aspect, the invention features a method to identify phosphopeptide-binding modules, the method involves the steps of: (a) providing an immobilized phosphopeptide library and an immobilized peptide library; (b) contacting the libraries with a polypeptide or polypeptide fragment; and (c) detecting preferential binding, where preferential binding to the phosphopeptide library in comparison to the peptide library identifies the polypeptide or polypeptide fragment as a phosphopeptide binding module.

In another aspect, the invention provides a method to identify non-phosphopeptide-binding modules, the method involves the steps of: (a) providing an immobilized degenerate phosphopeptide library and an immobilized peptide library; (b) contacting the libraries with a polypeptide or polypeptide fragment; and (c) detecting preferential binding, where preferential binding to the peptide library in comparison to the phosphopeptide library identifies the polypeptide or polypeptide fragment as a non-phosphopeptide binding module.

In another aspect, the invention provides a method to identify phosphopeptide-binding modules in the DNA damage response pathway, the method involves the steps of: (a) providing an immobilized pSer or pThr degenerate phosphopeptide library and an immobilized Ser or Thr peptide library; (b) contacting the libraries with a polypeptide or polypeptide fragment; and (c) detecting differential binding, where preferential binding to the phosphopeptide library in comparison to the peptide library identifies the polypeptide or polypeptide fragment as a phosphopeptide binding module. In one embodiment, the phosphopeptide or peptide libraries do not have the amino acids Arg, Lys, or His in a degenerate position in the libraries. In another embodiment, the polypeptides or polypeptide fragments are in vitro translated (IVT) polypeptides.

In another aspect, the invention features a degenerate phosphopeptide containing a pSer or pThr that binds a BRCT domain. In one embodiment, the phosphopeptide further comprises an aromatic or aliphatic residue in the pSer or pThr +3 position; aromatic or aliphatic residues in the pSer or pThr +3 or +5 positions; a Gln or an aromatic or an aliphatic residue in the +1 position; or the amino acid sequence Y-D-I-(pSer or pThr)-Q-V-F—P—F (SEQ ID NO: 10).

In another aspect, the invention features a phosphopeptide binding module containing a BRCT tandem domain. In one embodiment, the BRCT tandem domain comprises at least 100 amino acids of the 3rd and 4th BRCT domains of PTIP. In another embodiment, the BRCT pair comprises at least 100 amino acids of the BRCT domains of BRCA 1. In another embodiment, the tandem domain functions as a single module in phosphopeptide binding.

In another aspect, the invention features an isolated fragment (e.g, 50, 100, 150, 200, 250, or 300 amino acids) of tandem BRCT domains of PTIP or BRCA1 in complex with a phosphopeptide containing a pSer or pThr amino acid.

In another aspect, the invention features a complex containing a tandem BRCT phosphopeptide binding module and a phosphopeptide containing a pSer or pThr. In one embodiment, the tandem BRCT phosphopeptide binding module is a fragment of PTIP in complex with a phosphopeptide. In another embodiment, the phosphopeptide further comprises an aromatic or aliphatic residue in the (pSer or pThr)+3 position; an aromatic or aliphatic residues in the (pSer or pThr)+3 or +5 positions a Gln, or an aromatic or aliphatic residue in the +1 position; or the amino acid sequence Y-D-I-(pSer or pThr)-Q-V-F—P—F (SEQ ID NO: 10). In another aspect, the invention provides a method for identifying a candidate compound for the treatment or prevention of a neoplasia, the method containing detecting binding of the phosphopeptide binding module to a phosphopeptide in the presence of the candidate compound, where a candidate compound that modulates the binding is a compound useful for the treatment or prevention of a neoplasia. In one embodiment, binding is detected using an immunological assay, an enzymatic assay, or a radioimmunoassay. In another embodiment, the phosphopeptide binding module or fragment thereof is an isolated phosphopeptide binding module. In another embodiment, the phosphopeptide binding module or fragment thereof is an isolated phosphopeptide containing a pSer or pThr. In one embodiment, phosphopeptide is fixed to a solid support. In another embodiment, the phosphopeptide binding module is a tandem BRCT binding domain. In another embodiment, the phosphopeptide binding module is fixed to a solid support. In another embodiment, the binding is assayed using an immunological assay, an enzymatic assay, or a radioimmunoassay. In another embodiment, the candidate compound is preincubated with the phosphopeptide binding module. In another embodiment, the candidate compound is preincubated with the phosphopeptide. In another embodiment, the phosphopeptide binding module and the phosphopeptide form a complex prior to being contacted with the candidate compound. In another embodiment, the candidate compound, the phosphopeptide and the phosphopeptide binding module are contacted concurrently.

In another aspect, the invention features a method for identifying a candidate compound useful in treating or preventing a neoplasia in a subject, the method involves: (a) providing a cell expressing a phosphopeptide binding module or fragment thereof and a phosphopeptide containing a pSer or pThr; (b) contacting the cell with a candidate compound; and (c) comparing binding of the phosphopeptide binding module and the phosphopeptide in the cell contacted with the candidate compound to the binding in a control cell, where a modulation of the binding identifies the candidate compound as a compound useful to treat or prevent a neoplasia in a subject. In one embodiment, phosphopeptide binding moduleand the phosphopeptide are expressed in a prokaryotic or a eukaryotic cell in vitro. In another embodiment, the phosphopeptide binding module is expressed endogenously by the cell. In another embodiment, the phosphopeptide binding module is expressed as a recombinant protein. In another embodiment, the cell is a neoplastic cell. In another embodiment, the neoplastic cell is a mammalian cell. In another embodiment, the neoplastic cell is a human cell. In another embodiment, the candidate compound decreases the affinity of the binding.

In another aspect, the invention features a pharmaceutical composition containing (i) a phosphopeptide containing a pSer or pThr and (ii) a pharmaceutically acceptable carrier, where the phosphopeptide is present in amounts that, when administered to a subject, ameliorates a neoplastic disease. In one embodiment, the compositions comprises a second chemotherapeutic agent. In another embodiment, the second chemotherapeutic agent is selected from the group consisting of paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, mechlorethamine, melphalen, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, pentostatin, procarbazine, alemtuzumab, rituximab, streptozocin, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, trastuzumab, vincristine, vindesine, rofecoxib, celecoxib, etodolac and vinorelbine.

In another aspect, the invention provides a method for treating or inhibiting a cellular proliferative disorder in a patient, the method involves administering a pharmaceutical composition of the phosphopeptide of a previous aspect, where the phosphopeptide is in an amount sufficient to treat or inhibit the cellular proliferative disorder in the patient. In one embodiment, the method includes administering a second chemotherapeutic agent, the phosphopeptide and the chemotherapeutic agent are in amounts sufficient to treat or inhibit the cellular proliferative disorder in the patient, and where the chemotherapeutic agent is administered simultaneously or within fourteen days of administering the phosphopeptide. In another embodiment, the second chemotherapeutic agent is selected from the group consisting of paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, mechlorethamine, melphalen, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, pentostatin, procarbazine, alemtuzumab, rituximab, streptozocin, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, trastuzumab, vincristine, vindesine, rofecoxib, celecoxib, etodolac and vinorelbine. In another embodiment, the cellular proliferative disorder is a neoplasm.

In another aspect, the invention features a method for identifying a peptidomimetic compound that modulates BRCT biological activity, the method involves the steps of a) contacting the phosphopeptide of claim a previous aspect and a BRCT binding domain domain polypeptide to form a complex between the phosphopeptide and the BRCT; b) contacting the complex with a candidate compound; and c) measuring the displacement of the phosphopeptide from the BRCT binding domain, where the displacement of the phosphopeptide from the BRCT binding domain indicates that the candidate compound is a peptidomimetic compound that modulates BRCT binding domain biological activity.

In another aspect, the invention features a method for identifying a peptidomimetic compound that modulates BRCT binding domain biological activity, the method involves the steps of: a) contacting the phosphopeptide of a previous aspect and a BRCT binding domain in the presence of a candidate compound; and b) measuring binding of the phosphopeptide and the BRCT binding domain, where a reduction in the amount of binding relative to the amount of binding of the phosphopeptide and the polypeptide in the absence of the candidate compound indicates that the candidate compound is a peptidomimetic compound that modulates BRCT binding domain biological activity. In one embodiment, the phosphopeptide or the BRCT binding domain is detectably labeled. In another embodiment, the phosphopeptide and the BRCT binding domain are differentially labeled. In other embodiments, the BRCT binding domain is BRCA 1 or PTIP. In another embodiment, the BRCT binding domain is of human BRCA1. In one embodiment, BRCT binding domain is of human PTIP.

In another aspect, the invention features a kit containing (i) a small molecule that binds a BRCT binding domain and (ii) instructions for administering the small molecule to a patient diagnosed with or having a propensity to develop a neoplasia. In one embodiment, the kit further comprises a second chemotherapeutic compound.

In another aspect, the invention features a method of assessing a patient as having, or having a propensity to develop, a neoplasia, the method involves determining the level of expression of an a BRCT binding domain nucleic acid molecule or polypeptide in a patient sample, where an increased level of expression relative to the level of expression in a control sample, indicates that the patient has or has a propensity to develop a neoplasia. In one embodiment, the patient sample is a blood or tissue sample. In another embodiment, the method comprises determining the level of expression of the BRCT binding domain nucleic acid molecule. In another embodiment, the method comprises determining the level of expression of the BRCT binding domain polypeptide. In another embodiment, the level of expression is determined in an immunological assay. In another embodiment, the method is used to diagnose a patient as having neoplasia.

In another aspect, the invention features a method to identify a peptide-binding module, the method involves the steps of: (a) providing an immobilized modified peptide library and an immobilized peptide library; (b) contacting the libraries with a polypeptide or polypeptide fragment; and (c) detecting preferential binding, where preferential binding to the modified peptide library in comparison to the peptide library identifies the polypeptide or polypeptide fragment as a modified peptide binding module.

In another aspect, the invention features a method for identifying a binding pair consisting of a modified peptide and a peptide-binding domain, the method involves the steps of: a) providing a biased peptide library containing a collection of modified peptides fixed to a solid support, each peptide having one amino acid residues whose position is invariant; b) providing a pooled cDNA library, where the cDNA library is positioned for protein expression; c) expressing the pooled cDNA library in the presence of a detectable label; d) contacting the peptide library and the expressed cDNA library; and e) detecting a modified peptide and peptide-binding domain interaction, where an interaction identifies a modified peptide and peptide-binding domain binding pair. In one embodiment, the amino acid contains a modification that is natural or unnatural. In another embodiment, the modification is selected from the group consisting of methylation, acetylation, ubiquitination, glycosylation, sumolation, or arsenylation, or any other modification known to the skilled artisan.

In various embodiments of any of the above aspects, the peptide includes unnatural amino acids as described herein.

By “analog” is meant a molecule that is not identical but has analogous features. For example, a peptide analog retains the biological activity of a corresponding naturally-occurring peptide, while having certain biochemical modifications that enhance the analogs function relative to a naturally occurring peptide. Such biochemical modifications might increase the analogs protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog can include a non-natural amino acid.

In another example, a nucleic acid analog retains the ability to hybridize to a naturally-occurring corresponding nucleic acid sequence, while having certain biochemical modifications that enhance the analogs function relative to a naturally-occurring nucleic acid. In some nucleic acid analogs the sugar and/or the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. Peptide and nucleic acid modifications may be achieved by any of the techniques known in the art for derivatization of peptides or nucleic acids into fragments, analogs, or derivatives thereof. Such terms and in particular, “analog”, also specifically include peptide, non-peptide, peptide/nucleic acid hybrid molecules, small molecules and other compounds that function as Polo-like kinase nucleic acid or peptide mimics.

By “apoptosis” is meant the process of cell death where a dying cell displays at least one of a set of well-characterized biological hallmarks, including cell membrane blebbing, cell soma shrinkage, chromatin condensation, or DNA laddering.

By “biased phosphopeptide library” is meant a phosphoserine, phosphothreonine, and/or phosphotyrosine degenerate peptide library, wherein specific amino acid residues of the phosphopeptide are fixed so as to be expressed in all phosphopeptides in the specific library. For instance, a biased phosphopeptide library can be synthesized to contain the core sequence Ser-pSer-Pro or Ser-pThr-Pro. In a desirable embodiment, the amino acid residue adjacent to the phosphoserine, phosphothreonine, or phosphotyrosine residue is fixed.

By an “amino acid fragment” is meant an amino acid residue that has been incorporated into a peptide chain via its alpha carboxyl, its alpha nitrogen, or both. A terminal amino acid is any natural or unnatural amino acid residue at the amino-terminus or the carboxy-terminus. An internal amino acid is any natural or unnatural amino acid residue that is not a terminal amino acid.

As used herein, the terms “alkyl” and the prefix “alk-” are inclusive of both straight chain and branched chain groups and of cyclic groups, i.e., cycloalkyl and cycloalkenyl groups. Cyclic groups can be monocyclic or polycyclic and preferably have from 3 to 8 ring carbon atoms, inclusive. Exemplary cyclic groups include cyclopropyl, cyclopentyl, cyclohexyl, and adamantyl groups.

By “aromatic residue” is meant an aromatic group having a ring system with conjugated π electrons (e.g., phenyl or imidazole). The ring of the aryl group is preferably 5 to 6 atoms. The aromatic ring may be exclusively composed of carbon atoms or may be composed of a mixture of carbon atoms and heteroatoms. Preferred heteroatoms include nitrogen, oxygen, sulfur, and phosphorous. Aryl groups may optionally include monocyclic, bicyclic, or tricyclic rings, where each ring has preferably five or six members. The aryl group may be substituted or unsubstituted. Exemplary substituents include alkyl, hydroxyl, alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halo, fluoroalkyl, carboxyl, carboxyalkyl, amino, aminoalkyl, monosubstituted amino, disubstituted amino, and quaternary amino groups.

By “aryl” is meant a carbocyclic aromatic ring or ring system. Unless otherwise specified, aryl groups are from 6 to 18 carbons. Examples of aryl groups include phenyl, naphthyl, biphenyl, fluorenyl, and indenyl groups.

By “heteroaryl” is meant an aromatic ring or ring system that contains at least one ring hetero-atom (e.g., O, S, N). Unless otherwise specified, heteroaryl groups are from 1 to 9 carbons. Heteroaryl groups include furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, oxatriazolyl, pyridyl, pyridazyl, pyrimidyl, pyrazyl, triazyl, benzofuranyl, isobenzofuranyl, benzothienyl, indole, indazolyl, indolizinyl, benzisoxazolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, naphtyridinyl, phthalazinyl, phenanthrolinyl, purinyl, and carbazolyl groups.

By “heterocycle” is meant a non-aromatic ring or ring system that contains at least one ring heteroatom (e.g., O, S, N). Unless otherwise specified, heterocyclic groups are from 1 to 9 carbons. Heterocyclic groups include, for example, dihydropyrrolyl, tetrahydropyrrolyl, piperazinyl, pyranyl, dihydropyranyl, tetrahydropyranyl, tetrahydrofuranyl, dihydrothiophene, tetrahydrothiophene, and morpholinyl groups.

By “halide” or “halogen” or “halo” is meant bromine, chlorine, iodine, or fluorine.

The aryl, heteroaryl, and heterocyclyl groups may be unsubstituted or substituted by one or more substituents selected from the group consisting of C_1-5alkyl, hydroxy, halo, nitro, C_1-5alkoxy, C_1-5alkylthio, trihalomethyl, C_1-5acyl, arylcarbonyl, heteroarylcarbonyl, nitrile, C_1-5alkoxycarbonyl, oxo, arylalkyl (wherein the alkyl group has from 1 to 5 carbon atoms) and heteroarylalkyl (wherein the alkyl group has from 1 to 5 carbon atoms).

By an “amino acid fragment” is meant an amino acid residue that has been incorporated into a peptide chain via its alpha carboxyl, its alpha nitrogen, or both.

A terminal amino acid is any natural or unnatural amino acid residue at the amino-terminus or the carboxy-terminus. An internal amino acid is any natural or unnatural amino acid residue that is not a terminal amino acid.

By “BRCA1 nucleic acid” is meant a nucleic acid, or analog thereof, that encodes BRCA1 or is substantially identical to Gene Bank Accession No: 30039658 (SEQ ID NO: 11).

By “BRCA 1 polypeptide” is meant a polypeptide, or analog thereof, substantially identical to BRCA1 Genbank Accession NO. 30039659 (SEQ ID NO: 12) and having BRCA1 biological activity.

By “BRCA1 biological activity” is meant function in a DNA damage response pathway or phosphopeptide binding.

By “BRCT nucleic acid is meant a nucleic acid, or nucleic acid analog, that encodes tandem BRCT domains. For example, a nucleic acid substantially identical to PTIP BC033781[21707457] (SEQ ID NO: 13), or NM_—007349 (PAX transcription activation domain interacting protein 1 mRNA) (SEQ ID NO: 14) or Gene Bank Accession No: AY273801[30039658] (SEQ ID NO: 11).

By “tandem BRCT polypeptide is meant a protein having at least 2 tandem BRCT domains. For example, a protein substantially identical to AAH33781 (SEQ ID NO: 15), NP_—031375 (SEQ ID NO: 16), or Genbank Accession NO. 30039659 (SEQ ID NO: 12).

By “candidate compound” is meant any nucleic acid molecule, polypeptide, or other small molecule, that is assayed for its ability to alter gene or protein expression levels, or the biological activity of a gene or protein by employing one of the assay methods described herein. Candidate compounds include, for example, peptides, polypeptides, synthesized organic molecules, naturally occurring organic molecules, nucleic acid molecules, and components thereof.

By “detectably-labeled” is meant any means for marking and identifying the presence of a molecule, e.g., a PBD-interacting phosphopeptide, a PBD, a nucleic acid encoding the same, or a peptidomimetic small molecule. Methods for detectably-labeling a molecule are well known in the art and include, without limitation, radionuclides (e.g., with an isotope such as ³²P, ³³P, ¹²⁵I, or ³⁵S) and nonradioactive labeling (e.g., chemiluminescent labeling or fluorescein labeling).

If required, molecules can be differentially labeled using markers that can distinguish the presence of multiply distinct molecules. For example, a PBD domain-interacting phosphopeptide can be labeled with fluorescein and a PBD domain polypeptide can be labeled with Texas Red. The presence of the phosphopeptide can be monitored simultaneously with the presence of the PBD.

By “diseases or disorder characterized by inappropriate cell cycle control” is meant any pathological condition in which there is an abnormal increase or decrease in cell proliferation. Exemplary diseases or disorder characterized by inappropriate cell cycle control include cancer or neoplasms, inflammatory diseases, or hyperplasias (e.g. some forms of hypertension, prostatic hyperplasia).

By “disease or disorder characterized by inappropriate cell death” is meant any pathological condition in which there is an abnormal increase in apoptosis. Exemplary diseases or disorders characterized by inappropriate cell death include neurodegenerative diseases (e.g., Alzheimer's, Huntington's, and Parkinson's disease), cardiac disorders (e.g., congestive heart failure and myocardial infarction), diabetic retinopathy, and age-related macular degeneration.

By “fragment” is meant a portion of a protein (50, 100, 150, 175, 200, 300, or 400 amino acids) or nucleic acid (50, 100, 150, 175, 200, 300, or 400 nucleic acids) that is substantially identical to a reference protein or nucleic acid, and retains at least 50% or 75%, more preferably 80%, 90%, or 95%, or even 99% of the biological activity of the reference protein or nucleic acid using a molting assay as described herein.

By “halide” or “halogen” or “halo” is meant bromine, chlorine, iodine, or fluorine.

By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule which is transcribed from a DNA molecule, as well as a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

By “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components which naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By “modulate” is meant a change, such as a decrease or increase. Desirably, the change is either an increase or a decrease of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in expression or biological activity, relative to a reference or to control expression or activity, for example the expression or biological activity of a naturally occurring Polo-like kinase.

By “neoplasia” is meant a disease characterized by the pathological proliferation of a cell or tissue and its subsequent migration to or invasion of other tissues or organs. Neoplasia growth is typically uncontrolled and progressive, and occurs under conditions that would not elicit, or would cause cessation of, multiplication of normal cells. Neoplasias can affect a variety of cell types, tissues, or organs, including but not limited to an organ selected from the group consisting of bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, intestines, kidney, liver, lung, lymph node, nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, and vagina, or a tissue or cell type thereof. Neoplasias include cancers, such as sarcomas, carcinomas, or plasmacytomas (e.g., acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma Hodgkin's disease, Waldenstrom's macroglobulinemia, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenriglioma, schwannoma, meningioma, melanoma, neuroblastoma, or retinoplastoma).

By “nucleic acid” is meant an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid, or analog thereof. This term includes oligomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages as well as oligomers having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases.

Specific examples of some preferred nucleic acids envisioned for this invention may contain phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are those with CH₂—NH—O CH₂, CH₂—N(CH₃)—O—CH₂, CH₂—O—N(CH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂and O—N(CH₃)—CH₂—CH₂backbones (where phosphodiester is O—P—O—CH₂). Also preferred are oligonucleotides having morpholino backbone structures (Summerton, J. E. and Weller, D. D., U.S. Pat. No. 5,034,506). In other preferred embodiments, such as the protein-nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (P. E. Nielsen et al. Science 199: 254, 1997). Other preferred oligonucleotides may contain alkyl and halogen-substituted sugar moieties comprising one of the following at the 2′ position: OH, SH, SCH₃, F, OCN, O(CH₂)_nNH₂or O(CH₂)—CH₃, where n is from 1 to about 10; C₁to C₁₀lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a conjugate; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.

Other preferred embodiments may include at least one modified base form. Some specific examples of such modified bases include 2-(amino)adenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine, or other heterosubstituted alkyladenines.

By “Pax2 trans-activation domain-interacting protein (PTIP) nucleic acid” is meant a nucleic acid, or analog thereof, substantially identical to Genebank Accession No:21707457 (SEQ ID NO: 13) or NM_—007349 (SEQ ID NO: 14).

By “Pax2 trans-activation domain-interacting protein (PTIP)” is meant a polypeptide, or analog thereof, substantially identical to Genebank Accession No: AAH33781.1 (SEQ ID NO: 15) or NP_—031375 (SEQ ID NO: 16), and having PTIP biological activity.

By “PTIP biological activity” is meant function in a DNA damage response pathway or phosphopeptide binding.

By “pharmaceutically acceptable excipient” is meant a carrier that is physiologically acceptable to the subject to which it is administered and that preserves the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable excipient is physiological saline. Other physiologically acceptable excipients and their formulations are known to one skilled in the art and described, for example, in “Remington: The Science and Practice of Pharmacy” (20th ed., ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins).

By a “peptidomimetic” is meant a compound that is capable of mimicking or antagonizing the biological actions of a natural parent peptide. A peptidomimetic may include non-peptidic structural elements, unnatural peptides, synthesized organic molecules, naturally occurring organic molecules, nucleic acid molecules, and components thereof. Identification of a peptidomimetic can be accomplished by screening methods incorporating a binding pair and identifying compounds that displace the binding pair. Alternatively, a peptidomimetic can be designed in silico, by molecular modeling of a known protein-protein interaction, for example, the interaction of a phosphopeptide of the invention and a PBD. Desirably, the peptidomimetic will displace one member of a binding pair by occupying the same binding interface. More desirably the peptidomimetic will have a higher binding affinity to the binding interface.

By “Polo-like kinase (PLK) nucleic acid molecule” is meant a nucleic acid, or nucleic acid analog, that encodes a Polo-like kinase polypeptide. For example, a Plk-1 nucleic acid molecule is substantially identical to GenBank Accession Number X73458 (SEQ ID NO: 17) or NM_—005030 (SEQ ID NO: 18); a Plk-2/SNK nucleic acid molecule is substantially identical to NM_—006622 (SEQ ID NO: 19); a Plk-3 nucleic acid molecule is substantially identical to NM_—004073 (SEQ ID NO: 20); a Plx-1 nucleotide sequence is substantially identical to GenBank Accession Number U58205 (SEQ ID NO: 21); and a Polo nucleic acid molecule is substantially identical to GenBank Accession Number AY095028 (SEQ ID NO: 22) or NM 079455 (SEQ ID NO: 23).

By a “Polo-like kinase” is meant a polypeptide substantially identical to a Polo-like kinase amino acid sequence, having serine/threonine kinase activity, and having at least one Polo-box domain consisting of 2 Polo-boxes. Exemplary Polo-like kinase polypeptides include, Plk-1 (GenBank Accession Number NP 005021, SEQ ID NO:1); Plk-2 (GenBank Accession Number NP 006613, SEQ ID NO:4); and Plk-3 (GenBank Accession Number NP_—004064, SEQ ID NO:5). Additional Polo-like kinase polypeptides include GenBank Accession Numbers P53350 (SEQ ID NO: 24), and Q07832 (SEQ ID NO: 25).

Structurally, Polo or Polo-like kinases have a unique amino terminus followed by a serine/threonine kinase domain, a linker region, a Polo-box (PB1), a linker sequence, a second Polo-box (PB 2), and a small stretch of 12-20 amino acids at the carboxy terminus (see FIG. 2A).

In desirable embodiments, Polo-like kinases include Saccaromyces cereviseae, CdcS, Schizosaccaromyces pombe, Plo-1, Drosophila melanogaster, Polo, Xenopus laevis, Plx (Plx-1, -2, -3), and mammalian Plk-1, Prk/Fnk, Snk, and Cnk. The Polo-box is approximately 70 amino acids in length and is shown in FIG. 2B (indicated by the bold lines).

By “Polo-like kinase biological activity” is meant any biological activity associated with Polo-like kinases, such as serine/threonine kinase activity. Other biological activities of Polo-like kinases include the localization of the kinase to the centrosomes, spindle apparatus, and microtubular organizing centers (MOCs).

By “polypeptide” is meant any chain of at least two naturally-occurring amino acids, or unnatural amino acids (e.g., those amino acids that do not occur in nature) regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally-occurring or unnatural polypeptide or peptide, as is described herein. Naturally occurring amino acids are any one of the following, alanine (A or Ala), cysteine (C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H, or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), ornithine (O or Orn), proline (P or Pro), hydroxyproline (Hyp), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), or tyrosine (Y or Tyr).

By “peptide” is meant any compound composed of amino acids, amino acid analogs, chemically bound together. In general, the amino acids are chemically bound together via amide linkages (CONH); however, the amino acids may be bound together by other chemical bonds known in the art. For example, the amino acids may be bound by amine linkages. Peptide as used herein includes oligomers of amino acids, amino acid analog, or small and large peptides, including polypeptides.

Polypeptides or derivatives thereof may be fused or attached to another protein or peptide, for example, as a Glutathione-S-Transferase (GST) fusion polypeptide. Other commonly employed fusion polypeptides include, but are not limited to, maltose-binding protein, Staphylococcus aureus protein A, Flag-Tag, HA-tag, green fluorescent proteins (e.g., eGFP, eYFP, eCFP, GFP, YFP, CFP), red fluorescent protein, polyhistidine (6×His), and cellulose-binding protein.

By “phosphopeptide” or “phosphoprotein” means a peptide or protein in which one or more phosphate moieties are covalently linked to serine, threonine, tyrosine, aspartic acid, histidine amino acid residues, or amino acid analogs. A peptide can be phosphorylated to the extent of the number of serine, threonine, tyrosine, or histidine amino acid residues that is present. Desirably, a phosphopeptide is phosphorylated at 4 independent Ser/Thr/Tyr residues, at 3 independent Ser/Thr/Tyr residues, or at 2 independent Ser/Thr/Tyr residues. Most desirably, a phosphopeptide is phosphorylated at one Ser/Thr/Tyr residue regardless of the presence of multiple Ser, Thr, or Tyr residues.

Typically, a phosphopeptide is produced by expression in a prokaryotic or eukaryotic cell under appropriate conditions or in translation extracts where the peptide is subsequently isolated, and phosphorylated using an appropriate kinase. Alternatively, a phosphopeptide may be synthesized by standard chemical methods, for example, using N-α-FMOC-protected amino acids (including appropriate phosphoamino acids). In a desired embodiment, the use of non-hydrolysable phosphate analogs can be incorporated to produce non-hydrolysable phosphopeptides (Jenkins et al., J. Am. Chem. Soc., 124:6584-6593, 2002; herein incorporated by reference). Such methods of protein synthesis are commonly used and practiced by standard methods in molecular biology and protein biochemistry (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1994, J. Sambrook and D. Russel, Molecular Cloning: A Laboratory Manual, 3^rdEdition, Cold Spring Harbor Laboratory Press, Woodbury N.Y., 2000). Desirably, a phosphopeptide employed in the invention is generally not longer than 100 amino acid residues in length, desirably less than 50 residues, more desirably less than 25 residues, 20 residues, 15 residues. Most desirably the phosphopeptide is 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues long.

By “substantially identical” is meant a polypeptide or nucleic acid exhibiting at least 75%, but preferably 85%, more preferably 90%, most preferably 95%, or even 99% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 35 amino acids, preferably at least 45 amino acids, more preferably at least 55 amino acids, and most preferably 70 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 60 nucleotides, preferably at least 90 nucleotides, and more preferably at least 120 nucleotides.

Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). This software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine, methionine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

By “unnatural amino acid” is meant an organic compound that has a structure similar to a natural amino acid, where it mimics the structure and reactivity of a natural amino acid. The unnatural amino acid as defined herein generally increases or enhances the properties of a peptide (e.g., selectivity, stability, binding affinity) when the unnatural amino acid is either substituted for a natural amino acid or incorporated into a peptide.

Unnatural amino acids and peptides including such amino acids are described in U.S. Pat. Nos. 6,566,330 and 6,555,522.

Other features and advantages of the invention will be apparent from the following description of the desirable embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains drawings executed in color (FIGS. 10, 11, 12, 14, and 21). Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee.

FIGS. 1A and 1B depict a novel phospho-motif-based library vs. library screen to identify phosphoserine/threonine binding domains. FIG. 1A depicts a library of phosphothreonine-proline oriented phosphopeptides, biased toward the phosphorylation motifs for cyclin-dependent kinases and MAP kinases and toward the epitope of the monoclonal antibody MPM-2, and immobilized on Streptavidin beads. This library and its unphosphorylated counterpart were screened against 680 pools of in vitro translated ³⁵S-Met labeled proteins. pT denotes phosphothreonine. B represents a biased mixture of the amino acids P, L, I, V, F, M, W. FIG. 1B is a set of four SDS-PAGE/autoradiographs. The WW-domain containing protein Pin1 and a fragment of the mitotic kinase Plk-1, denoted by asterisks, were isolated from two pools as clones that associated preferentially with the phosphorylated form of the immobilized peptide library. In each panel, the first lane shows 10% of the input radiolabeled protein pool, while the second and third lanes show binding of proteins within this pool to the phosphorylated and unphosphorylated immobilized libraries, respectively. Identification of Pin1 and Plk1 occurred through progressive subdivision of their respective pools to single clones (panels on right). Arrowheads indicate partial translation or proteolytic breakdown products of Plk1 that exhibit more dramatic phospho-discrimination than the full-length transcript of the isolated Plk1 fragment, suggesting that the full-length transcript likely contains a smaller discrete phospho-binding domain.

FIG. 2A is a schematic diagram showing various C-terminal truncations of Plk-1, translated in vitro, and assayed for selective binding to the phosphorylated peptide library of FIG. 1A over its unphosphorylated counterpart. The two shaded regions in the C-terminus of Plk-1 correspond to its polo boxes (PB1 and PB2) as defined by Pfam. Truncated constructs were designed according to boundaries of sequence homology within the polo-like kinase family rather than boundaries of the Pfam-delineated polo boxes. Clone 407-C6 is the fragment of Plk-1 isolated from the screen depicted in FIGS. 1A and B.

FIG. 2B shows an amino acid sequence alignment of the C-terminal noncatalytic region of human Plk-1 (SEQ ID NO: 77), Xenopus Plx-1 (SEQ ID NO: 78), and Drosophila Polo (SEQ ID NO: 79). Bold lines indicate the designated polo boxes (PB1 and PB2) of Plk-1 as defined by Pfam.

FIGS. 3A-3D are histograms showing the binding ratios of the Plk-1 polo-box domain (PBD). The Polo-box Domain (PBD, residues 326-603) of Plk-1 was expressed as a GST fusion protein, immobilized on Glutathione-agarose beads, and incubated with phosphothreonine/serine-oriented degenerate peptide libraries consisting of the sequences MAXXXXpTPXXXXAKK (SEQ ID NO: 26) (3A), MAXXXXpSPXXXXAKK (SEQ ID NO: 27) (3B), MAXXXXSpTXXXXAKK (SEQ ID NO: 28) (3C), or MAXXXXSpSXXXXAKK (SEQ ID NO: 29) (3D) where X indicates all amino acids except Cys. Following extensive washing, bound peptides were eluted and sequenced. The bar graphs show the relative abundance of each amino acid at a given cycle of sequencing compared to its abundance in the starting peptide library mixture. The Plk-1 PBD selects for serine in the pThr/Ser-1 position strongly (5.9 or 8.1) and for proline in the pThr/Ser+1 position moderately (1.6 or 1.8).

FIG. 3E is an autoradiograph. Pin1 (3E) shows an absolute requirement for proline in the pThr+1 position, whereas the PBD of Plk-1 does not. Full-length Pin1 and the PBD (residues 326-603) of Plk-1 were translated in vitro in the presence of ³⁵S-methionine and tested for binding to four immobilized peptide libraries that differed by phosphorylation status and/or the presence of proline in the pThr+1 position.

pTP=biotin-ZGZGGAXXBXpTPXXXXAKKK (SEQ ID NO: 30),

TP=biotin-ZGZGGAXXBXTPXXXXAKKK (SEQ ID NO: 31),

pT=biotin-ZGZGGAXXXXpTXXXXXAKKK (SEQ ID NO: 32),

T=biotin-ZGZGGAXXXXTXXXXXAKKK (SEQ ID NO: 33),

where pT is phosphothreonine, Z indicates aminohexanoic acid, X denotes all amino acids except Cys, and B is a biased mixture of the amino acids P, L, I, V, F, M, W.

FIGS. 4A-D shows isothermal titration calorimetry results. These results show that Plk1 PBD binds its optimal phosphopeptide ligand with high affinity and high specificity.

FIG. 4E is a table. Isothermal titration calorimetry (ITC) was used to determine binding constants (K_d) for the association of the Plk-1 PBD (residues 326-603) with its optimal phosphopeptide ligand and with nine mutated versions of this peptide. All observed binding stoichiometries were consistent with a 1:1 complex of PBD and phosphopeptide. N.D.B indicates no detectable binding by ITC for a Plk-1 PBD concentration of at least 150 μM. pT, pS, and pY denote phosphothreonine, phosphoserine, and phosphotyrosine, respectively. The following sequences are shown: PoloBoxtide-optimal (SEQ ID NO:3), PoloBoxtide-8T (SEQ ID NO:34), PoloBoxtide-8pS (SEQ ID NO:65), PoloBoxtide-8pY (SEQ ID NO:66), PoloBoxtide-7V (SEQ ID NO:67), PoloBoxtide-7A (SEQ ID NO:68), PoloBoxtide-7G (SEQ ID NO:69), PoloBoxtide-7C (SEQ ID NO:70), PoloBoxtide-7T (SEQ ID NO:71), and PoloBoxtide-9N (SEQ ID NO:72).

FIG. 5A upper panel shows a FACS (fluorescence activated cell sorter) trace of human cells used in the pull-down assays shown below. The upper left panel shows the FACS profile of the cells arrested with aphidocolin in G1 (so the total DNA content is 1N where N=the normal amount of DNA in a diploid human cell) and verifies that the cells were in G1. The right trace shows the FACS profile of the cells arrested with nocadozole to trap them in G2/M, and shows that their DNA content is 2N, verifying that they are arrested in G2/M. FIG. 5A (lower panel) and 5B are immunoblots showing that the Plk-1 PBD associates with mitotic phosphoproteins in HeLa cells. Lysates from HeLa cells, arrested at interphase with aphidicolin or in G2/M with nocodazole, were incubated with GST, GST-Pini, and the GST-Plk-1 PBD (residues 326-603; FIG. 5A). Mitotic phosphoproteins co-precipitated with these GST fusions were detected by blotting with the pSer-Pro specific monoclonal antibody MPM-2. Interaction of the GST-Plk-1 PBD (residues 326-603) with mitotic phosphoproteins from nocodazole-arrested HeLa cells was disrupted by pre-incubation of GST-Plk-1 PBD with its optimal phosphopeptide ligand, MAGPMQ-S-pT-P-LNGAKK (SEQ ID NO: 2) (PoloBoxtide-optimal), but not with an unphosphorylated equivalent peptide, MAGPMQ-S-T-P-LNGAKK (SEQ ID NO:34) (PoloBoxtide-8T), nor a phosphopeptide whose serine at pThr-1 was mutated to valine (PoloBoxtide-7V; FIG. 5B).

FIGS. 6A, 6C, and 6D are immunoblots showing that Plk-1 PBD interacts with Thr₁₃₀of mitosis-dependent phosphorylated Cdc25C from HeLa cells. FIG. 6A is an anti-CDC25 western blot on lysates from HeLa cells arrested in interphase with aphidicolin or in G2/M with nocodazole, incubated with a GST fusion of the Plk-1 PBD (residues 326-603). Endogenous Cdc25C from mitotic lysates was precipitated with GST-Plk-1 PBD and detected by anti-Cdc25C (Santa Cruz Biotechnology). Interaction of GST-Plk-1 PBD with Cdc25C was disrupted as in FIG. 5B by pre-incubation of GST-Plk-1 PBD with its optimal phosphopeptide ligand (PoloBoxtide-optimal) but not with the PoloBoxtides-8T or -7V. FIG. 6B is a sequence alignment showing that a consensus motif for the Polo-box Domain of Plk-1 is conserved between human (SEQ ID NO: 80) and Xenopus Cdc25C (SEQ ID NO: 81). T130 and T138 of human and Xenopus Cdc25C, respectively, are known to be phosphorylated during mitosis (FIG. 6B). Lysates were prepared from HeLa cells transfected with either wild type, T130A, or S129V HA-Cdc25C (human), arrested in G2/M with nocodazole, and normalized for equal loading of the mitotically up-shifted form. Interaction of GST-Plk-1 PBD (residues 326-603) with mitotically phosphorylated Cdc25C from these lysates was detected by pull-down with glutathione beads, separation by 11.4% SDS-PAGE and anti-HA blotting (FIG. 6C). FIG. 6D shows lysates, analyzed by 9% SDS-PAGE to enhance separation of the hyper-phosphorylated (P) form of Cdc25C from partially phosphorylated and unphosphorylated (U) forms.

FIG. 7A is a set of micrographs visualized using fluorescence microscopy. FIG. 7B is a histogram showing the ratio of centrosomal localization by the GST-PBD relative to centrosomal γ-tubulin. U2OS cells were arrested in G2/M with nocodazole and then incubated with 4 μM GST-Plk-1 PBD (residues 326-603) in cell permeabilization buffer containing 1 U/ml Streptolysin-O in the presence of no peptide (upper panel), 250 μM of the optimal phosphopeptide (optimal, middle panel), or 250 μM of the corresponding unphosphorylated analogue (8T, lower panel). Following incubation, the cells were washed extensively, fixed with paraformaldehyde, extracted with Triton X-100, immunostained for GST and γ-tubulin, and counterstained with DAPI to visualize the nucleus. Overlap of the GST (Alexa Fluor 488) and γ-tubulin (Texas Red) signals is shown in the merged figure in the far right column (FIG. 7A). The ratio of centrosomal localization by the GST-PBD relative to centrosomal γ-tubulin levels is shown in FIG. 7B.

FIG. 8 is a schematic diagram showing a model for 2-step activation of Cdc25 and Cdc2/Cyclin B auto-activation through Plk-1. Phosphorylation of a few molecules of Cdc25, either by a small amount of de-repressed Cdc2/Cyclin B or another proline-directed kinase early in mitosis, primes those Cdc25 molecules for binding of Plk-1 through its PBD. Activation of the Plk-1 kinase domain by Plkkl generates the first wave of Cdc25 activation, dephosphorylating more Cdc2/Cyclin B, which, in turn, phosphorylates additional Cdc25 molecules for interaction with the Plk-1 PBD. The net result is a positive feedback loop for Cdc2/Cyclin B activation (circled).

FIG. 9A is a table showing the conservative mutations at the pT-1 serine that abolish Plk1 PBD/peptide binding in solution. Isothermal titration calorimetry was used to determine binding affinities. The Plk1 PBD (residues 326-603) was expressed in E. coli as a GST fusion, purified on glutathione agarose, proteolytically digested from GST, and further purified by anion exchange chromatography. N.D.B. indicates no detectable binding for a Plk1 PBD concentration of at least 150 μM. pT denotes phosphothreonine. Throughout FIGS. 9A and 9B, the domains are depicted as follows: kinase: white; PC: gray; PB1: red; PB2: blue; The following sequences are shown: PoloBoxtide-optimal (SEQ ID NO:3), PoloBoxtide-7A (SEQ ID NO:68), PoloBoxtide-7G (SEQ ID NO:69), PoloBoxtide-7C (SEQ ID NO:70), and PoloBoxtide-7T (SEQ ID NO:71).

FIG. 9B is a filter array that shows binding of GST-Plk1 PBD (residues 326-603) to peptide spots, comprising single point mutants of the Plk1 PBD optimal phosphopeptide (right column). Bound GST-Plk1 was detected by blotting with HRP-conjugated anti-GST antibody.

FIG. 10A is a schematic diagram showing the boundaries of the PBD by limited proteolysis. Domain architecture of full-length Plk1 and stable fragments (left) are shown together with the time-course of V8 protease digestion (right). Molecular weight and amino acid boundaries of the limiting domain were determined by mass spectroscopy.

FIG. 10B is a schematic diagram showing the Polo-box 1 and Polo-box 2 β₆α structures, colored as in (A), are shown superimposed.

FIG. 10C is a RIBBONS representation (Carson, 1991) of the structure of the Plk1 PBD in complex with a phosphothreonine-containing peptide shown as a ball and stick representation in yellow. The Polo-boxes and Polo-cap region are colored as in (A). The phosphopeptide binds at one end of a pocket formed between the two polo boxes.

FIG. 11A shows a structure-based sequence alignment of the Polo-box Domain family. The following sequences are shown: HsPlk1 (SEQ ID NO: 82), MmPlk1 (SEQ ID NO: 83), RnPlk1 (SEQ ID NO: 84), CePlk1 (SEQ ID NO: 85), DmPolo (SEQ ID NO: 86), XlP1X1 (SEQ ID NO: 87), HpPlk1 (SEQ ID NO: 88), HsPlk2 (SEQ ID NO: 89), MmPlk2 (SEQ ID NO: 90), RnPlk2 (SEQ ID NO: 91), CePlk2 (SEQ ID NO: 92), XlP1x2 (SEQ ID NO: 93), HsPlk3 (SEQ ID NO: 94), MmPlk3 (SEQ ID NO: 95), RnPlk3 (SEQ ID NO: 96), XlP1X3 (SEQ ID NO: 97), SpPlol (SEQ ID NO: 98), and ScCdc5 (SEQ ID NO: 99). Residues with 100% conservation are shaded purple while highly conserved residues are shaded cyan.

FIG. 11B is an image of the molecular surface of the PBD based on the structure determined by X-ray crystallography. The surface positions corresponding to the conserved residues are colored as in FIG. 11A. The most highly conserved residues within the Plk1 PBD are located exclusively on the peptide-binding face of the PBD. The most highly conserved residues within the Plk1 PBD are located exclusively on the peptide binding face of the PBD. The coloring scheme is as in 11A.

FIG. 11C is a schematic diagram depicting the electrostatic potential of the PBD phosphopeptide pocket, calculated using GRASP (Nicholls et al., 1991), with the phosphopeptide superimposed in stick representation (oxygen atoms, red; nitrogen atoms, blue). Negative potential of the PBD surface is colored red and positive potential blue.

FIG. 11D is a schematic representation of the interactions between the phosphopeptide (blue) and the Plk1 PBD. Hydrogen bonds, van der Waals interactions, and water molecules are denoted by dotted lines, purple crescents, and green circles, respectively.

FIG. 11E is a schematic representation of direct and indirect hydrogen bonds (dotted lines) between the phosphate and the Plk1 PBD. Hydrogen bond lengths are given in angstroms.

FIG. 12A is a schematic diagram showing a comparison of the β-sandwich folds of the Plk1 PBD and the Sak polo-box dimer. Tertiary structures are shown on the top together with secondary structure topology (triangles, β strands; rectangles, α-helices) on the bottom. PB1 and PB2 of Plk1 are denoted by red and purple colors, respectively, while the Pc of Plk1 is shown in green. Polo-boxes from separate Sak molecules within the dimer are likewise denoted by red and purple. The Sak β sandwich involves strand swapping between separate polo-boxes within the dimer.

FIG. 12B is a sequence alignment of the Polo-boxes from Plk1 (HsPlk1_pb1, SEQ ID NO: 100; and HsPlk1_pb2, SEQ ID NO: 101) and Sak (SEQ ID NO: 102). Plk1 has a β6α secondary topology while Sak has a circularly altered β5αβ topology. β-sheet and α-helix notation follows PB1; the corresponding elements for PB2 are β7 through β12 and αC. A conserved salt-bridging interaction initially observed in the Sak structural analysis (Leung et al., Nat. Struct. Biol. 9:719-724, 2002) is shown by the blue bracket. Conserved non-polar residues are highlighted in blue and residues conserved between Sak and at least one of the Plk1 PBDs are boxed.

FIG. 13A is an autoradiograph. Wild type and mutant Plk1 PBD (residues 326-603) were translated in vitro in the presence of ³⁵S-methionine and examined for binding to an immobilized pThr-Pro-oriented library and its unphosphorylated counterpart. pTP=biotin-ZGZGGAXXBXpTPXXXXAKKK SEQ ID NO: 30, TP=biotin-ZGZGGAXXBXTPXXXXAKKK SEQ ID NO: 31, where pT is phosphothreonine, Z is aminohexanoic acid, X is all amino acids except Cys, and B denotes a biased mixture of the amino acids P, L, I, V, F, M, W.

FIG. 13B is a diagram showing isothermal titration calorimetry results. A H538A/K540M mutation of the Plk1 PBD abolishes binding to its optimal phosphopeptide as measured by isothermal titration calorimetry.

FIG. 13C is a Western blot showing that mutation of the H538/K540 pincer disrupts interaction of the isolated Plk1 PBD with Cdc25 in vivo. HeLa cells were transfected with wild type and mutant versions of a His-Xpress-tagged Plk1 PBD construct (residues 326-603) or with a control Plk1PBD construct lacking the second Polo-box (residues 326-506) and arrested in G2/M with nocodazole. The Plk PBD was pulled down with Ni²⁺ beads and bound endogenous proteins analysed by SDS-PAGE and blotted for Cdc25.

FIG. 13D is a Western blot showing that mutation of the H538/K540 pincer in the Plk1 PBD disrupts interaction of full-length Plk1 with Cdc25 in vivo. HeLa cells were transfected with wild type and mutant versions of full-length myc-tagged Plk1 and arrested in G2/M with nocodazole. Plk-myc was immunoprecipitated with anti-myc-conjugated beads and Cdc25 binding to Plk1 analyzed as in 13C.

FIG. 14 is a series of photomicrographs showing that mutation of the H538/K540 pincer sequence abolishes centrosomal localization of the Plk1 PBD in HeLa Cells. U2OS cells were arrested in G2/M with nocodazole and then incubated with 4 μM wild-type or mutant GST-Plk1 PBD (residues 326-603) in cell permeabilization buffer containing 1 U/ml Streptolysin-O. Following incubation, the cells were washed extensively, fixed with paraformaldehyde, extracted with Triton X-100, immunostained for GST and γ-tubulin, and counterstained with DAPI to visualize the nucleus. Overlap of the GST (Alexa Fluor 488) and γ-tubulin (Texas Red) signals is shown in the merged figure in the far right column.

FIG. 15 is a series of diagrams showing the results of FACS analysis. HeLa cells were transfected with wild type and mutant GFP-tagged Plk1 (residues 326-603) for 32 hours. Cells were harvested, stained with Hoechst 33342, and analyzed by FACS to determine DNA content in the total cell populations (left panels). Similar analysis limited to the transfected cell population was performed by gating only on the GFP expressing cells (right panels). G2/M population percentages are averages from three independent experiments.

FIG. 16A is a Western blot that phosphopeptide binding by full-length Plk1 is reduced relative to that for the isolated Plk1 PBD. Approximately 10% of input full length Plk1 (residues 1-603) interacted with an immobilized pThr-Pro oriented library with slight preference over the unphosphorylated library analogue. The phosphorylation-dependent component of binding arose from the PBD, as it was eliminated by mutation of the His538/K540M pincer. In contrast, phosphopeptide binding by the isolated PBD (FIG. 13A) was 10-fold greater and considerably more phospho-dependent.

FIG. 16B is a graph showing that the optimal PBD phosphopeptide stimulates full-length Plk1 kinase activity. GST-Plk1 (prepared in SF9 cells) was preincubated without peptide (closed circles), with 250 μM of the optimal PBD phosphopeptide (open squares) or with 250 μM of the non-phosphorylated optimal peptide counterpart (closed squares) for 5 minutes at room temperature prior to initiating the kinase reaction by addition of ATP. [32P]-incorporation into casein was determined by SDS-PAGE electrophoresis, autoradiography, and densitometry. Pre-incubation with the optimal PBD phosphopeptide ligand enhanced the rate of casein phosphorylation by Plk1 by a factor of 2.6 as determined from three independent experiments.

FIG. 16C is a schematic diagram depicting a model for Plk1 regulation by the PBD. PB1 and PB2 are shaded orange, kinase domain cyan, phosphopeptide purple with phosphate in red. Inhibitory interactions between the PBD and the kinase domain in the basal state (left) are relieved by phosphopeptide binding, which may also stabilize association of the two Polo-boxes (right).

FIG. 17A is an autoradiograph showing the identification of phosphoSer/Thr-binding domains using an ATM/ATR-motif library. An oriented (pSer/pThr) phosphopeptide library, biased toward the phosphorylation motifs for ATM/ATR kinases, was immobilized on Streptavidin beads. This phosphopeptide library [pSQ=biotin-ZGZGGAXXXB(pS/pT)QJXXXAKKK (SEQ ID NO:35)] and its non-phosphorylated counterpart were screened against in vitro translated ³⁵S-Met labeled proteins. (pS/pT) denotes 50% phosphoserine and 50% phosphothreonine; Z indicates aminohexanoic acid; B represents a biased mixture of the amino acids A, I, L, M, N, P, S, T, V; and J represents a biased mixture of 25% E, 75% X, where X denotes all amino acids except Arg, Cys, His, and Lys. PTIP, denoted by arrow, was isolated from pool EE11 as a clone that associated preferentially with the phosphorylated form of the immobilized peptide library. In each panel, the first and second lanes show binding of proteins within the pool to the phosphorylated and non-phosphorylated libraries, respectively. Identification of PTIP occurred through progressive subdivision of the EE11 pool to a single clone (panel on right denoted by asterisk). Longer exposures revealed partial translation or proteolytic breakdown products of PTIP that also exhibit phospho-discrimination, suggesting that the full-length transcript likely contains a smaller discrete phospho-binding domain. The uppermost band is a fusion artifact of PTIP with vector sequences resulting from translation initiation at an upstream ATG in the vector.

FIG. 17B is an autoradiograph showing deletion mapping of the phospho-binding domain of PTIP. Truncations of PTIP were translated in vitro and assayed for selective binding to the phosphorylated peptide library as in FIG. 17A. Shaded regions in the C-terminus of PTIP correspond to its BRCT domains. Truncation constructs were designed according to boundaries of sequence homology within the BRCT domain, boundaries from sequence alignments, and from the Pfam-delineated BRCT domains (Bateman et al., Nucleic Acids Res 27: 260-2, 1999).

FIG. 18A is an autoradiograph. PTIP, BRCA1, MDC1, 53BP1 and Rad9 tandem BRCT domains were translated in vitro in the presence of 35S-methionine and tested for binding to immobilized phosphopeptide and non-phosphopeptide libraries as described in FIG. 17A. The peptide libraries used were pSQ as defined in FIG. 17A. pS=biotin-ZGZGGAXXXXpSXXXXXAKKK SEQ ID NO: 36; pT=biotin-ZGZGGAXXXXpTXXXXXAKKK SEQ ID NO: 32, where pS is phosphoserine, pT is phosphothreonine, Z indicates aminohexanoic acid, and X denotes all amino acids except Cys. Both PTIP and BRCA1 tandem BRCT domains display stronger binding to the pSQ and pS libraries as compared to the non-phospho libraries. Domain boundaries: PTIP as indicated in FIG. 1 (SEQ ID NO: 15); BRCT1 and 2: amino acids 1634-1863 of SEQ ID NO: 12; BRCT1 alone: amino acids 1634-1751 of SEQ ID NO: 12; BRCT2 alone: 1725-1863 of SEQ ID NO: 12; MDC1: amino acids 1880-2089 of SEQ ID NO: 37(NP_—055456.1); 53BP1: amino acids 1700-1972 of SEQ ID NO: 38 (NP 005648.1); Rad9: amino acids 1025-1309 of SEQ ID NO: 39 (NP_—010503.1).

FIGS. 18B and C are autoradiographs showing that the PTIP and BRCA1 BRCT domains show strong selection for Phe at the (pSer/pThr)Gln +3 position (7.0 or 7.5), respectively. Tandem BRCT domains of PTIP and BRCA1 were immobilized as glutathione-S-transferase (GST) fusion proteins on glutathione beads and incubated with non-biotinylated versions of the oriented degenerate phosphopeptide libraries described in FIG. 17A (XXXB-pS/pT-QJXXX, SEQ ID NO: 118). Following extensive washing, bound peptides were eluted and sequenced. Bar graphs show the relative abundance of each amino acid at a given cycle of sequencing compared to its abundance in the starting peptide library mixture, as described (Yaffe et al., Methods Enzymol 328:157-70, 2000).

FIGS. 18D, 18E, 18F, and 18G show binding of GST-PTIP and BRCA1 tandem BRCT domains to a filter array of peptide spots, comprising single point mutants of the optimal BRCT domain phosphopeptide (left column). Bound GST-BRCT domains were detected by blotting with HRP-conjugated anti-GST antibody. The resulting consensus binding motif is indicated in the right column, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, and SEQ ID NO: 122 for FIGS. 18D, 18E, 18F, and 18G, respectively; wherein X denotes no dominant selection, φ denotes residues with aliphatic or aromatic side chains, and letters enclosed in square brackets are specifically de-selected. The top row indicates the amino acid that was substituted for the optimal amino acid. Substitution of pSer for pThr enhanced binding for both PTIP and BRCA1 BRCT domains, consistent with the ITC results. Substitution of pTyr for pThr eliminated binding altogether, verifying that tandem BRCT domains are pSer/pThr-specific binding modules. Replacement of pThr with Thr, Ser or Tyr abrogated tandem BRCT domain binding. The pTQ oriented blots on the left show strong selection at several positions for both PTIP and BRCA1 BRCT domains; especially for Phe in the +3 position in agreement with the oriented peptide library screening data. The pS oriented blots on the right show that the +3 position is the most important position for peptide selection.

FIG. 19A is a Western blot. Lysates from U2OS cells were obtained prior to and 2 hours after the cells were exposed to 10 Gy of ionizing radiation (IR). The lysates were incubated with GST-PTIP tandem BRCT domains, and bound proteins were detected by blotting with the anti-ATM/ATR phosphoepitope motif antibody. Interaction of the PTIP BRCT domains with these phosphoproteins from IR treated cells was disrupted by pre-incubation with the pSQ peptide library, but not with the SQ peptide library or the pTP library.

FIG. 19B is a Western blot showing that the interaction of the PTIP BRCT domains with DNA damage induced phosphoproteins from IR treated U2OS cells was disrupted by pre-treating the cells with caffeine (25 mM) prior to IR exposure or by pre-incubating the beads with an optimal BRCT-binding peptide (BRCTtide-opt), but not by preincubating the beads with the peptide's non-phosphorylated counterpart (BRCTtide-7T).

FIG. 19C is a Western blot showing that tandem BRCT domains of PTIP interact with 53BP 1 following DNA damage. Endogenous 53BP 1 from IR treated U2OS cells was precipitated with GST-PTIP tandem BRCT domains and detected by incubating with an anti-53BP1 antibody. Interaction of GST-PTIP tandem BRCT domains with HA-tagged 53BP1, was then detected by anti-HA blotting. This interaction was abolished by treating the lysates with lambda phosphatase, by pre-incubating the beads with an optimal BRCT-binding peptide (BRCTtide-opt), but not with its non-phosphorylated counterpart (BRCTtide-7T), or by preincubating the beads with the pSQ library, but not by preincubating with the SQ library or the pTP library. Treatment of the cells with 25 mM caffeine also disrupted the interaction.

FIG. 19D is a Western blot. Lysates from U2OS cells 2 hours following IR were incubated with GST-BRCA1 tandem BRCT domains. DNA damage-induced phosphoproteins were detected by blotting with the anti-ATM/ATR phosphoepitope motif antibody. The interaction of the GST-BRCA1 tandem BRCT domains with the phosphoproteins were disrupted as in panel B. These results show that tandem PTIP and BRCA1 BRCT domains associate with DNA damage-induced phosphoproteins through their phosphopeptide-binding pockets.

FIGS. 20A-C are photomicrographs showing immunofluorescence in U2OS cells demonstrating that full length PTIP forms DNA damage induced foci and co-localizes with (pSer/pThr)-Gln proteins, 53BP1, and γ-H2AX. FIG. 20A shows U2OS cells transfected with a full length PTIP-GFP construct (PTIP-FL residues 1-757). FIG. 20B shows U2OS cells transfected with a PTIP deletion construct in which the last two BRCT domains were removed (PTIP-ABRCT, residues 1-550). FIG. 20C shows U2OS cells transfected with a PTIP construct containing only the last two BRCT domains (BRCT)₂, residues 550-757). In FIGS. 20A-20C, 24 hours following transfection cells were either treated with 10 Gy of ionizing radiation or mock irradiated, allowed to recover for 2 hours, stained, and analyzed by immunofluorescence microscopy.

FIGS. 21A and B are photomicrographs showing immunofluorescence in U2OS cells demonstrating that caffeine attenuates recruitment of PTIP to DNA damage foci in response to ionizing radiation. U2OS cells transfected with full-length PTIP-GFP cDNA were mock treated or pretreated with 10 mM caffeine for 70 minutes before exposure to 10Gy ionizing radiation. (A) In response to IR, mock-treated U2OS cells formed nuclear foci containing PTIP (in green) and H2AXp (in red); these two proteins co-localize at sites of DNA damage (merge). (B) In response to IR, caffeine treated U2OS cells formed reduced numbers of nuclear foci; PTIP was mislocalized and did not form discrete nuclear foci (in green) and there were reduced numbers of H2AXp (in red) containing foci; pretreatment with caffeine effectively abolished co-localization of PTIP and H2AXp (merge).

FIG. 22 shows the PTIP amino acid sequence (SEQ ID NO: 15).

FIG. 23 shows the PTIP nucleic acid sequence (SEQ ID NO: 13).

FIG. 24 shows the BRCA1 amino acid sequence (SEQ ID NO: 12).

FIGS. 25A-C show the BRCA1 nucleic acid sequence (SEQ ID NO: 11).

FIG. 26 shows the MDC1 amino acid sequence (SEQ ID NO: 37).

FIGS. 27A-C show the MDC1 nucleic acid sequence (SEQ ID NO: 40).

FIG. 28 shows the 53BP1 amino acid sequence (SEQ ID NO: 38).

FIGS. 29A-C show the 53BP1 nucleic acid sequence (SEQ ID NO: 41).

FIG. 30 shows the Rad9 amino acid sequence (SEQ ID NO: 39).

FIGS. 31A-B show the Rad9 nucleic acid sequence (SEQ ID NO: 42).

DESCRIPTION OF THE INVENTION

The present invention features a method for identifying kinase targets, an exemplary kinase target, the Polo box domain of the Polo-like kinase, and exemplary peptide mimetics that interfere with signaling by the Polo-like kinase.

We have developed a proteomic approach that allows us to identify virtually any peptide-binding domain by simultaneously screening a polypeptide expression library with a biased peptide library. We have used this method to identify, for example, targets downstream of kinases in signaling pathways. This strategy involves using an immobilized library of partially degenerate phosphopeptides, biased toward a kinase phosphorylation motif, to isolate interacting effector proteins targeted by substrates of that kinase. Using this approach for cyclin-dependent kinases, we identified the Polo-box Domain (PBD) of the mitotic kinase Plk-1 as a phosphoserine/threonine binding domain. Polo-like kinases (Plks) perform crucial functions in cell-cycle progression and multiple stages of mitosis. Plks are characterized by the presence of a C-terminal non-catalytic region containing two tandem Polo-boxes, termed the Polo-box domain (PBD).

In addition, we have discovered that the PBDs of human, Xenopus, and yeast Plks all recognize similar phosphoserine/threonine-containing motifs. The 1.9 Å X-ray structure of a human Plk1 PBD-phosphopeptide complex shows that the Polo-boxes β₆α structures. They associate to form a novel 12-stranded β-sandwich domain, to which the phosphopeptide-binds within a conserved, positively-charged cleft located at the edge of the Polo-box interface. Mutations designed to specifically disrupt phosphodependent interactions abolish cell-cycle dependent localization and provide compelling phenotypic evidence that PBD-phospholigand binding is necessary for proper mitotic progression. In addition, phosphopeptide-binding to the PBD stimulates kinase activity in full-length Plk1, suggesting a conformational switching mechanism for Plk regulation and a dual functionality for the PBD. Together, our data reveal a central role for PBD-phosphoprotein interactions in many, if not all, cellular functions of Plks. This finding provides a structural explanation for how Plk-1 localizes to specific sites within cells in response to Cdk phosphorylation at those sites.

Activation of signaling cascades in eukaryotic cells involves the directed assembly of protein-protein complexes at specific locations within the cell. This process is controlled by protein phosphorylation on serine, threonine and/or tyrosine residues that directly or indirectly regulate protein-protein interactions, often through the actions of modular binding domains. Historically, studies of phospho-binding domains have focused on SH2 and PTB domains, which bind to specific phosphotyrosine-containing sequence motifs. Until recently, it was thought that phosphorylation of proteins on serine and threonine residues was not responsible for direct interactions with modular binding domains but instead induced conformational changes to regulate function. However, a number of domains (14-3-3 proteins, FHA domains, WD40 repeats of F-box proteins, MH2 domains and the WW domain of the prolyl isomerase Pin1) have been identified that bind directly to short phosphoserine or phosphothreonine-containing sequences to control cell cycle progression, coordinate the response to DNA damage, and regulate apoptosis.

The vast majority of intracellular proteins are phosphorylated on serine or threonine residues at some point during their lifetime. Furthermore, known phosphoserine/threonine binding domains comprise a diverse structural group, demonstrating that many divergent tertiary folds have acquired a phospho-dependent binding function through evolution. Approximately one-third of the modular protein domains identified by Pfam and SMART on the basis of sequence homology have no known function. Our technique enables the identification of additional phosphopeptide binding modules that target serine/threonine residues.

2×2 Biased Library Screening

To design a general proteomic screen capable of identifying novel phosphoserine/threonine binding modules, we took advantage of the observation that protein kinases and phosphopeptide binding domains seem to have co-evolved to recognize overlapping sequence motifs (Yaffe et al., Nat. Biotechnol. 19:348-353, 2001; Obata et al., J. Biol. Chem. 275:36108-36115, 2000). For example, the basophilic protein kinase, Akt, phosphorylates substrates at sites that contain the core motif RXRSX[S/T] (SEQ ID NO: 43) and 14-3-3 proteins bind to a subset of these phosphorylated sites that have the optimal motif RSX[pS/pT]XP (SEQ ID NO: 44). Cyclin-dependent kinases (Cdks) phosphorylate substrates at [S/T]PXR (SEQ ID NO: 45) motifs, and the WW domain of the proline isomerase Pin1 recognizes the phosphorylated forms of these [pS/pT]P sites to mediate isomerization of the proline residue. Importantly, this apparent overlap between kinase and phospho-binding motifs is not perfect. Instead, limited overlap allows combinatorial interactions between substrates of particular kinases and downstream binding modules.

Our motif-based strategy for identifying pSer/Thr-binding domains involved biasing a library of partially degenerate phosphopeptides towards the phosphorylation motif of a kinase and then using an immobilized form of this library as bait in a screen for interacting proteins translated in vitro from a cDNA library.

Using a library of phosphopeptides biased towards motifs phosphorylated by cyclin-dependent kinases (Cdks), we identified the C-terminal Polo-box containing region of the human Polo-like kinase, Plk-1, as a specific phosphopeptide recognition module. It has been previously shown that this non-catalytic region is critical both for Polo kinase subcellular localization and for proper mitotic progression in yeast and human cells. Our findings provide the first description of a biochemical mechanism through which Plk-1 performs these essential mitotic functions. Furthermore, the identification of the conserved Plk-1 PBD as the latest member of the growing superfamily of pSer/Thr-binding domains suggests that phospho-specific docking may be a general mechanism for Ser/Thr kinase signaling in eukaryotic biology.

To identify pSer/Thr-binding domains involved in cell cycle regulation, we designed a pThr-Pro-oriented peptide library biased to resemble the motif that would be generated by the action of cyclin-dependent kinases and MAP kinases, as well as that recognized by the mitotic phosphoprotein-specific monoclonal antibody MPM-2, whose pSer/Thr-binding motif we had determined previously (Yaffe et al., Science 278:1957-1960, 1997). The library was constructed with a flexible linker and an N-terminal biotin tag, allowing an immobilized form of this library to be used as bait in an interaction screen against a library of proteins produced by in vitro expression cloning (Lustig et. al., Methods Enzymol 283:83-99, 1997; FIG. 1A).

This library vs. library screening approach is the reverse of a traditional peptide library screen in which a single purified domain is assayed against a degenerate peptide library to reveal the optimal binding motif. In the approach presented here, a degenerate but motif-biased peptide library is used to screen for novel binding domains. By using a collection of peptides biased towards the motif of a protein kinase superfamily, the screen casts a larger net than would be possible if only a single peptide were used as bait. To control for phospho-independent peptide binding, an identical library was constructed with Thr substituted for the fixed pThr residue (FIG. 1A).

The pThr-Pro-oriented peptide library, and its non-phosphorylated Thr-Pro library counterpart were immobilized on Streptavidin beads and screened in parallel against 680 individual pools of in vitro translated [³⁵S]-labeled proteins. Each pool contains ˜30 radiolabeled proteins/pool that are detectable by SDS-PAGE/autoradiography (FIG. 1B, “pool” lanes). As shown in FIG. 1B, proteins produced by in vitro translation often failed to bind either library at all or bound more strongly to the non-phosphorylated peptide library-containing beads. However, we identified 7 distinct pools containing radiolabeled translation products that bound preferentially to the pThr-Pro library compared with the Thr-Pro library (asterisks in FIG. 1B).

Plasmid pools containing these positively scoring hits were progressively subdivided and re-screened for phospho-binding until individual clones were isolated and sequenced. Of the 7 positive clones, 3 were successfully recovered, two of which are reported here. One of the clones, 109-B7, was found to encode the prolyl isomerase Pin1, which is known to bind and isomerize pThr-Pro motifs recognized by the monoclonal antibody MPM-2. Its isolation, therefore, validated the feasibility of our screening approach.

A second positively scoring hit, clone 407-C6, was found to encode the C-terminal 80% of the mitotic kinase Plk-1 (polo-like kinase-1, amino acids 95-603). This clone was missing critical components of the Plk-1 kinase domain, including the glycine rich loop (amino acids 60-66) and the invariant lysine (K82), implying that phosphopeptide binding was independent of Plk-1 kinase activity. Phospho-specific binding by the full-length transcript of this incomplete Plk-1 clone was less pronounced than binding by Pin1 (FIG. 1B). Partial translation products or proteolytic breakdown fragments arising from this clone (FIG. 1B, arrowheads) showed strong discrimination for the phosphorylated peptide library, suggesting that these fragments included a functional phosphopeptide binding domain.

Identification of Polo-Box Domain as a Phosphopeptide Recognition Module

A hallmark feature of the Polo kinase family is the presence of a highly conserved C-terminal region downstream from a conserved amino-terminal kinase domain (FIGS. 2A and B). This region includes two blocks of strong homology, termed Polo Boxes. To define the limiting fragment of Plk-1 responsible for phosphospecific binding, we generated a series of deletion constructs based on an alignment of the C-terminal regions of human Plk-1, Xenopus Plx-1 and Drosophila Polo (FIG. 2B), and analyzed these deletion fragments for phosphopeptide-specific binding. As shown in FIG. 2A, a construct that began immediately after the kinase domain and extended to the last residue of the protein (residues 326-603) demonstrated strong and specific binding to the phosphothreonine-proline peptide library compared with the non-phosphorylated control. Notably, this construct was superior to the parent clone 407-C6 in discriminating for phosphopeptides. Neither of the individual Polo Boxes alone (denoted PB1 and PB2), nor a construct containing both Polo Boxes but lacking the linker region between the kinase domain and PB1, was capable of phosphopeptide binding (FIG. 2A). Furthermore, a construct that included the linker region and PB1 but not PB2 was also unable to bind phosphopeptides. Thus, it appears that the linker region together with both Polo-boxes functions together as a single phosphopeptide-binding module, and we therefore propose that this segment be called the Polo-box Domain (PBD). Intriguingly, this region encompassing both Polo-boxes has been previously shown to regulate the localization of Plk-1 to centrosomes and kinetochores during prophase and to the midbody during late stages of mitosis. Significantly, neither Polo-box alone was sufficient for this localization function, though mutations within PB1 were sufficient to disrupt it.

The Plk-1 Polo-Box Domain Consensus Motif

A central feature of our screen for phosphopeptide-binding domains is that any pSer/Thr-binding domain identified through interaction with phosphopeptide library-immobilized beads is amenable to subsequent determination of its optimal binding motif using a standard “forward” peptide library screening approach. A GST fusion protein of the Plk-1 PBD was therefore expressed in bacteria, immobilized on glutathione beads, and incubated with degenerate phosphopeptide libraries oriented on a fixed pThr-Pro (FIG. 3A) or pSer-Pro motif (FIG. 3B). Following extensive washing, the PBD-bound peptides were eluted and sequenced, and the amount of each amino acid in every degenerate position was compared to that present in the starting library mixture to derive amino acid selectivity ratios. Surprisingly, the Plk-1 PBD displayed an extraordinarily strong and novel selection for Ser in the pThr-1 position when the pThr-Pro library was used. Extremely strong selection for Ser was also observed in the −1 position when the PBD was assayed using the fixed pSer-Pro library. Binding of the PBD to a phosphoserine-containing peptide library is noteworthy in itself, since at least one other family of phosphopeptide-binding modules, FHA domains, appear to bind only to phosphothreonine-containing motifs. The relative selection values observed for Ser in either the pThr-1 or pSer-1 position, 5.9 and 8.1 respectively, are among the largest we have observed for any domain whose specificity has been previously determined by peptide library screening.

Since the Plk-1 PBD was isolated in a screen for domains that bind to pThr-Pro motifs, it was important to determine the relative importance of Pro in the pThr+1 position for PBD recognition. To accomplish this, peptide library screens were performed with libraries containing a fixed pThr residue, a fixed pSer residue, fixed Ser-pThr residues, or fixed Ser-pSer residues (Table 1, FIGS. 3C, and 3D). Little selection was observed for proline in the pThr/pSer+1 position when serine was not fixed in the pThr/pSer-1 position (Table 1). Inclusion of serine at this position in a Ser-pThr oriented library, however, unmasked a moderate selection (1.7) for proline at pThr+1 (FIG. 3C and Table 1). Proline selection (1.8) was also uncovered at this position when a Ser-pSer oriented library was used (FIG. 3D and Table 1). Notably, synergistic selection between serine and proline was also observed in reverse such that inclusion of a fixed Pro residue in the peptide libraries led to a higher selection for serine (Table 1).

Table 1, below, summarizes the results obtained from phosphopeptide motif selection screening (SEQ ID NO: 103).

TABLE 1

pT and pS Peptide Motif Selection by Plk-1 Polo Box Domain

−3
−2
−1

+1

M (1.3)
A (1.4)

S (5.9)

pT
P

Y (1.3)
H (1.4)
A (1.6)

H (1.3)
M (1.4)

F (1.2)

T (1.3)

K (1.2)

F (1.3)

I (1.4)
A (1.5)

S (3.7)

pT
X

K (1.4)
Q (1.3)
A (1.6)

T (1.2)
G (1.3)

M (1.5)
Q (1.5)
S
pT
P (1.6)

F (1.4)
A (1.5)

M (1.3)

L (1.2)
H (1.5)

M (1.4)

F (1.3)

T (1.2)

M (1.7)

T (1.9)

S (8.1)

pS
P

Y (1.5)
H (1.7)

H (1.4)
M (1.5)

F (1.3)

F (1.4)

K (1.2)

F (1.4)

T (1.9)

S (6.0)

pS
X

M (1.3)
H (1.4)

Y (1.3)
M (1.3)

A (1.3)

M (1.6)
M (1.6)
S
pS
P (1.8)

F (1.3)
Q (1.5)

M (1.3)

Y (1.3)
H (1.5)

L (1.2)
A (1.3)

T (1.3)

A GST fusion of the Plk-1 Polo Box Domain was screened for binding to six phosphopeptide libraries, which contained the sequences MAXXXXpTPXXXXAKKK SEQ ID NO: 46, MAXXXXpTXXXXAKKK SEQ ID NO: 47, MAXXXXSpTXXXXAKKK SEQ ID NO: 48, MAXXXpSPXXXAKKK SEQ ID NO: 49, MAXXXXpSXXXXAKKK SEQ ID NO: 50, and MAXXXXSpTXXXXAKKK SEQ ID NO: 48, where X indicates all amino acids except Cys. Residues showing strong enrichment are underlined. Selection for Pro (1.4) was observed in the −4 position in the X₄SpTX₄and X₄SpSX₄screens. Slight selection for aliphatic and aromatic residues was observed in the +2 position in most screens. Little or no selection was observed in the −5, +3, +4, or +5 positions in any of the screens.

These results suggested that the presence of Pro in the pThr/pSer+1 position, while helpful, was not absolutely required for binding. In agreement with this, the Plk-1 PBD bound in a phospho-specific manner to bead-immobilized peptide libraries containing either a fixed pThr-Pro dipeptide or an isolated pThr alone (FIG. 3E). In contrast, the other protein isolated in our screen, full-length Pin1, bound only to the pThr-Pro peptide library beads.

To verify the results of oriented peptide library screening, binding of individual phosphopeptides to the Plk-1 PBD was measured by isothermal titration calorimetry (FIGS. 4A and 4B). The optimal phosphopeptide ligand (PoloBoxtide-optimal), containing the core sequence Met-Gln-Ser-phoshoThr-Pro-Leu (SEQ ID NO: 51) derived from peptide library screening, bound tightly to the Plk-1 PBD with a dissociation constant of 280 nM. Furthermore, it formed a 1:1 protein/peptide complex, indicating that separate phosphopeptides were not interacting simultaneously with each of the two polo boxes within the PBD. Substitution of threonine for phosphothreonine (PoloBoxtide 8T) resulted in complete loss of binding, reiterating the absolute dependence of interaction on the presence of a phosphate group. Substitution of phosphoserine for phosphothreonine within the optimal PBD motif maintained peptide binding to the Plk-1 PBD in agreement with the peptide library screening results, albeit with a seven-fold drop in affinity. In contrast, substitution of phosphotyrosine for phosphothreonine completely abrogated binding, demonstrating conclusively that the Plk-1 PBD is a pThr/pSer-specific binding domain. The extraordinarily strong selection observed for Ser in the pThr/pSer-1 position within the Plk-1 PBD binding motif was confirmed using a series of mutant peptides. When this Ser was replaced with either of the sterically small amino acids Ala or Gly, with the hydroxyl containing amino acid Thr, or with the homologous amino acid Cys, no peptide binding was detectable. Moderate selection for Pro in the pThr/pSer+1 position was verified by a greater than five-fold increase in K_dwhen another β-turn forming residue, Asn, was substituted for Pro in this position. Based on the oriented peptide library screening data (FIG. 3, Table 1) and these ITC results, we therefore propose that the core consensus motif recognized by the Plk-1 PBD is S-[pT/pS]-(P/X) (SEQ ID NO: 52).

Physiological Substrates of PBD

The monoclonal antibody MPM-2 (Mitotic Phosphoprotein Monoclonal-2), originally raised against mitotic HeLa cell extracts, recognizes a conserved pSer/pThr-Pro epitope present on ˜50 phosphoproteins that are localized to various mitotic structures. The initial screen from which the Plk-1 PBD was identified used a peptide library that was partially biased to resemble the MPM-2 epitope. A number of important mitotic regulators that are recognized by this antibody, including Cdc25, Weel, Mytl, Topoisomerase II alpha and inner centromere proteins (INCENP), contain one or more exact matches of the S-[pS/pT]-P PBD-binding motif We therefore investigated whether the Plk-1 PBD bound to MPM-2 reactive proteins. HeLa cells were treated with aphidocolin to induce a G1/S arrest or with nocodazole to induce a G2/M arrest and cell lysates were analyzed by immunoblotting (FIG. 5A). As expected, the number of MPM-2 reactive proteins was greatly enhanced in the mitotically-arrested cells. Many of these MPM-2 reactive mitotic phosphoproteins were specifically bound by the Plk-1 PBD, suggesting that phosphorylation of these proteins by proline-directed mitotic kinases generated a PBD-binding site. Furthermore, the Plk-1 PBD bound to a different and somewhat smaller subset of MPM-2 epitope-containing proteins than those that bound to Pin1 (FIG. 5A), which was expected given that the MPM-2 epitope motif more closely resembles the optimal consensus motif for Pin1 than that of the Plk-1 PBD.

To determine whether the Plk-1 PBD associates with MPM-2 epitopes through its phosphopeptide binding pocket, peptide competition assays were performed. Pre-incubation of the Plk-1 PBD with its optimal phosphopeptide ligand dramatically inhibited the binding of MPM-2 epitopes (FIG. 5B, ‘opt’). In contrast, the non-phosphorylated analogue (‘8T’) or a peptide with Val substituted for Ser in the pT-1 position (‘7V’) had no effect.

One particular MPM-2 antigen that is also known to be phosphorylated and regulated by Plk-1 and its Xenopus homologue is the cell-cycle regulated protein phosphatase Cdc25. We therefore investigated whether Cdc25C associated with the Plk-1 PBD in a cell-cycle-regulated and phospho-specific manner. During mitosis, Cdc25C undergoes a dramatic reduction in gel mobility due to extensive phosphorylation at its N-terminus. The Plk-1 PBD was found to interact only with this mitotically up-shifted form of Cdc25C (FIG. 6A). Pre-incubation of the Plk-1 PBD with its optimal phosphopeptide ligand, but not with the 8T or 7V mutant peptides, completely prevented this association, demonstrating that it was mediated through the phosphopeptide binding pocket of Plk-1. During mitosis, Cdc25C is known to be phosphorylated on five conserved Ser/Thr-Pro sites within its N-terminus. One of these sites, Thr₁₃₀(corresponding to Thr₁₃₈in Xenopus Cdc25C) contains a conserved Plk-1 PBD consensus motif (FIG. 6B). To investigate whether this site was important for the Cdc25C-Plk-1 interaction, HeLa cells were transfected with HA-tagged wild-type Cdc25C, or with Thr₁₃₀Ala or Ser₁₂₉Val point mutants of Cdc25C expected to disrupt the PBD-binding motif. Following mitotic arrest with nocodazole, the Plk-1 PBD bound strongly only to the wild-type protein, but only very weakly to either of the point mutants, indicating direct interaction between the Plk-1 PBD phosphopeptide-binding pocket and a mitotically-phosphorylated PBD consensus motif in Cdc25C (FIG. 6C). Furthermore, both of these point mutants had a decreased electrophoresis mobility shift when analyzed on lower percentage gels (FIG. 6D), suggesting that mutations which impair Plk-1 PBD binding result in incomplete Cdc25C phosphorylation in vivo.

Centrosomal Localization of the Plk-1 PBD Occurs Through its Phosphopeptide-Binding Pocket.

Plk-1 localizes to centrosomes and kinetochores in prophase and to the spindle mudstone during late stages of mitosis. Centrosomal localization has been shown to require both the PB1 and PB2 regions, but not kinase activity, since localization is maintained when Lys₈₂, which is mediates phosphate transfer, is mutated to Met. To investigate whether the phosphopeptide binding function of the Plk-1 PBD was critical for its centrosomal localization, U2OS cells were mitotically arrested with nocodazole, permeablized with Streptolysin-O, and incubated with GST-Plk-1 PBD in the absence or presence of peptide competitors. The Plk-1 PBD was observed to localize to the centrosomes of late prophase-arrested cells (FIG. 7A), as verified by co-staining with an anti-γ-tubulin antibody.

This centrosomal localization was significantly disrupted in the presence of an optimal Plk-1 PBD phosphopeptide but was unaffected when the assay was performed using the same concentration of the non-phosphorylated peptide analogue (FIGS. 7A and 7B). This observation, together with published data showing that the C-terminus of Polo-like kinases is essential for their function in vivo, strongly suggests that intracellular targeting of Plk-1 to critical substrates is mediated through interaction of the PBD phosphopeptide pocket with phosphorylated motifs in mitotic structures.

The Plk-1 PBD and Regulation of Mitotic Progression by Cyclin-Dependent Kinase Priming

Our identification of the Plk-1 PBD as a novel phosphoserine/threonine-binding domain adds another member to the growing superfamily of pSer/Thr-binding modules and demonstrates the general utility of our phospho-motif-based affinity screen for discovering and functionally characterizing novel signaling domains that function downstream of protein kinases. This screening technique can be used to identify binding modules interacting with substrates of any kinase whose phosphorylation motif is known. Other techniques that identify protein-protein and protein-peptide interactions, such as yeast 2-hybrid and phage display approaches cannot be used in screens for phospho-binding domains since reliable and constitutive phosphorylation of a diverse collection of bait sequences is required. A further strength of our technique is that any domain isolated through screening with bead-immobilized peptide libraries yields an optimal consensus binding motif when the domain is subsequently analyzed by traditional peptide library screening. This allows the motif for the pSer/Thr-binding domain to be combined with that of the potential phosphorylating kinase(s) in database searching and protein sequence analysis and should facilitate the proteome-wide prediction of ligands within a common signaling pathway.

The C-terminal region of Polo-like kinases has long been recognized as essential for their in vivo function in mitosis and cytokinesis, but its structural mechanism has remained mysterious. Mutations within this region of Plk-1 and its S. cereviseae homologue, CdcS, abolish their ability to rescue a temperature-sensitive mutant of cdc5 despite the presence of a fully functional kinase domain. When expressed alone, the C-terminal domain of Polo-like kinases localizes to centrosomes and the spindle midzone similar to the full-length kinase, and its overexpression causes mitotic and cytokinetic arrest.

We have shown that the C-terminal domain of Plk-1 is a phosphoserine/threonine-binding module whose phospho-binding pocket binds to known Polo substrates and mediates localization to subcellular sites where endogenous Polo kinases are found. In the basal state the PBD binds to the kinase domain, inhibiting its phosphotransferase activity. In addition to overcoming this inhibition, maximal activation of the kinase domain also requires phosphorylation in its activation loop by upstream kinases such as xPlkk1/SLK. This requirement for both priming phosphorylation of substrates and activation loop phosphorylation provides a molecular switch that regulates Plk-1 kinase function at discrete stages of the cell cycle. In addition, it provides a potential means for mitotic checkpoint control, since neither phosphorylation of the activation loop nor substrate priming phosphorylation alone would be sufficient for proper activation of Polo kinases in vivo.

A number of striking parallels between the PBD of Plk-1, SH2 domains in Src family kinases, and FHA domains in the Rad53/Chk2 family of checkpoint kinases are apparent. Like the Plk-1 PBD, SH2 domains of Src-family kinases both inhibit kinase activity in the inactive state and facilitate substrate targeting when Src kinases have been activated by phosphorylation on their activation loops. In Src kinases, the mechanism of inhibition involves intramolecular binding of the SH2 domain to a pTyr motif at the end of the kinase domain. It remains unknown whether Polo kinase family inhibition by the PBD involves a similar interaction with internal pSer/pThr sites, or whether an alternative PBD surface is involved. Members of the Chk2 kinase family contain one or more pThr-binding FHA domains in addition to the kinase module. The FHA domain(s) are critical for proper Chk2 function in response to DNA damage and for the phospho-dependent targeting of Chk2 into larger multimolecular complexes where activation occurs.

We found the optimal motif for Plk-1 PBD binding to be S-[pS/pT]-P/X. Differences in PBD selectivity for amino acids flanking the pSer/Thr position are likely to be biologically important for the interaction of Polo kinases with their substrates in vivo. The primary role of the +1 Pro may be to link phospho-dependent PBD binding to activation of cyclin-dependent kinases that phosphorylate the motif, providing a means to temporally and spatially regulate the action of Polo-like kinases during mitosis. The absolute requirement for Ser in the −1 position provides strong discrimination for Plk-1 binding to only a limited subset of mitotic kinase substrates. In addition, we found that the motif recognized by the Plk-1 PBD partially overlaps with the proline-directed sequence motif recognized by the monoclonal antibody MPM-2 which reacts against a large number of mitotically phosphorylated proteins, and we demonstrated a direct interaction between the PBD phosphobinding pocket and MPM-2 reactive proteins in pull-down experiments with mitotic cell extracts. This finding provides an elegant explanation for the progressive accumulation of MPM-2 immuno-reactivity and Polo kinase localization observed at maturing centrosomes, and suggests that generation of MPM-2 epitopes by Cdks and other mitotic kinases triggers PBD-mediated recruitment of Polo kinases to specific mitotic structures.

Both Cdks and Polo kinases have been implicated in activating the phosphatase Cdc25, leading to desphosphorylation and activation of Cdc2/Cyclin B and progression through mitosis. The relative roles of Cdks and Polo kinases in Cdc25 activation, however, remains controversial. Our finding that the Plk-1 PBD binds to one or more critical Cdk sites on Cdc25C suggests a molecular rationale for 2-step activation of Cdc25 that has been postulated to drive auto-amplification of Cdc2/CyclinB activity. In prophase, low levels of Cdc2/CyclinB activity are insufficient to fully activate Cdc25, but provide priming phosphorylation of Cdc25 for interaction with the PBD. Subsequent activation of Polo kinases later in mitosis by activation loop kinases such as Plkkl/SLK leads to an initial wave of Cdc25 activation, which generates more Cdc2/Cyclin B activity, primes additional Cdc25 molecules for activation by Polo-like kinases, and results in a positive feedback loop for the production of additional Cdc2/Cyclin B activity (FIG. 8). This model is able to explain the result of Toyoshima-Morimoto et al. (EMBO Rep., 3:341-348, 2002) that maximal intracellular targeting and activation of Cdc25, even in the presence of constitutively active Plk-1, still requires the co-expression of Cyclin B1.

Increased levels of Plk expression have been detected in a variety of human tumors and tumor cell lines, and high levels of expression correlate with poor prognosis. The PBD would be an attractive target for the design of anti-proliferative chemotherapeutics since its compact tripeptide binding motif may be particularly amenable to the design of small molecule peptidomimetics.

Optimal phosphopeptide-binding motifs for the PBDs from all members of the human Plk family, Xenopus Plx1 and Saccharomyces cerevesiae Cdc5p were determined by oriented peptide library screening as described above. Since we initially isolated the Plk1 PBD in a search for domains that recognize a pThr-Pro-containing motif, primary screens were performed using peptide libraries containing a fixed pThr-Pro core flanked on both sides by four degenerate positions. As seen in Tables 2 and 3, the five PBD's examined each selected for distinct but largely overlapping motifs: Plk1 (SEQ ID NO: 104), Plk2 (SEQ ID NO: 105), Plk3 (SEQ ID NO: 106), Plx 1 (SEQ ID NO: 107), and CdcS (SEQ ID NO: 108).

TABLE 2

Phosphothreonine Peptide Motif Selection by

Human Polo Kinase Family PBDs

−5
−4
−3
−2
−1

+1
+2

Plk1

M (1.5)
M (1.3)
A (1.4)

S (5.9)

pT
P
F (1.2)

F (1.1)
Y (1.3)
H (1.4)
A (1.6)

I (1.2)

H (1.3)
M (1.4)

K (1.2)

F (1.2)
T (1.3)

K (1.2)
F (1.3)

P (1.4)
P (1.5)
M (1.5)
Q (1.5)
S
pT
P (1.6)
L (1.2)

F (1.1)
F (1.3)
F (1.4)
A (1.5)

M (1.3)
K (1.1)

M (1.3)
L (1.2)
H (1.5)

V (1.1)

L (1.2)

M (1.4)

I (1.1)

F (1.3)

T (1.2)

Plk2

F (1.9)
Q (1.9)
T (2.1)

S (7.5)

pT
P
F (1.5)

I (1.6)
M (1.8)
H (2.1)

L (1.5)

M (1.5)
H (1.6)
Q (1.2)

I (1.3)

L (1.4)
F (1.3)

V (1.1)

P (1.1)

P (2.4)

M (1.5)
Q (1.9)

T (2.8)

S
pT
P (1.7)
K (1.5)

F (1.4)
F (1.5)
T (1.6)
H (2.0)

L (1.2)

I (1.2)
P (1.4)
M (1.6)
Q (1.7)

I (1.1)

L (1.4)
H (1.6)

I (1.3)
F (1.2)

V (1.2)

Plk3

I (1.5)
M (1.6)
T (1.6)

SP (3.0)

pT
P
K (1.3)

L (1.4)
L (1.3)
H (1.4)

V (1.2)

V (1.3)
F (1.3)

F (1.2)

F (1.2)

P (1.2)

P (1.2)
L (1.2)
A (1.5)

T (2.6)

S
pT
P (1.6)
K (1.4)

I (1.2)
M (1.2)
H (1.6)

D (1.4)

F (1.2)

E (1.3)

I (1.2)

GST fusions of the Polo-box Domains (PDBs) from hPlk1, hPlk2, and hPlk3 were screened for binding to phosphopeptide libraries containing the sequences MAXXXXpTPXXXXAKKK (SEQ ID NO: 46) and MAXXXXSpTXXXXAKKK (SEQ ID NO: 48), where X indicates all amino acids except Cys. Residues showing strong enrichment are underlined.

TABLE 3

Phosphothreonine Peptide Motif Selection

by Polo Kinase PBD Orthologs

−5
−4
−3
−2
−1

+1
+2

Plx1

F (2.1)
F (1.5)
T (2.1)

S (7.3)

pT
P
I (1.6)

I (1.6)
L (1.5)
H (1.7)

L (1.5)

L (1.3)
M (1.5)

V (1.1)

M (1.2)

P (1.8)
P (1.6)
F(1.6)

T (3.0)

S
pT
P (1.9)
K (1.4)

F (1.4)
F (1.5)
M (1.5)
H (1.6)

I (1.3)

L (1.5)
L(1.4)
Q (1.3)

L (1.2)

I (1.4)

M (1.3)

Cdc5

M (1.9)

A (2.5)

T (2.4)

S (5.3)

pT
P
X

L (1.5)
M (1.5)
A (1.8)

I (1.4)
F (1.1)
Q (1.5)

F (1.2)

M (1.4)

H (1.4)

P (2.8)

L (2.2)

A (3.4)

A (2.1)
S
pT
P (1.4)
L (1.3)

F (1.3)
M (1.7)
V (1.3)
Q (1.7)

I (1.1)

I (1.5)
I (1.2)
T (1.6)

F (1.5)

H (1.6)

V (1.1)

M (1.3)

GST fusions of the Polo-box Domains (PDBs) from Xenopus Plx1 and S. Cerevisiae Cdc5p were screened for binding to phosphopeptide libraries containing the sequences MAXXXXpTPXXXXAKKK (SEQ ID NO: 46) and MAXXXXSpTXXXXAKKK (SEQ ID NO: 48), where X indicates all amino acids except Cys. Residues showing strong enrichment are underlined.

All of the PBDs showed unequivocal selection for Ser in the pThr-1 position with selectivity ratios (i.e. the mol % of Ser in the PBD-bound peptides at the pThr-1 position divided by the mol % of Ser in the starting library mixture at the pThr-1 position) ranging from 3.0 to 7.5. Motif similarity occurs even though these PBDs vary considerably in amino-acid sequence and the respective human Plks perform divergent cellular functions. The PBDs as a group consistently demonstrated moderate selection for Thr, His, Gln, and Met in the pThr-2 position. There was general selection amongst all PBDs for aliphatic and aromatic residues in the pThr-3, pThr-4 and pThr+2 positions, although Cdc5p showed a particularly strong and unique selection for Ala in the pThr-3 position, while Plk2 showed strong and unique selection for Gln at this position. All PBDs except Cdc5p also selected for Pro in the pThr-4 position and Lys in the pThr+2 position

Based on these data, secondary peptide libraries containing a fixed Ser-pThr core were used to further refine the motifs and investigate the relative importance of Pro in the pThr+1 position. These screens revealed modest selection for Pro at pThr+1 for all PBDs, with selectivity ratios ranging from 1.4 to 1.9 (Tables 2 and 3). Selection at other motif positions for each PBD was consistent with those obtained using the pThr-Pro library, though we were now able to observe significant and conserved selection for Pro and Phe in the pThr-5 position. (pT-5 was degenerate in the Ser-pThr library, but was a fixed Ala residue in the pThr-Pro-oriented library.) Thus, it appears that the PBDs of all Plks investigated, including all conventional human Plk homologues, select a similar motif that can be most generally represented by the consensus sequence: [Pro/Phe]-[φ/Pro]-[φ/Ala_Cdc5p/Gln_Plk2]-[Thr/Gln/His/Met]-Ser-[pThr/pSer]-[Pro/X] SEQ ID NO: 2, where φ represents hydrophobic amino acids.

The striking selection observed for Ser in the pThr-1 position in all PBDs was examined in detail for the human Plk1 PBD, which binds to its optimal motif, Pro-Met-Gln-Ser-pThr-Pro-Leu (SEQ ID NO:6) (Table 2), with a K_dof 280 nM (FIG. 9A).

A variety of small side-chain amino-acids were therefore substituted in the pThr-1 position, and peptide binding to the Plk1 PBD measured using isothermal titration calorimetry (ITC) (FIG. 9A). Surprisingly, replacement of Ser with Gly, Ala, the hydroxyl-containing amino-acid Thr, or the Ser isostere Cys, completely abrogated Plk1 PBD-phosphopeptide binding. We had previously observed that replacement of Ser at the pThr-1 position with Val, the amino-acid showing the lowest selection in this position, was sufficient to eliminate peptide binding (Elia et al., Science 299:1228-1231, 2003). Nevertheless, the finding that replacement of Ser with a variety of chemically similar amino acids also completely disrupted the interaction between the PBD and free phosphopeptides in solution was unexpected.

To extend this analysis, each amino acid in the eight positions flanking the phosphothreonine within the optimal Plk1 PBD binding motif was substituted with each of the remaining nineteen naturally occurring amino acids using a solid phase array of immobilized phosphopeptides (FIG. 9B). This conclusively demonstrated that only Ser was tolerated in the pThr-1 position (FIG. 9B). Selectivities at other positions were generally consistent with the results of oriented peptide library screening. Cys and Gly, however, were selected at the pThr+1 position at least as strongly as Pro in the immobilized phosphopeptide assay. Cys is routinely omitted during construction of oriented peptide libraries to minimize cross-linking and oxidation effects. Higher relative selection for Gly in the context of immobilized peptides than in solution phase peptide library assays may be due, in part, to the greater entropic penalties associated with ordering Gly residues compared with Pro residues when both ends of a peptide are free. Alternatively, these subtle differences may reflect the fact that the peptide filter assay examines individual point mutations in the context of a single amino-acid sequence, while oriented peptide library screening samples an entire ensemble of sequence motifs simultaneously. Regardless, Pro probably represents the most ‘physiological’ amino acid in the pThr+1 position, since the phosphorylation event necessary for PBD binding is likely to be catalyzed primarily by Pro-directed kinases such as Cdks and MAP kinases.

Overall Structure of the Plk1 PBD

The boundaries of the minimal PBD within the C-terminal regions of both Plk1 and Cdc5p were determined using limited proteolysis and mass-spectrometry. Studies using V8 protease (FIG. 10A) and trypsin (data not shown) indicated that only the last 45 residues of the linker between the kinase domain and the first Polo-box were structured as part of the PBD (FIG. 10A). Similar results were obtained using the C-terminal segment of Cdc5p (data not shown). We refer to the beginning of this additional region as the Polo-cap (Pc). For both Plk1 and Cdc5p, we found no significant difference in the phosphopeptide-binding affinities of fragments encompassing the entire C-terminal regions or the proteolytically-defined PBDs, indicating that the first ˜40 amino acids between the kinase and the Pc plays no major role in peptide binding. Shorter fragments of both Plk1 and Cdc5p encompassing just the Polo boxes, but lacking the Pc, were insoluble in E. coli, indicating a clear structural role for the Pc in both proteins, despite the absence of any extensive sequence homology between the two proteins in this region.

The X-ray structure of a recombinant form of the proteolytically-defined Plk1 PBD (residues 367-603) in complex with its ‘optimal’ phosphopeptide was solved by multiwavelength anomalous diffraction (MAD) using Se-Met-containing protein, and refined against native data extending to 1.9 Å resolution (Table 4).

TABLE 4

Crystallographic analysis

Data Collection

Dataset (λÅ)
Native (0.98)
Se (0.97838)
Se (0.97887)
Se (0.95)

14.1 - SRS

14.2 - SRS

d (Å)
20.0-1.9
20.0-3.5
20.0-3.5
20.0-3.5

Completeness (%)
97.7
99.9
99.0
99.2

Redundancy¹
3.6
3.7³
~1.9³
~1.9³

R text missing or illegible when filed

(%)²
5.3
5.4³
5.2³
4.9³

Phasing analysis

Resol bin (Å)
20-11.2
11.2-7.5
7.5-6.0
6.0-5.2
5.2-4.6
4.6-4.2
4.2-3.9
3.9-3.6

FOM
0.79
0.83
0.79
0.70
0.59
0.53
0.48
0.44

Mean FOM
0.60

Refinement

R text missing or illegible when filed

(%)⁴
R text missing or illegible when filed

(%)⁵

(Å)
rmx text missing or illegible when filed

(deg.)

24.0
26.8
0.007
1.2

¹N text missing or illegible when filed

²R

− S_j|<I> − I_j|/S<I> where I_jis the intensity of the jth reflection and <I> is the average intensity.

³Calculated with Bijvoets seperated

⁴R text missing or illegible when filed

= S

|F_res− F_calc|/S text missing or illegible when filed

⁵R

= as for R text missing or illegible when filed

but calculated on 5% of the data excluded from the refinement calculation.

text missing or illegible when filed

indicates data missing or illegible when filed

The structure (FIG. 10B) shows that the PBD contains two β₆α motifs that comprise the two Polo-box regions (PB1 & 2) identified by sequence profiling. The atomic structural coordinates of this structure are provided in Table 5. In spite of the fact that the amino-acid sequences of the two Polo-boxes within any one Plk exhibit only ˜20-25% sequence identity, the structures of the two motifs are quite similar (root mean square (rms) deviation of 77 Cα atoms of 1.6 Å; FIG. 10B). The two Polo-boxes pack together to form a 12-stranded β-sandwich flanked by three α-helical segments (FIG. 10C). Although motifs resembling the Polo-box structure are represented in the Protein Databank, the overall domain structure represents a new protein fold.

The Pc consists of an α-helical segment αA, loop, and short 3₁₀helix which connects to the N-terminal β-strand of Polo-box 1 (β1) through a ˜10 residue linker region (L1). The Pc wraps around Polo-box 2 like a hook tethering it to Polo-box 1. αA packs against αC from PB2 in an anti-parallel coiled-coil arrangement, while the 3₁₀helix packs against the shorter αC′. The two Polo-boxes are connected by a second ˜30 residue linker sequence (L2) that is partially conserved. L1 and L2 run in anti-parallel directions between the two Polo-box β-sheets. Thus, the hydrophobic core is formed from direct interactions of highly conserved non-polar residues predominantly located on β1/β2 from PB1 and β6/β7 from PB2, together with an array of interactions with the intercalating linker regions.

Novel PBD-Phosphopeptide Interactions are Crucial for Specificity

The phosphopeptide binds in a largely extended conformation to a region of positive charge, located at one end of a shallow cleft formed between the two Polo-boxes (FIG. 10). In all, ˜1000 Å²of solvent accessible surface are buried by binding of the seven phosphopeptide residues that are visible in our electron density maps. Binding involves part of an extensive, highly conserved surface that is located exclusively on the peptide-binding face of the PBD (FIG. 11A, 11B). This conserved surface coincides with the only significant region of positive electrostatic potential within the entire PBD (FIG. 11C). Overall, the phosphopeptide interacts predominantly with β1 from PB1, the N-terminal end of L2 and β8 and 9 from PB2. Hydrogen bonding interactions formed with the peptide side- and main-chain atoms alternate to some degree between residues within the two Polo-boxes, forming a zipper-like structure at the edge of the PB1/PB2 interface (FIG. 11D).

PBD binding to the phosphate moiety involves a combination of direct contacts with protein side-chains together with extensive indirect interactions through a well-defined lattice of water molecules, many of which are fully hydrogen-bonded (FIG. 11E). In total, the phosphate group participates in eight hydrogen-bonding interactions explaining the critical dependence on peptide phosphorylation for binding (Elia et al., Science 299:1228-1231, 2003). The only residues that contact the phosphate group directly are His-538 and Lys-540 from PB2, whose side chains form a pincer-like arrangement that chelates the O1, O3, and Oγ phosphate oxygens.

The structural basis for the extraordinarily high selectivity for serine at the pThr-1 position results from a major difference in orientation of the bound phosphopeptide when compared with phosphopeptide complexes of 14-3-3 proteins and FHA domains, the two major classes of pSer/pThr binding proteins (Durocher et al., Mol. Cell. 6:1169-82, 2000; Yaffe et al., Cell 91:961-971, 1997). In these structures, the pThr-1 side-chain is solvent exposed and little selection is observed at this position. In contrast, the peptide orientation in the Plk1 complex is inverted such that the Ser-1 side-chain is directed towards the Plk1 surface (FIG. 11B). In this orientation, it engages in two hydrogen bonding interactions with Trp-414 main-chain atoms, and one with the Leu-491 main-chain carbonyl via a water molecule (FIG. 11C). Significantly, the Ser-1 Cβ atom makes favourable van der Waals interactions with Cδ1 from the Trp-414 indole side-chain. This explains why even a conservative replacement of Ser with Thr at this position abrogates peptide binding (FIG. 9A), presumably due to a steric clash of the threonine γ-methyl substituent with Trp-414.

The critical role of Trp-414 in ligand binding revealed by our crystal structure (FIG. 11D) explains the observation that a W414F mutation eliminates both centrosomal localization of Plk1 and its ability to complement the cdc5-1 is mutation (Lee et al., Proc. Natl. Acad. Sci. USA 95:9301-9306, 1998). Both of these effects are likely to be at least partly attributable to disruption of critical Ser-1 interactions with the PBD. In agreement with this, a mutant PBD containing the W414F substitution is severely compromised in phosphopeptide binding, with an affinity of >100 μM as determined by ITC. Loss of binding is unlikely to result from gross structural perturbation of the Polo-box fold, since the mutant PBD exhibits similar secondary structural content to the wild-type protein as judged from far UV CD spectra (data not shown). Furthermore, Trp-414 in Polo-box 1 is replaced by tyrosine in PB2 of both wild-type S. pombe Plol and S. cerevisiae Cdc5p PBD's, (FIG. 11A), showing that similar substitutions are naturally tolerated in a related structural context.

Consistent with the oriented library selection, the protein-peptide interface is dominated by interactions of the PBD with the pThr and Ser-1 (FIG. 11C, 11D). Although we observed modest selection for Pro at the pThr+1 position, it appears from the structure that it does not contribute greatly to the binding interface, and multiple substitutions at this position are tolerated for peptide binding (FIG. 9B). In the PBD structure, the trans-proline introduces a kink after the Ser-pThr directing the peptide backbone back toward the binding surface, allowing the pThr+2 main chain amino group to contact the PBD. Thus, the +1 Pro likely increases binding affinity by diminishing the entropic penalty for making this favorable backbone contact. This contrasts with structures of pSer-Pro peptide complexes of both the Pin1 WW and the Cdc4 WD40 domains in which the Pro+1 side chain inserts into a hydrophobic pocket and makes coplanar interactions with a buried tryptophan (Leung et al., Nat. Struct. Biol. 9:719-724, 2002; Verdecia et al., Nat Struct Biol 7:639-643, 2000).

Plk1 and Sak Polo-boxes are Structurally Distinct—One Motif, Two Folds

The human Plk family encompasses the canonical kinases (Plks 1-3) and Sak, which contains a highly homologous Ser/Thr kinase domain but only a single divergent Polo-box. Recent structural data has shown that the isolated Polo-box from murine Sak forms an intermolecular dimer, leading to the suggestion that tandem Polo-boxes in Plk1-related Plks may form a related, intra-molecular ‘dimeric’ architecture (Leung et al., Nat. Struct. Biol. 9:719-724, 2002). Our structure shows that this notion is broadly correct. In each case, the Polo-box repeat comprises a six-stranded β-sheet and α-helix. This structural unit associates with a second Polo-repeat via intra- or intermolecular interactions in Plk1 and Sak respectively, to form β-sandwich domain structures. However, closer examination reveals profound differences between the organizations of the two structures (FIGS. 12A and 12B). The β₆α topology of the Plk1 Polo-box is replaced by a circularly-permuted β₅αβ topology in Sak. Consequently, Plk1 β1 has no equivalent in the Sak Polo-box sequence, and instead overlaps structurally with Sak β6. In addition, the Sak β-sheet is completed by a ‘segment-swap’ of β4 & 5 between monomers. Most strikingly, the association of the two Polo-boxes differs completely such that residues forming the interface between Polo-repeats in the Sak homodimer are located largely on the exterior of the Plk1 β-sandwich, where they partially form the interface with the flanking α-helical segments.

Mutation of the His-Lys Pincer Abolishes Phosphopeptide Binding in vitro, Cdc25 Binding in vivo, and Centrosomal Localization of the Plk1 PBD

To verify that the key phosphothreonine-interacting residues identified in the X-ray crystal structure were indeed responsible for mediating phospho-dependent interactions in vitro and in vivo, we mutated His-538 and Lys-540 of the pThr pincer motif, to either Ala and Met, or Glu and Met, respectively. These mutations severely disrupt phosphopeptide binding in solution as judged by the reduced binding of in vitro translated Plk1 PBD to a bead-immobilized pThr-Pro oriented library (FIG. 13A) and by ITC (FIG. 13B).

During mitotic entry, Cdc2/Cyclin-B and Plk1 cooperate to activate the dual specificity phosphatase Cdc25 through extensive phosphorylation of its N-terminus as part of an amplification loop for Cdc2/Cyclin-B activation (Abrieu et al., J. Cell. Sci. 111:1751-1757, 1998; Hoffmann et al., EMBO J. 12:53-63, 1993; Izumi et al., Mol. Biol. Cell 4:1337-1350, 1993; Izumi et al., Mol. Biol. Cell 6:215-226, 1995; Kumagai et al., Cell 70:139-151, 1992; Kumagai et al., Science 273:1377-1380, 1996; Qian et al., Mol. Cell. Biol. 19:8625-8632, 1999; Qian et al., Mol. Biol. Cell 12:1791-1799, 2001). Mitotically phosphorylated Cdc25C exhibits a large mobility shift on SDS-PAGE (Kumagai et al., Cell 70:139-151, 1992). Cdc25C is phosphorylated on at least five Ser/Thr-Pro sites by Cdc2/Cyclin-B in vitro (Izumi et al., Mol. Biol. Cell 4:1337-1350, 1993; Strausfeld et al., J. Biol. Chem. 269:5989-6000, 1994). One of these sites, Thr-130, occurs within a near-optimal PBD binding motif, Leu-Leu-Cys-Ser-pThr-Pro-Asn (SEQ ID NO: 53). We previously observed that a GST-fusion of the isolated PBD could pull-down wild-type Cdc25C, but not a T130A or S129V Cdc25C mutant, from mitotically-arrested HeLa cell lysates. These data strongly suggested that Cdk priming of Thr-130 generates a binding site for the Plk1 PBD to facilitate full activation of Cdc25C by subsequent Plk1-mediated phosphorylation (Elia et al., Science 299:1228-1231, 2003). As shown in FIG. 13C, expression of His-Xpress-tagged wild-type Plk1 PBD in vivo results in a strong interaction with the mitotically phosphorylated form of endogenous Cdc25C in nocodazole-arrested HeLa cells. However, expression of the His-538/Lys-540 pincer mutants eliminates Cdc25C binding as also observed in cells transfected with a PBD construct lacking the second Polo-box.

To investigate whether the PBD plays a similar substrate-targeting role in the context of full-length Plk1, HeLa cells were transfected with myc-tagged wild-type or mutant constructs of full-length Plk1, and interactions between Plk1 and endogenous Cdc25C examined in nocodazole-arrested cells using immunoprecipitation and Western blotting (FIG. 13D). We observed a strong in vivo interaction between the mitotically upshifted form of endogenous Cdc25C with full-length Plk1 in arrested cells that, somewhat surprisingly, was not increased when a kinase-dead Plk1 mutant (K82R) or a double mutant incorporating a T210D mutation in the T-loop to further expose the kinase-binding cleft were employed as substrate traps. Conversely, mutation of the His-538/Lys-540 phosphate pincer mechanism in full-length Plk1 completely disrupted the in vivo interaction between Plk1 and Cdc25C demonstrating that the interaction of full-length Plk1 with full-length Cdc25 in G2/M-arrested cells is mediated primarily through the PBD, rather than its associated the kinase domain. This result is important since it directly demonstrates a requirement for PBD phosphopeptide-binding in substrate targeting in the context of the full-length Plk1 molecule.

Finally, we observed that mutation of the His-538/Lys-540 pincer eliminates targeting of the Plk1 PBD to centrosomes in permeabilized prophase-arrested cells (FIG. 6). This finding suggests that the localization of Plk1 to centrosomes observed in vivo (Jang et al., Proc. Natl. Acad. Sci. USA 99:1984-1989, 2002; Lee et al., Proc. Natl. Acad. Sci. USA 95:901-9306, 1998) results from direct interactions between the PBD and phosphorylated centrosomal components. In summary, the results in FIGS. 13 and 14 show conclusively that the structurally defined His-538/Lys-540 pincer mechanism that is responsible for mediating phosphopeptide binding in vitro, plays a similar critical role in substrate targeting in vivo.

Phosphodependent Substrate Recognition is Necessary for the Disruption of Mitotic Progression by the Isolated Plk1 PBD

Since the PBD is necessary for targeting Plk1 to primed substrates, its overexpression might be expected to act in a dominant-negative fashion to inhibit correct localization of endogenous Plk1 and, therefore, disrupt Plk1 function in vivo. Indeed, overexpression of the C-terminus of Plk1 has been shown to cause mitotic arrest and induce formation of randomly oriented, disorganized spindles (Jang et al., Proc. Natl. Acad. Sci. USA 99:1984-1989; Seong et al., J. Biol. Chem. 277:32282-32293, 2002). The X-ray structure of the PBD-phosphopeptide complex now enables us to dissect the role of phospho-specific binding in this phenotype. In agreement with previous studies, we found that overexpression of a GFP-fusion of the Plk1 PBD in HeLa cells caused a dramatic increase in the population of cells in G2/M (60% for PBD-GFP— vs. 17% for GFP-expressing cells) (FIG. 15). Importantly, this accumulation of mitotic cells was abolished by mutation of His-538 and Lys-540 (23% in G2/M). In addition, expression of the wild-type PBD-GFP construct induced aneuploidy in HeLa cells, evident as a peak of cells with DNA content >4N, in agreement with anti-Plk1 antibody microinjection studies reported by Lane and Nigg (Lane et al., J. Cell. Biol. 135:1701-1713, 1996). However, this effect was completely lost when the His/Lys pincer mutant was employed. The dominant negative effects strongly suggest that phosphopeptide-binding by the PBD in full-length Plk1 normally plays a role in both proper mitotic progression and in the establishment of a functional bipolar spindle to ensure equal chromosome segregation.

Phosphopeptide Binding to the PBD Stimulates Plk1 Kinase Activity

Lee and Erikson (Lee et al., Mol. Cell. Biol. 17:3408-3417, 1999) and Mundt et al. (Biochem. Biophys. Res. Commun. 239:377-385, 1997) observed that deletion of the C-terminus of Plk1 increased the kinase activity ˜3-fold while Jang et al (Jang et al., Proc. Natl. Acad. Sci. USA 99:1984-1989, 2002) found that the isolated Plk1 C-terminus interacts with and inhibits the activity of the isolated kinase domain towards the exogenous substrate casein. We observed the complementary result, namely that the kinase domain appears to inhibit phosphopeptide binding by the PBD. While the isolated Plk1 PBD binds strongly and specifically to pSer/pThr-containing peptides (FIG. 13A), phosphopeptide binding by the PBD within full-length Plk1 is reduced at least 10-fold, and is considerably less phospho-dependent (FIG. 16A, wt lanes). The phospho-specific binding component of full-length Plk1 is clearly mediated by the PBD (FIG. 16A, compare wt pTP and TP lanes with H538A/K540M pTP and TP lanes). This suggested that a mutually inhibitory interaction exists between the Plk1 PBD and the kinase domain in full-length Plk1.

We wondered whether binding of the PBD to phosphopeptides was sufficient to relieve this intramolecular interaction and stimulate the activity of the kinase domain towards exogenous substrates. Baculovirally-produced Plk1 was therefore incubated with either the optimal PBD phosphopeptide or its non-phosphorylated counterpart and kinase activity towards casein measured by SDS-PAGE/autoradiography. As shown in FIG. 16B, addition of the optimal PBD phosphopeptide increased Plk1 kinase activity by a factor of 2.6, while addition of the non-phosphorylated peptide had no effect. This result compares quite favourably with the ˜2.5-fold stimulation of Src and Hck kinase activity that is observed when these full-length Src family kinases are incubated with their optimal SH2-binding phosphotyrosine peptides to relieve SH2-mediated inhibition of the kinase domain (Liu et al., Oncogene 8:1119-1126, 1993; Moarefi et al., Nature 385:650-653, 1997). Thus, our results for Plk1 suggested that binding of the PBD to primed phosphorylation sites not only serves to target the kinase domain to substrates but also simultaneously activates the kinase domain for substrate phosphorylation by relieving an inhibitory intramolecular interaction (FIG. 16C).

In this study, we have elucidated a conserved phosphopeptide-binding motif that is recognized by the PBDs of all canonical members in the human Plk family, Xenopus Plx1 and S. cerevesiae Cdc5p. The high-resolution X-ray structure of the Polo-box domain bound to an optimal phosphothreonine peptide, provides a molecular rationale for motif selection, defines a new protein fold, and illustrates a unique mechanism for phospho-dependent ligand binding involving the participation of ordered solvent molecules, together with a conserved His/Lys pincer motif. We have identified a pSer/Thr-dependent mechanism of Plk activation in which intramolecular inhibition of the kinase by the PBD is relieved by PBD interaction with pre-phosphorylated binding targets.

Structural Definition of the Polo-Box Domain: A General Phosphoprotein Recognition Module

Previous reports have described the presence of 1-3 Polo-boxes within the C-terminal regions of Polo-like kinases (Glover et al., Genes Dev. 12:3777-3787, 1998; Glover et al., J. Cell. Biol. 135:1681-1684, 1996; Nigg, Curr. Opin. Cell. Biol. 10:776-783, 1998; Seong et al., J. Biol. Chem. 277:32282-32293, 2002). Our structure now definitively shows that the PBD consists of two structurally homologous regions corresponding to two conserved Polo-box sequences. Phosphopeptide binding occurs at the interface of the two Polo-boxes, rationalizing both the observed 1:1 stoichiometry of PBD/ligand binding (FIG. 5B) and the requirement for both Polo-boxes for efficient subcellular localization of Plk1 in vivo (Seong et al., J. Biol. Chem. 277:32282-32293, 2002). Polo-box Domains (PBDs) now join an expanding family of phosphoserine/phosphothreonine binding domains that includes 14-3-3 proteins, WW, FHA, WD40, and Smad MH2 domains (Yaffe et al., Curr Opin Cell Biol 13:131-138, 2001; Yaffe et al., Structure 9:R33-38, 2001). In contrast to other more ubiquitous phosphodependent binding modules, PBDs occur only in Polo-like kinases where they localize Plks to specific subcellular organelles and mitotic structures (Jang et al., 2002; Lee et al., Proc. Natl. Acad. Sci. USA 95:9301-9306, 1998; (Lee et al., Mol Cell Biol 17, 3408-3417, 1999) and target the kinase to substrates that have been primed by prior phosphorylation.

Common Phosphopeptide Motif Selection by the PBD Family

In higher eukaryotes, different Plk family members function at different points in the cell cycle (Donaldson et al., 2001; Glover et al., Genes Dev 12:3777-3787, 1998; Glover et al., J Cell Biol 135, 1681-1684, 1996; Ma et al., Mol Cancer Res 1, 376-384, 2003; Nigg, Curr Opin Cell Biol 10:776-783, 1998) or play antagonistic roles in response to DNA damage (Bahassi et al., Oncogene 21, 6633-6640, 2002; Smits et al., Nat Cell Biol 2:672-676, 2000; Xie et al., Cell Cycle 1:424-429, 2002). Given the similarity in the selected motifs with a Ser-pSer/pThr-Pro/X core for these three proteins, potential mechanisms to separate Plks within a single organism achieve substrate specificity might include different substrate selectivities by their respective kinase domains, spatially and temporally restricted activation of Plks by upstream kinases, or the well documented cell-cycle regulation of Plk1 and 2 expression (Golsteyn et al., Cell Sci 107:1509-1517, 1994; Lee et al., 1995; Ma et al., Mol Cancer Res 1:376-384, 2003). One pathway in which such specificity must be vital is the DNA damage response, since Plk1 is inhibited by DNA damage (Smits et al., Nat Cell Biol 2:672-676, 2000), while Plk3 appears to be activated (Xie et al., Cell Cycle 1:424-429, 2002).

In addition to pThr-1 selectivity for serine, all PBDs that we have examined exhibit moderate specificity for proline at the pThr+1 position, emphasizing a central role for CDKs and other proline-directed kinases in priming substrates for Plk1 targeting. Several lines of evidence support this model. For example, maximal Plk1-induced activation and nuclear translocation of Cdc25 has been shown to require cyclin B coexpression (Toyoshima-Morimoto et al., EMBO Rep. 3:341-348, 2002). Furthermore, full reconstitution of purified APC activity requires prior synergistic phosphorylation of the APC by both Cdc2 and Plk1 (Golan et al., J. Biol. Chem. 277:15552-15557, 2002). Interestingly, the backbone torsion angles of the trans-proline in the Plk1-bound phosphopeptide are very similar to those of the equivalent Pro residue in the ternary cyclinA3/CDK2/peptide complex structure (Brown et al., Nat. Cell. Biol. 1:438-443, 1999). Thus, the conformation of the peptide in the PBD complex reflects not only the structural requirements for Plk interaction but also the requirements for the initial priming phosphorylation.

Nevertheless, a clear tolerance for residues other than proline demonstrates that other mitotic kinases may also serve as priming agents. In this regard, the NIMA-related kinase Finl has been recently shown to increase Plol affinity for spindle pole bodies in S. pombe (Grallert et al., EMBO J. 21:3096-3107, 2002). Identification of substrates for Plk family members, as well as the kinases involved in substrate priming is, therefore, important.

The Structural Basis of Phosphopeptide Binding

The PBD binds to phosphorylated epitopes in a way that is distinct from that observed previously in structures of other protein-phosphopeptide complexes (Yaffe et al., Structure 9:R33-38, 2001). These differences include the His/Lys pincer, a significant contribution from bridging water molecules and an unusual orientation of the pThr-1 residue that is directed toward the protein-binding surface. Although stereospecific, solvent-mediated binding has been described in other systems, ‘solvent-bridged’ interactions with the phosphoryl group have not been observed in any structures of protein-phosphopeptide complexes reported to date. Rather, the phospho moiety is always held by direct interactions, most often with highly conserved arginine side-chains (Eck et al., Nature 362:87-91, 1993; Waksman et al., Nature 358:646-653, 1992; Yaffe et al., Structure 9:R33-38, 2001). The importance of the His/Lys pincer in the Plk1 PBD structure is exemplified by our observations that its mutation abrogates phosphopeptide binding by the PBD in vitro, targeting of Plk1 to Cdc25C in vivo, and centrosomal localization, as well as disrupt the ability of the isolated PBD to induce G2/M arrest and aberrant spindle function.

Structure-based sequence alignments (FIG. 12B) show that the binding surface formed at the interface of the two Polo-boxes is the only totally conserved region in the PBD, further supporting our finding that the PBDs from different Plks generally select very similar optimal phosphopeptide binding motifs. Crucial hydrogen-bond interactions and van der Waals contacts with Trp-414 of Plk1 rationalize both the strong serine selection at the (pThr/pSer)-1 position and the fact that mutation of Trp-414 disrupts Plk1 function in vivo (Lee et al., Proc. Natl. Acad. Sci. USA 95:9301-9306, 1998). The absolute conservation of Trp-414 predicts that all family members should exhibit the same serine preference, and we now show that this is the case. Historically, the 10 amino acid sequence surrounding Trp-414 was considered the signature motif for the non-catalytic region of Polo-family kinases (Golsteyn et al., Cell Sci. 107:1509-1517, 1994).

Comparison of the Plk1 PBD and Sak Polo-Box Structures

The Plk1 PBD and Sak Polo-box structures emphasize how related sequence motifs are able to form markedly different protein folds. Significant structural differences between homologous proteins have been observed only rarely and most prominently in the KH family of small RNA-binding domains (Grishin, Nucleic Acids Res. 29:638-643, 2001 and references therein). In this case, two distinct sub-families of structures are distinguishable by different topologies of α and β secondary structural elements although all share a related hydrophobic core and similar overall tertiary structure. The differences between the Plk1 PBD and Sak Polo-box are more extreme and emphasize how related sequence motifs are able to form markedly different protein folds. This, in turn, has considerable implications for both motif-based structure prediction and efforts to delineate biological function from structures of apparently homologous proteins.

How do these unexpected structural differences relate to PBD function in Plk1 and Polo-box function in Sak subfamily Plks? The grossly different architectures argue against conservation of the phosphoprotein-binding function since residues most intimately involved in phosphopeptide binding by Plk1 (e.g. His-538/Lys-540, Trp-414) are not conserved in Sak. Furthermore, examination of the electrostatic potential surface of the Sak Polo-box dimer shows no significant regions of positive charge (data not shown), a property otherwise common to phospho-dependent binding proteins.

A Model for Phospholigand-Induced Stimulation of Plk Kinase Activity

Two alternative models for intramolecular regulation of kinase activity by a phosphopeptide binding domain are exemplified by the mechanisms of SH2 domain-mediated inhibition in Src family kinases and SHP-family tyrosine phosphatases. In the Src-type model, the phosphopeptide binding cleft of the SH2 domain engages an internal phosphotyrosine motif at the C-terminus of the molecule to hold the kinase domain in an inactive conformation (Sicheri et al., Nature 385:602-609, 1997; Xu et al., Nature 385:595-602, 1997). We believe that Plk1 does not operate through this mechanism since it does not possess an internal optimal PBD binding site, and interaction of the PBD with the Plk1 kinase domain is not dependent on phosphorylation (Jang et al., Proc. Natl. Acad. Sci. USA 99:1984-1989, 2002). In fact, mutation of Thr-210 to Asp as a mimic of kinase activation loop phosphorylation, actually abolishes PBD binding (Jang et al., Proc. Natl. Acad. Sci. USA 99:1984-1989, 2002). Furthermore, mutation of Trp-414 in Polo-box 1 has been shown to have no effect on the basal level of Plk1 kinase activity (Lee et al., Proc. Natl. Acad. Sci. USA 95:9301-9306, 1998). Since mutations at this position disrupt phosphodependent PBD interactions, it would seem that kinase regulation occurs through a phospho-independent binding function of the PBD.

In the SHP2 model, binding of the back surface of the N-terminal SH2 domain to the phosphatase domain partially occludes the catalytic cleft and simultaneously deforms the SH2 domain's binding pocket to reduce its affinity for phosphopeptide ligands (Hof et al., Cell 92:441-450, 1998). This is entirely consistent with the reduced phosphopeptide binding that we observe for the PBD in the context of full-length Plk 1 (FIG. 8A, 8C). In the case of SHP2, high local concentrations of phosphotyrosine ligands are able to bind to the N-terminal SH2 domain, inducing a concomitant conformational rearrangement of the SH2 binding cleft that is transmitted to its phosphatase-interacting surface and releases the catalytically competent phosphatase domain. We believe Plks may be regulated by a related mechanism (FIG. 8C). Some support for the SHP-like mechanism arises from our observation that the N-terminal Polo-box of one molecule in the crystallographic asymmetric unit that is not involved in extensive lattice contacts displays significantly higher temperature factors than its C-terminal counterpart (58 Å²vs 37 Å²). This implies a rather dynamic association of the two Polo-boxes that is likely to be more pronounced in the absence of the phosphopeptide ligand. In our current model, binding of the phosphopeptide between the N- and C-terminal Polo motifs acts as a structural switch, stabilizing a conformation of the PBD that is inappropriate for association with the kinase domain. Subsequent T210D phosphorylation by upstream kinases would then serve to maintain the active state by preventing re-binding of the PBD to the kinase. Definitive proof of this mechanism will require the determination of structures of full-length Plk's and their complexes. This work is in progress.

It is clear that proper mitotic progression requires the highly regulated interplay between CDK's and a variety of other proteins kinases such as Aurora, NIMA, and Polo-like kinases, yet the molecular events that underlie the activity of many of these enzymes are largely unknown. The results of our integrated biochemical, structural and cell-biological approach now provide a framework within which the cellular function of the Polo-box motif can be understood. Plk1 is overexpressed in a variety of human tumors (Strebhardt et al., JAMA 283:479-480, 2000; Takai et al., Cancer Lett. 169:41-49, 2001), and down-regulation of human Plk1 has been shown to inhibit proliferation of cultured tumor cells (Elez et al., Biochem. Biophys. Res. Commun. 269:352-356, 2000; Liu et al., Proc. Natl. Acad. Sci. USA 100:5789-5794, 2003), suggesting that Plks are potentially important targets for therapeutic intervention. Here, we have shown that the Plk1 PBD binds to phosphorylated epitopes in a way that is distinct from any observed previously in structures of other protein-phosphopeptide complexes. The unique pattern of interactions with the Ser-pThr dipeptide suggest this motif may be employed as a useful template for the design of anti-proliferative inhibitors specifically directed against Polo-box domains. The experiments described above were carried out using the following methods.

Phospho-Motif Screen for Phosphoserine/Threonine Binding Domains

A phospho-motif-biased peptide library and its unphosphorylated counterpart were constructed as follows: biotin-Z-Gly-Z-Gly-Gly-Ala-X-X-B-X-pThr-Pro-X-X-X-X-Ala-Lys-Lys-Lys SEQ ID NO: 30 and biotin-Z-Gly-Z-Gly-Gly-Ala-X-X-B-X-Thr-Pro-X-X-X-X-Ala-Lys-Lys-Lys SEQ ID NO: 31, where pThr is phosphothreonine, Z indicates aminohexanoic acid, X denotes all amino acids except Cys, and B is a biased mixture of the amino acids P, L, I, V, F, M, W. Streptavidin beads (Pierce, 75 pmol/μL gel) were incubated with a five-fold molar excess of each biotinylated library in 20 mM Tris/HCl (pH7.5), 125 mM NaCl, 0.5% NP-40, 1 mM EDTA and washed four times with the same buffer to remove unbound ligand. The bead-immobilized libraries (30 μL gel) were added to 6 μL of an in vitro translated [³⁵S]-labeled protein pool in 200 μL binding buffer (20 mM Tris/HCl (pH7.5), 125 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1 mM DTT, 4 μg/mL pepstatin, 4 μg/mL aprotinin, 4 μg/mL leupeptin, 200 μM Na₃VO₄, 50 mM NaF). Each pool consisted of ˜30 radiolabeled proteins produced by coupled in vitro transcription/translation (Promega) of a plasmid pool containing ˜100 cDNA clones from a unidirectional and oligo dT-primed human HeLa cell library in pcDNA3.1 (Kanai et al., EMBO J. 19:6778-6791, 2000). After incubation at 4° C. for 2-3 hours, the beads were rapidly washed four times with binding buffer prior to separation on SDS-PAGE (11.4%) and autoradiography. Positively scoring hits within pools were recognized as protein bands that interacted more strongly with the phosphorylated immobilized library than its unphosphorylated counterpart. Pools containing positively scoring clones were progressively subdivided using a 96-well format and re-screened for phospho-binding until single clones were isolated and identified by DNA sequencing.

Cloning, Expression, and Purification of Plk-1 PBD Proteins

For deletion mapping of the PBD, C-terminal fragments of Plk-1 were generated by PCR and cloned into the EcoRI and XhoI sites of pcDNA3.1 (Invitrogen). For production of recombinant PBD as a GST fusion in bacteria, the 326-603 fragment of Plk-1 was ligated into the EcoRI and XhoI sites of pGEX-4T (Pharmacia), transformed into BL21, and induced in late log-phase cells at 37° C. for 3.5 hours in the presence of 0.4 mM IPTG. For measurements of peptide binding affinity by ITC, GST-Plk-1 (326-603) was isolated from bacterial lysates using glutathione agarose, cleaved from GST using thrombin (10 U/mL), and purified by anion exchange chromatography (Q Sepharose HP, Pharmacia).

Peptide Library Screening

Phosphothreonine- and phosphoserine-oriented degenerate peptide libraries containing the sequences Met-Ala-X-X-X-X-pThr-Pro-X-X-X-X-Ala-Lys-Lys-Lys SEQ ID NO: 46(theoretical degeneracy (td)=1.7×10¹⁰), Met-Ala-X-X-X-X-pThr-X-X-X-X-Ala-Lys-Lys-Lys SEQ ID NO: 47(td=1.7×10¹⁰), Met-Ala-X-X-X-X-Ser-pThr-X-X-X-X-Ala-Lys-Lys-Lys SEQ ID NO: 48(td=1.7×10¹⁰), Met-Ala-X-X-X-pSer-Pro-X-X-X-Ala-Lys-Lys-Lys SEQ ID NO: 49(td=4.7×10⁷), Met-Ala-X-X-X-X-pSer-X-X-X-X-Ala-Lys-Lys-Lys SEQ ID NO: 50(td=1.7×10¹⁰), and Met-Ala-X-X-X-X-Ser-pSer-X-X-X-X-Ala-Lys-Lys-Lys SEQ ID NO: 54(td=1.7×10¹⁰) were synthesized using N-α-FMOC-protected amino acids and standard BOP/HOBt coupling chemistry. Peptide library screening was performed using 100 μl of glutathione beads containing saturating amounts of GST-Plk-1 (residues 326-603) fusion protein (−1-1.5 mg) as described in Yaffe & Cantley (Methods Enzymol., 328:157-170, 2000). Beads were packed in a 1 mL column and incubated with 0.5 mg of the peptide library mixture for 10 minutes at room temperature in PBS (150 mM NaCl, 3 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.2). Unbound peptides were removed from the column by two rapid washes with PBS containing 0.5% NP-40 and two subsequent washes with PBS. Bound peptides were eluted with 30% acetic acid for 10 minutes at room temperature, lyophilized, resuspended in H₂O, and sequenced by automated Edman degradation on a Procise protein microsequencer. Selectivity values for each amino acid were determined by comparing the relative abundance (Mole percentage) of each amino acid at a particular sequencing cycle in the recovered peptides to that of each amino acid in the original peptide library mixture at the same position.

Isothermal Titration Calorimetry

Peptides were synthesized by solid phase technique with two C-terminal lysines to enhance solubility, purified by reverse phase HPLC following deprotection, and confirmed by MALDI-TOF 9 Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. Some peptides contained an additional tyrosine residue to facilitate concentration determination by optical absorbance. calorimetry measurements were performed using a VP-ITC microcalorimeter (MicroCal Inc., Studio City, Calif.). Experiments involved 10 μL injections of peptide solutions (150 μM-180 μM) into a sample cell containing 15 μM Plk-1 PBD (residues 326-603) in 50 mM Tris/HCl (pH 8.1), 200 mM NaCl, 2 mM TCEP. Thirty injections were performed with a spacing of 240 s and a reference power of 25 μCal/s. Binding isotherms were plotted and analyzed using Origin Software (MicroCal Inc. Studio City, Calif.).

Plk-1 PBD Binding to Cellular Substrates

HeLa cells were arrested in interphase or G2/M by treatment with aphidicolin (5 μg/mL) or nocodazole (50 ng/mL), respectively, for 16 hours. Cells were lysed in 25 mM Tris/HCl (pH 7.5) containing 125 mM NaCl, 0.5% NP-40, 5 mM EDTA, 2 mM DTT, 4 μg/mL pepstatin, 4 μg/mL aprotinin, 4 μg/mL leupeptin, 1 mM Na₃VO₄, 50 mM NaF, and 1 viM microcystin, and 150 μgs of lysate incubated with 10 μL of glutathione agarose beads containing 2-5 μg of GST-Plk-1 (residues 326-603), GST-Pin1, or GST for 30 minutes at 4° C. Beads were washed four times with lysis buffer. Precipitated proteins were eluted in sample buffer and detected by blotting with monoclonal MPM-2 (Upstate Biotechnology, Inc.) or polyclonal anti-Cdc25C (Santa Cruz Biotechnology, Santa Cruz, Calif.). For peptide competition experiments, GST-Plk-1 (residues 326-603) was immobilized on glutathionine beads and preincubated with 320 μM of PoloBoxtide-optimal, −8T, or −7V for 45 minutes at 4° C. For binding experiments involving mutant cdc25C, HeLa cells were transfected with wild-type and mutated versions of HA-tagged Cdc25C in pECE using Superfect (Qiagen, Valencia, Calif.). Nocodazole (50 ng/mL) was added seventeen hours after transfection and cells incubated for an additional 14 hours to arrest them in G2/M. Point mutations of Cdc25C were constructed using the QuickChange site-directed mutagenesis system (Stratagene) and verified by DNA sequencing.

Centrosomal Localization of the Plk-1 PBD

U2OS cells were cultured in 8-well chamber slides and arrested at G2/M by treatment with nocodazole (50 ng/mL) for 14 hours. After rinsing with PBS, cells were incubated with 4 μM GST-Plk-1 PBD (residues 326-603) and Streptolysin-O (1 U/ml) in permeabilization buffer (25 mM HEPES (pH 7.9), 100 mM KCl, 3 mM NaCl, 200 mM sucrose, 20 mM NaF, 1 mM NaOVO₄) for 20 minutes at 37° C. Cells were fixed in 3% paraformaldehyde/2% sucrose for 10 minutes at room temperature and extracted with a 0.5% Triton X-100 solution containing 20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 300 mM sucrose, and 3 mM MgCl₂for 10 minutes at RT. Slides were stained with Alexa Fluor 488-conjugated anti-GST (Molecular Probes, Eugene, Oreg.) and monoclonal anti-γ-tubulin (Sigma, St. Louis, Mo.) antibodies at 4° C. overnight, then stained with a Texas Red conjugated anti-mouse secondary antibody for 60 minutes at room temperature and counterstained with 4 μg/ml DAPI. Cells were examined using a Nikon Eclipse E600 fluorescence microscope equipped with a SPOT RTcamera and software (Diagnostic Instruments, Livingston, Scotland). Images were analyzed using NIH Image. For peptide competition experiments, the GST-Plk-1 PBD solution was preincubated with 250 μM of its optimal phosphopeptide ligand (PoloBoxtide-optimal) or its unphosphorylated counterpart (PoloBoxtide-8T) for 15 minutes at room temperature prior to use.

To quantitate centrosomal localization of the GST-Plk-1 PBD relative to γ-tubulin, black and white images of single cells showing comparable overall intensity for Alexa Fluor and Texas Red were selected and scaled to an average grayscale value of 200 (1=white, 255=black). The normalized intensity of centrosome-specific Alexa Fluor 488 staining (N.I._AF488) or Texas Red staining (N.I._TR) above background was defined as ([I_centrosome−1_cell]/I_cell) where I_centrosomeindicates the fluorescence intensity of either Alexa-Fluor 488 or Texas Red averaged over the centrosome and I_cellindicates the overall fluorescence intensity averaged over the entire cell. The relative GST-PBD/γ-tubulin specific staining was then calculated as N.I._AF488/NI_TR.

Screens to Identify Novel Binding Pairs

Novel binding pairs can be identified by the methods of the invention. For example, phosphopeptides are generated that are biased to include MAP kinase and Cell-cycle dependent kinase (Cdks) consensus phosphorylation sites (i.e., pSer-Pro), for use in screening for novel pSer-Pro binding polypeptides. Such a screen can be easily adapted to identify additional binding pairs. By taking advantage of the observation that protein kinases and phosphopeptide binding domains appear to co-evolve to recognize overlapping sequence motifs, phosphopeptides can be generated to follow specific protein kinase substrates. Thus, basophilic phosphopeptides having a core sequence including RXRSX[pS/pT] (SEQ ID NO: 55) (where R is arginine, pS is phosphoserine, pT is phosphothreonine, and X is any amino acid) can be used to identify novel binding partners dependent on the kinase, Akt. Other potential basophilic kinase substrates based on consensus phosphorylation sequences of protein kinase C (PKC), cAMP-dependent protein kinase (PKA), G-protein coupled receptor kinases such as β-ARK may also be used.

Several methods are known in the art to identify consensus kinase substrates, for example, in U.S. Pat. No. 5,532,167, U.S. Pat. No. 6,004,757, and WO 98/54577. Thus, degenerate phosphopeptides can be generated based on consensus kinase substrate peptide motifs. Exemplary kinase substrate peptide motifs that can be used include, without limitation, phosphopeptides derived from the consensus sequences of the serine/threonine kinases, Ca²⁺/calmodulin dependent kinases (CaMKs), check point kinases (e.g. CHK, Rad53), myosin light chain kinases, DRAK, Trio, casein kinase 1, cell cycle dependent kinases (CDKs, e.g., Cdc2, Cdk4, Cdk6), glycogen synthase kinases (GSK), MAP kinases (e.g., Jnk, Erk, p38), STE family kinases (e.g., PAK, GCK/MAP4K), MAP kinase activated kinases (e.g., Mnk), eIF2a kinases (e.g., PERK, PKR, HRI, GCN2), Raf kinases (e.g., A-Raf, B-Raf), casein kinase II, aurora/Polo kinases, mixed lineage kinases (e.g., MLK1, -2, -3), AKAP, Activin-receptor like kinase (Kir4), CAK, Mos, Pim, and Ksr. Other kinase substrate-derived phosphopeptide sequences that can be used in the invention include those derived from the dual specificity kinases, WEE-1, MEKs, DYRKs, Tesk, Clk, HIPK, Mps-1, TSK, and C-TAK. Dual specificity kinases also include polypeptides related to the lipid kinases FRAP, p110 PI3 Kinase, ATM, ATR, and DNA-PK.

Protein tyrosine kinase substrate peptide motifs can also be used in the invention and include phosphopeptides derived from the consensus substrate sequences of the receptor tyrosine kinases, which include the EGF-R family (e.g., EGF-R, Her2/Neu), PDGF-R, CSF—R, IGF-R, VEGF-R (e.g., Flk/Kdr, Flt), HGF-R (Met), NGF-R (e.g., TrkA, -B, -C), FGF-R, ROR, Tie-1, Tie-2/Tek, Eph (e.g., EphA_1-8, EphB_1-6), Rik, Ron, Ros, Ret, and from the cytoplasmic tyrosine kinases, which include, the Src family (e.g., Src, Lck, Lyn, Fyn, Hck, Yes), Abl, Csk, CTK, JAKs, FAK, ITK, BTK, Ack/Pyk, Tec, Tyk, Syk, Zap70, Fer, and Fes/Fps.

Binding pairs identified are not limited to those that include phosphopeptide binding domains. The methods of the invention may be used to identify virtually any peptide-binding domain in which the domain is identified by simultaneous screening of a protein/polypeptide expression library with a biased peptide library. For example, a screen for binding pairs is carried out to identify a peptide-binding domain, for example, a PDZ, SH3, or WW peptide binding domain. The “bait” peptide library contains a degenerate collection of peptides oriented around at least two or more fixed residues. A working example of such a screen is provided in the upper left panel of FIG. 9B, where there is a band at ˜24 kDa that binds the non-phosphopeptide library but not the phosphopeptide library., suggesting that it is specific for binding to BxTP motifs.

Cloning and Expression of PBD Proteins

C-terminal fragments of human Plk1 (residues 326-603), human Plk2 (residues 355-685), human Plk3 (residues 335-646), Xenopus Plx1 (residues 317-598), and Saccharomyces cerevesiae Cdc5p (residues 357-705) were amplified from IMAGE cDNA clones or directly from S. cerevisiae chromosomal DNA by PCR and ligated into suitably digested pGEX4T-3 or pGEX-6P1 (Pharmacia). Proteins were expressed in E. coli BL21(DE3) cells and purified by glutathione-affinity chromatography. For measurements of peptide binding affinity and domain mapping experiments, proteins were cleaved from GST with either thrombin or viral protease 3C (Pharmacia-LKB, Peapack, N.J.) and further purified by anion exchange chromatography (Q Sepharose HP, Pharmacia) or gel filtration (Superdex S-75, Pharmacia, Peapack, N.J.).

Oriented Peptide Library Screening

Phosphothreonine-oriented degenerate peptide libraries containing the sequences Met-Ala-X-X-X-X-pThr-Pro-X-X-X-X-Ala-Lys-Lys-Lys SEQ ID NO: 46(theoretical degeneracy (td)=1.7×10¹⁰) and Met-Ala-X-X-X-X-Ser-pThr-X-X-X-X-Ala-Lys-Lys-Lys SEQ ID NO: 48(td=1.7×10¹⁰) were synthesized using N-α-FMOC-protected amino acids and standard BOP/HOBt coupling chemistry. Peptide library screening was performed using 100 μl of glutathione beads containing saturating amounts (−1-1.5 mg) of GST-hPlk1, GST-hPlk2, GST-hPlk3, GST-Plx1, or GST-Cdc5p as described previously (Yaffe et al., Methods Enzymol 328:157-170, 2000).

Peptide Binding Measurements

Peptides were synthesized by solid phase technique with two C-terminal lysines to enhance solubility. Some peptides contained an additional tyrosine residue to facilitate concentration determination by optical absorbance. Isothermal titration calorimetry was performed using a VP-ITC microcalorimeter (MicroCal Inc. Studio City, Calif.) by titration of 15-40 μM solutions of PBD proteins with 30×10 μl injections of 150-400W peptide in a starting volume of 1.4-2.0 ml. Binding isotherms were plotted and analyzed using Origin Software (MicroCal Inc. Studio City, Calif.). Binding of in vitro translated Plk1 PBD (wild type and mutants) to bead-immobilized pTP and TP peptide libraries was performed as described previously (Elia et al., Science 299:1228-1231, 2003). pTP and TP indicate the peptide libraries biotin-Z-Gly-Z-Gly-Gly-Ala-X-X-B-X-pThr-Pro-X-X-X-X-Ala-Lys-Lys-Lys SEQ ID NO: 30 biotin-Z-Gly-Z-Gly-Gly-Ala-X-X-B-X-Thr-Pro-X-X-X-X-Ala-Lys-Lys-Lys SEQ ID NO: 31, respectively, where pThr is phosphothreonine, Z is aminohexanoic acid, X denotes all amino acids except Cys, and B is a biased mixture of the amino acids P, L, I, V, F, M, W.

Peptide Spot Array

An ABIMED peptide arrayer with a computer controlled Gilson diluter and liquid handling robot was used to synthesize peptides onto an amino-PEG cellulose membrane using N-α-FMOC-protected amino acids and DIC/HOBT coupling chemistry. The membrane was blocked in 5% milk/TBS-T (0.1%) for 2 hours at room temperature, incubated with 0.1 μM GST-Plk1 PBD (residues 326-603) in 5% milk, 50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 2 mM DTT for 1 hour at room temperature and washed with TBS-T (0.1%). It was then incubated with anti-GST conjugated HRP in 5% milk/TBS-T (0.1%) for 1 hour at room temperature, washed with TBS-T (0.1%), and subjected to chemiluminescence.

Domain Mapping and Protein Purification

Limited proteolysis of Plk1 (residues 326-603) and Cdc5p were performed using trypsin or endoproteinase Glu-C (Promega). N- and C-terminal limits were determined by Edman sequencing and electrospray mass spectrometry. DNA sequences encoding the proteolytically-defined domains were amplified by PCR and cloned into pGEX-6P1 (Cdc5p) or a version modified to allow ligation-independent cloning that also permits fusion-protein cleavage with TEV protease (Stols et al., Pro. Expr. Purif. 25:8-15 2002) (SJS—unpublished data). Recombinant PBDs were then expressed and purified as above.

Crystallization and Structure Determination

For crystallization, the phosphopeptide MAGPMQSpTPLNGAYKK (SEQ ID NO: 56) was mixed with the Plk1 PBD fragment in a 1.5:1 stoichiometric excess and concentrated to ˜0.2 mM in a buffer containing 20 mM Tris.HCT pH 8.0/500 mM NaCl, 1 mM EDTA, 3 mM DTT. Crystals were grown by microbatch methods at 18° C. using a Douglas Instruments IMPAX 1-5 crystallization robot and belong to monoclinic space-group P2₁(a=62.4 Å, b=79.5 Å, c=62.0 Å, β=93.26° with two complexes per asymmetric unit. Native data were collected on Station 14.1 at the SRS Daresbury using cryopreserved crystals at a temperature of 100° K. All data were reduced using the HKL suite of processing software (Otwinowski et al., Meth. Enzymol. 276:307-326, 1997). Phase information was derived from a three wavelength MAD experiment, using a single crystal of Se-methionine substituted PBD in complex with the phosphopeptide. Data for each wavelength were collected to a nominal 3.0 Å spacing on Station 14.2 at the SRS, Daresbury, UK. Ten Se sites corresponding to five sites per monomer in the asymmetric unit were located, and the phases refined using SOLVE (Terwilliger et al., Acta Crystallogr. D. Biol. Crystallogr 55:849-861, 1999). Phases were extended to ˜2.5 Å against the native data using real-space non-crystallographic symmetry averaging with solvent flattening in RESOLVE (Terwilliger et al., Acta Crystallogr. D. Biol. Crystallogr 55:849-861, 1999). These maps were readily interpretable allowing a partial model of the PBD, together with seven residues of the phosphopeptide to be built using ‘O’ (Jones et al., Acta Crystallogr. A 47:110-119, 1991). Subsequent refinement using native data to 1.9 Å was carried out using CNS (Brunger et al., Acta Crystallogr. D Biol. Crystallogr. 54:905-921, 1998) and REFMAC 5.0-ARP/wARP from the CCP4 suite. A summary of statistics for the structure solution and refinement are shown in Table 5 (amino acids 20-241 of Plk1, SEQ ID NO: 109). Residues in bold: His 538, Lys540, Trp414, and Leu491.

TABLE 5

Plk1-PBD.pdb

????----

┌
┘ o ~
î custom-character

CRYST1
62.352

79.518
61.993 90.00 93.26 90.00 P 1 21 1

SCALE1
0.016038
0.000000
0.000914
0.00000

SCALE2
0.000000
0.012576
0.000000
0.00000

SCALE3
0.000000
0.000000
0.016157
0.00000

ATOM
1
N
ALA
A
209
36.401
10.634
1.405
1.00
33.71
7
N

ATOM
2
CA
ALA
A
20
37.156
9.417
1.828
1.00
32.78
6
C

ATOM
3
CB
ALA
A
20
38.634
9.615
1.623
1.00
32.91
6
C

ATOM
4
C
ALA
A
20
36.862
9.066
3.284
1.00
31.86
6
C

ATOM
5
O
ALA
A
20
36.468
9.924
4.069
1.00
32.15
8
O

ATOM
6
N
LEU
A
21
37.062
7.804
3.631
1.00
31.41
7
N

ATOM
7
CA
LEU
A
21
36.766
7.324
4.979
1.00
31.14
6
C

ATOM
8
CB
LEU
A
21
36.948
5.812
5.061
1.00
31.43
6
C

ATOM
9
CG
LEU
A
21
35.921
4.969
4.306
1.00
32.63
6
C

ATOM
10
CD1
LEU
A
21
36.274
3.499
4.379
1.00
32.84
6
C

ATOM
11
CD2
LEU
A
21
34.520
5.215
4.881
1.00
32.61
6
C

ATOM
12
C
LEU
A
21
37.637
8.010
6.018
1.00
31.08
6
C

ATOM
13
O
LEU
A
21
37.163
8.368
7.096
1.00
30.32
8
O

ATOM
14
N
SER
A
22
38.912
8.200
5.687
1.00
30.80
7
N

ATOM
15
CA
SER
A
22
39.852
8.854
6.589
1.00
31.39
6
C

ATOM
16
CB
SER
A
22
41.244
8.902
5.948
1.00
31.77
6
C

ATOM
17
OG
SER
A
22
42.200
8.363
6.833
1.00
35.33
8
O

ATOM
18
C
SER
A
22
39.378
10.264
6.935
1.00
31.00
6
C

ATOM
19
O
SER
A
22
39.403
10.669
8.094
1.00
30.62
8
O

ATOM
20
N
ASP
A
23
38.959
11.012
5.919
1.00
30.36
7
N

ATOM
21
CA
ASP
A
23
38.404
12.341
6.135
1.00
30.45
6
C

ATOM
22
CB
ASP
A
23
38.129
13.027
4.805
1.00
30.88
6
C

ATOM
23
CG
ASP
A
23
39.394
13.545
4.149
1.00
33.47
6
C

ATOM
24
OD1
ASP
A
23
40.452
13.591
4.819
1.00
34.01
8
O

ATOM
25
OD2
ASP
A
23
39.418
13.915
2.961
1.00
36.44
8
O

ATOM
26
C
ASP
A
23
37.126
12.293
6.974
1.00
29.75
6
C

ATOM
27
O
ASP
A
23
36.922
13.105
7.875
1.00
29.61
8
O

ATOM
28
N
MET
A
24
36.249
11.355
6.662
1.00
29.28
7
N

ATOM
29
CA
MET
A
24
35.024
11.224
7.432
1.00
28.49
6
C

ATOM
30
CB
MET
A
24
34.134
10.133
6.852
1.00
28.64
6
C

ATOM
31
CG
MET
A
24
32.785
10.050
7.547
1.00
29.20
6
C

ATOM
32
SD
MET
A
24
31.750
8.750
6.855
1.00
32.07
16
S

ATOM
33
CE
MET
A
24
31.461
9.420
5.196
1.00
29.51
6
C

ATOM
34
C
MET
A
24
35.335
10.920
8.897
1.00
28.13
6
C

ATOM
35
O
MET
A
24
34.693
11.451
9.793
1.00
27.58
8
O

ATOM
36
N
LEU
A
25
36.313
10.059
9.139
1.00
28.15
7
N

ATOM
37
CA
LEU
A
25
36.694
9.740
10.516
1.00
28.97
6
C

ATOM
38
CB
LEU
A
25
37.779
8.665
10.539
1.00
28.90
6
C

ATOM
39
CG
LEU
A
25
38.345
8.310
11.915
1.00
29.83
6
C

ATOM
40
CD1
LEU
A
25
37.224
7.836
12.841
1.00
29.82
6
C

ATOM
41
CD2
LEU
A
25
39.421
7.240
11.787
1.00
30.16
6
C

ATOM
42
C
LEU
A
25
37.158
10.988
11.261
1.00
28.74
6
C

ATOM
43
O
LEU
A
25
36.769
11.219
12.406
1.00
28.86
8
O

ATOM
44
N
GLN
A
26
37.971
11.812
10.602
1.00
28.76
7
N

ATOM
45
CA
GLN
A
26
38.480
13.026
11.236
1.00
28.88
6
C

ATOM
46
CB
GLN
A
26
39.463
13.764
10.309
1.00
29.72
6
C

ATOM
47
CG
GLN
A
26
40.667
12.948
9.920
1.00
33.49
6
C

ATOM
48
CD
GLN
A
26
41.649
13.722
9.050
1.00
38.46
6
C

ATOM
49
OE1
GLN
A
26
41.310
14.150
7.939
1.00
41.83
8
O

ATOM
50
NE2
GLN
A
26
42.864
13.898
9.546
1.00
39.82
7
N

ATOM
51
C
GLN
A
26
37.336
13.953
11.589
1.00
27.91
6
C

ATOM
52
O
GLN
A
26
37.307
14.528
12.675
1.00
27.39
8
O

ATOM
53
N
GLN
A
27
36.395
14.098
10.660
1.00
26.30
7
N

ATOM
54
CA
GLN
A
27
35.246
14.968
10.848
1.00
25.97
6
C

ATOM
55
CB
GLN
A
27
34.419
15.035
9.553
1.00
26.10
6
C

ATOM
56
CG
GLN
A
27
35.155
15.752
8.396
1.00
25.54
6
C

ATOM
57
CD
GLN
A
27
34.598
15.402
7.022
1.00
25.17
6
C

ATOM
58
OE1
GLN
A
27
33.521
14.808
6.903
1.00
25.56
8
O

ATOM
59
NE2
GLN
A
27
35.337
15.760
5.979
1.00
25.43
7
N

ATOM
60
C
GLN
A
27
34.366
14.489
12.005
1.00
25.75
6
C

ATOM
61
O
GLN
A
27
33.896
15.292
12.819
1.00
25.65
8
O

ATOM
62
N
LEU
A
28
34.135
13.184
12.055
1.00
25.72
7
N

ATOM
63
CA
LEU
A
28
33.317
12.590
13.121
1.00
26.50
6
C

ATOM
64
CB
LEU
A
28
32.975
11.134
12.778
1.00
26.13
6
C

ATOM
65
CG
LEU
A
28
31.914
10.996
11.687
1.00
26.32
6
C

ATOM
66
CD1
LEU
A
28
31.749
9.549
11.289
1.00
25.22
6
C

ATOM
67
CD2
LEU
A
28
30.580
11.563
12.173
1.00
26.83
6
C

ATOM
68
C
LEU
A
28
34.027
12.674
14.472
1.00
26.87
6
C

ATOM
69
O
LEU
A
28
33.417
13.019
15.488
1.00
27.36
8
O

ATOM
70
N
HIS
A
29
35.318
12.373
14.488
1.00
27.61
7
N

ATOM
71
CA
HIS
A
29
36.063
12.458
15.740
1.00
28.39
6
C

ATOM
72
CB
HIS
A
29
37.530
12.070
15.579
1.00
28.75
6
C

ATOM
73
CG
HIS
A
29
38.329
12.314
16.819
1.00
31.08
6
C

ATOM
74
ND1
HIS
A
29
38.125
11.598
17.978
1.00
31.37
7
N

ATOM
75
CE1
HIS
A
29
38.939
12.045
18.917
1.00
32.66
6
C

ATOM
76
NE2
HIS
A
29
39.647
13.041
18.417
1.00
31.82
7
N

ATOM
77
CD2
HIS
A
29
39.279
13.236
17.107
1.00
32.90
6
C

ATOM
78
C
HIS
A
29
35.989
13.870
16.283
1.00
28.68
6
C

ATOM
79
O
HIS
A
29
35.781
14.076
17.474
1.00
28.88
8
O

ATOM
80
N
SER
A
30
36.135
14.849
15.396
1.00
28.42
7
N

ATOM
81
CA
SER
A
30
36.122
16.241
15.810
1.00
28.05
6
C

ATOM
82
CB
SER
A
30
36.479
17.148
14.628
1.00
28.85
6
C

ATOM
83
OG
SER
A
30
36.538
18.498
15.053
1.00
30.19
8
O

ATOM
84
C
SER
A
30
34.811
16.685
16.452
1.00
27.75
6
C

ATOM
85
O
SER
A
30
34.812
17.298
17.521
1.00
26.57
8
O

ATOM
86
N
VAL
A
31
33.683
16.396
15.807
1.00
27.10
7
N

ATOM
87
CA
VAL
A
31
32.415
16.802
16.396
1.00
26.70
6
C

ATOM
88
CB
VAL
A
31
31.227
16.754
15.377
1.00
27.14
6
C

ATOM
89
CG1
VAL
A
31
31.125
15.396
14.732
1.00
26.15
6
C

ATOM
90
CG2
VAL
A
31
29.904
17.116
16.063
1.00
26.78
6
C

ATOM
91
C
VAL
A
31
32.095
15.979
17.658
1.00
26.15
6
C

ATOM
92
O
VAL
A
31
31.607
16.529
18.647
1.00
25.84
8
O

ATOM
93
N
ASN
A
32
32.375
14.677
17.632
1.00
25.70
7
N

ATOM
94
CA
ASN
A
32
32.050
13.827
18.789
1.00
25.94
6
C

ATOM
95
CB
ASN
A
32
32.251
12.348
18.486
1.00
25.11
6
C

ATOM
96
CG
ASN
A
32
31.242
11.800
17.473
1.00
25.06
6
C

ATOM
97
OD1
ASN
A
32
30.221
12.410
17.196
1.00
25.48
8
O

ATOM
98
ND2
ASN
A
32
31.550
10.645
16.924
1.00
24.23
7
N

ATOM
99
C
ASN
A
32
32.875
14.188
20.022
1.00
26.29
6
C

ATOM
100
O
ASN
A
32
32.378
14.153
21.142
1.00
26.53
8
O

ATOM
101
N
ALA
A
33
34.142
14.517
19.806
1.00
26.41
7
N

ATOM
102
CA
ALA
A
33
35.035
14.890
20.918
1.00
27.37
6
C

ATOM
103
CB
ALA
A
33
36.468
15.007
20.435
1.00
27.30
6
C

ATOM
104
C
ALA
A
33
34.595
16.187
21.584
1.00
27.63
6
C

ATOM
105
O
ALA
A
33
34.921
16.447
22.743
1.00
27.93
8
O

ATOM
106
N
SER
A
34
33.834
16.994
20.858
1.00
27.83
7
N

ATOM
107
CA
SER
A
34
33.347
18.251
21.397
1.00
28.41
6
C

ATOM
108
CB
SER
A
34
33.075
19.252
20.268
1.00
28.21
6
C

ATOM
109
OG
SER
A
34
31.807
19.031
19.670
1.00
27.66
8
O

ATOM
110
C
SER
A
34
32.105
18.089
22.290
1.00
28.78
6
C

ATOM
111
O
SER
A
34
31.643
19.069
22.882
1.00
28.93
8
O

ATOM
112
N
LYS
A
35
31.597
16.857
22.397
1.00
28.68
7
N

ATOM
113
CA
LYS
A
35
30.425
16.523
23.229
1.00
29.44
6
C

ATOM
114
CB
LYS
A
35
30.795
16.531
24.711
1.00
29.93
6
C

ATOM
115
CG
LYS
A
35
31.934
15.594
25.089
1.00
31.61
6
C

ATOM
116
CD
LYS
A
35
32.098
15.557
26.612
1.00
34.33
6
C

ATOM
117
CE
LYS
A
35
32.129
16.969
27.205
1.00
36.89
6
C

ATOM
118
NZ
LYS
A
35
32.313
16.996
28.699
1.00
39.71
7
N

ATOM
119
C
LYS
A
35
29.261
17.475
22.987
1.00
29.56
6
C

ATOM
120
O
LYS
A
35
28.822
18.180
23.894
1.00
29.19
8
O

ATOM
121
N
PRO
A
36
28.746
17.459
21.762
1.00
29.62
7
N

ATOM
122
CA
PRO
A
36
27.742
18.428
21.311
1.00
29.86
6
C

ATOM
123
CB
PRO
A
36
27.509
18.018
19.849
1.00
29.74
6
C

ATOM
124
CG
PRO
A
36
27.873
16.537
19.841
1.00
29.62
6
C

ATOM
125
CD
PRO
A
36
29.099
16.493
20.706
1.00
29.42
6
C

ATOM
126
C
PRO
A
36
26.424
18.435
22.079
1.00
30.15
6
C

ATOM
127
O
PRO
A
36
25.743
19.461
22.046
1.00
29.59
8
O

ATOM
128
N
SER
A
37
26.056
17.335
22.742
1.00
30.35
7
N

ATOM
129
CA
SER
A
37
24.796
17.310
23.482
1.00
30.73
6
C

ATOM
130
CB
SER
A
37
24.096
15.950
23.337
1.00
30.91
6
C

ATOM
131
OG
SER
A
37
24.788
14.951
24.059
1.00
30.05
8
O

ATOM
132
C
SER
A
37
24.988
17.653
24.963
1.00
31.78
6
C

ATOM
133
O
SER
A
37
24.028
17.746
25.717
1.00
31.53
8
O

ATOM
134
N
GLU
A
38
26.234
17.860
25.358
1.00
32.67
7
N

ATOM
135
CA
GLU
A
38
26.562
18.138
26.743
1.00
34.86
6
C

ATOM
136
CB
GLU
A
38
27.696
17.206
27.183
1.00
34.88
6
C

ATOM
137
CG
GLU
A
38
27.227
15.750
27.139
1.00
37.05
6
C

ATOM
138
CD
GLU
A
38
28.344
14.733
26.972
1.00
40.86
6
C

ATOM
139
OE1
GLU
A
38
29.059
14.473
27.960
1.00
40.91
8
O

ATOM
140
OE2
GLU
A
38
28.496
14.175
25.852
1.00
42.71
8
O

ATOM
141
C
GLU
A
38
26.875
19.622
26.931
1.00
35.55
6
C

ATOM
142
O
GLU
A
38
27.772
20.009
27.672
1.00
36.66
8
O

ATOM
143
N
ARG
A
39
26.091
20.442
26.244
1.00
36.48
7
N

ATOM
144
CA
ARG
A
39
26.224
21.887
26.286
1.00
37.25
6
C

ATOM
145
CB
ARG
A
39
26.169
22.456
24.865
1.00
37.41
6
C

ATOM
146
CG
ARG
A
39
27.186
21.845
23.903
1.00
38.11
6
C

ATOM
147
CD
ARG
A
39
28.580
22.457
24.002
1.00
38.15
6
C

ATOM
148
NE
ARG
A
39
29.559
21.725
23.204
1.00
38.73
7
N

ATOM
149
CZ
ARG
A
39
29.706
21.866
21.893
1.00
37.81
6
C

ATOM
150
NH1
ARG
A
39
28.941
22.718
21.228
1.00
37.87
7
N

ATOM
151
NH2
ARG
A
39
30.616
21.153
21.249
1.00
37.30
7
N

ATOM
152
C
ARG
A
39
25.058
22.433
27.079
1.00
37.01
6
C

ATOM
153
O
ARG
A
39
24.022
21.783
27.198
1.00
37.60
8
O

ATOM
154
N
GLY
A
40
25.207
23.636
27.607
1.00
36.91
7
N

ATOM
155
CA
GLY
A
40
24.123
24.232
28.365
1.00
36.46
6
C

ATOM
156
C
GLY
A
40
22.905
24.597
27.533
1.00
35.96
6
C

ATOM
157
O
GLY
A
40
21.769
24.456
27.975
1.00
37.22
8
O

ATOM
158
N
LEU
A
41
23.134
25.097
26.331
1.00
34.84
7
N

ATOM
159
CA
LEU
A
41
22.045
25.476
25.452
1.00
33.51
6
C

ATOM
160
CB
LEU
A
41
21.947
27.000
25.353
1.00
33.41
6
C

ATOM
161
CG
LEU
A
41
20.995
27.589
24.315
1.00
33.40
6
C

ATOM
162
CD1
LEU
A
41
19.561
27.317
24.719
1.00
33.71
6
C

ATOM
163
CD2
LEU
A
41
21.232
29.095
24.155
1.00
33.45
6
C

ATOM
164
C
LEU
A
41
22.353
24.903
24.085
1.00
33.07
6
C

ATOM
165
O
LEU
A
41
23.431
25.131
23.548
1.00
33.54
8
O

ATOM
166
N
VAL
A
42
21.419
24.146
23.532
1.00
32.14
7
N

ATOM
167
CA
VAL
A
42
21.617
23.570
22.208
1.00
31.38
6
C

ATOM
168
CB
VAL
A
42
21.141
22.101
22.170
1.00
31.56
6
C

ATOM
169
CG1
VAL
A
42
21.051
21.591
20.724
1.00
31.57
6
C

ATOM
170
CG2
VAL
A
42
22.086
21.241
22.991
1.00
30.77
6
C

ATOM
171
C
VAL
A
42
20.912
24.406
21.148
1.00
31.02
6
C

ATOM
172
O
VAL
A
42
19.771
24.820
21.340
1.00
31.11
8
O

ATOM
173
N
ARG
A
43
21.619
24.692
20.055
1.00
30.35
7
N

ATOM
174
CA
ARG
A
43
21.061
25.422
18.927
1.00
30.73
6
C

ATOM
175
CB
ARG
A
43
21.636
26.843
18.839
1.00
30.57
6
C

ATOM
176
CG
ARG
A
43
21.148
27.756
19.974
1.00
32.57
6
C

ATOM
177
CD
ARG
A
43
21.173
29.237
19.630
1.00
33.26
6
C

ATOM
178
NE
ARG
A
43
22.519
29.784
19.645
1.00
33.72
7
N

ATOM
179
CZ
ARG
A
43
22.929
30.792
18.880
1.00
33.34
6
C

ATOM
180
NH1
ARG
A
43
22.106
31.358
18.006
1.00
34.75
7
N

ATOM
181
NH2
ARG
A
43
24.169
31.228
18.986
1.00
34.75
7
N

ATOM
182
C
ARG
A
43
21.325
24.641
17.640
1.00
30.19
6
C

ATOM
183
O
ARG
A
43
22.000
25.114
16.731
1.00
30.53
8
O

ATOM
184
N
GLN
A
44
20.794
23.427
17.595
1.00
30.12
7
N

ATOM
185
CA
GLN
A
44
20.953
22.529
16.453
1.00
30.34
6
C

ATOM
186
CB
GLN
A
44
20.082
21.289
16.681
1.00
30.51
6
C

ATOM
187
CG
GLN
A
44
20.483
20.058
15.907
1.00
32.20
6
C

ATOM
188
CD
GLN
A
44
19.725
18.832
16.380
1.00
33.37
6
C

ATOM
189
OE1
GLN
A
44
19.786
18.488
17.549
1.00
34.97
8
O

ATOM
190
NE2
GLN
A
44
19.001
18.184
15.476
1.00
34.78
7
N

ATOM
191
C
GLN
A
44
20.571
23.181
15.132
1.00
29.89
6
C

ATOM
192
O
GLN
A
44
21.191
22.925
14.097
1.00
29.81
8
O

ATOM
193
N
ALA
A
45
19.543
24.022
15.155
1.00
29.90
7
N

ATOM
194
CA
ALA
A
45
19.060
24.636
13.920
1.00
30.12
6
C

ATOM
195
CB
ALA
A
45
17.754
25.393
14.155
1.00
30.53
6
C

ATOM
196
C
ALA
A
45
20.095
25.532
13.259
1.00
30.05
6
C

ATOM
197
O
ALA
A
45
20.044
25.762
12.054
1.00
29.93
8
O

ATOM
198
N
GLU
A
46
21.051
26.018
14.039
1.00
29.96
7
N

ATOM
199
CA
GLU
A
46
22.078
26.895
13.501
1.00
30.20
6
C

ATOM
200
CB
GLU
A
46
22.767
27.675
14.628
1.00
30.43
6
C

ATOM
201
CG
GLU
A
46
21.879
28.715
15.287
1.00
32.59
6
C

ATOM
202
CD
GLU
A
46
21.397
29.779
14.324
1.00
33.11
6
C

ATOM
203
OE1
GLU
A
46
22.124
30.091
13.354
1.00
34.58
8
O

ATOM
204
OE2
GLU
A
46
20.290
30.319
14.537
1.00
35.13
8
O

ATOM
205
C
GLU
A
46
23.112
26.121
12.687
1.00
29.91
6
C

ATOM
206
O
GLU
A
46
23.984
26.717
12.053
1.00
29.50
8
O

ATOM
207
N
ALA
A
47
23.007
24.795
12.699
1.00
29.19
7
N

ATOM
208
CA
ALA
A
47
23.948
23.958
11.957
1.00
28.42
6
C

ATOM
209
CB
ALA
A
47
24.384
22.768
12.804
1.00
28.21
6
C

ATOM
210
C
ALA
A
47
23.339
23.473
10.641
1.00
28.77
6
C

ATOM
211
O
ALA
A
47
24.010
22.818
9.843
1.00
27.99
8
O

ATOM
212
N
GLU
A
48
22.071
23.802
10.423
1.00
28.97
7
N

ATOM
213
CA
GLU
A
48
21.373
23.409
9.196
1.00
29.55
6
C

ATOM
214
CB
GLU
A
48
19.886
23.769
9.292
1.00
29.77
6
C

ATOM
215
CG
GLU
A
48
19.116
23.003
10.360
1.00
31.36
6
C

ATOM
216
CD
GLU
A
48
17.644
23.379
10.405
1.00
33.36
6
C

ATOM
217
OE1
GLU
A
48
17.200
24.140
9.524
1.00
33.45
8
O

ATOM
218
OE2
GLU
A
48
16.930
22.917
11.324
1.00
34.99
8
O

ATOM
219
C
GLU
A
48
21.975
24.062
7.949
1.00
29.78
6
C

ATOM
220
O
GLU
A
48
22.231
25.265
7.921
1.00
28.44
8
O

ATOM
221
N
ASP
A
49
22.188
23.260
6.911
1.00
30.40
7
N

ATOM
222
CA
ASP
A
49
22.754
23.765
5.666
1.00
31.48
6
C

ATOM
223
CB
ASP
A
49
24.268
23.563
5.663
1.00
32.13
6
C

ATOM
224
CG
ASP
A
49
24.990
24.508
4.716
1.00
34.05
6
C

ATOM
225
OD1
ASP
A
49
24.342
25.064
3.807
1.00
35.90
8
O

ATOM
226
OD2
ASP
A
49
26.215
24.752
4.813
1.00
36.69
8
O

ATOM
227
C
ASP
A
49
22.112
22.973
4.531
1.00
31.75
6
C

ATOM
228
O
ASP
A
49
22.643
21.949
4.106
1.00
31.48
8
O

ATOM
229
N
PRO
A
50
20.966
23.445
4.054
1.00
32.06
7
N

ATOM
230
CA
PRO
A
50
20.224
22.748
2.994
1.00
32.74
6
C

ATOM
231
CB
PRO
A
50
18.970
23.615
2.810
1.00
33.04
6
C

ATOM
232
CG
PRO
A
50
18.897
24.474
4.020
1.00
32.80
6
C

ATOM
233
CD
PRO
A
50
20.301
24.689
4.475
1.00
32.50
6
C

ATOM
234
C
PRO
A
50
21.003
22.673
1.689
1.00
33.09
6
C

ATOM
235
O
PRO
A
50
20.700
21.823
0.839
1.00
33.20
8
O

ATOM
236
N
ALA
A
51
21.994
23.540
1.519
1.00
33.00
7
N

ATOM
237
CA
ALA
A
51
22.807
23.506
0.305
1.00
32.95
6
C

ATOM
238
CB
ALA
A
51
23.614
24.780
0.159
1.00
33.32
6
C

ATOM
239
C
ALA
A
51
23.728
22.277
0.274
1.00
32.90
6
C

ATOM
240
O
ALA
A
51
24.358
21.972
−0.748
1.00
32.85
8
O

ATOM
241
N
CYS
A
52
23.791
21.571
1.395
1.00
31.55
7
N

ATOM
242
CA
CYS
A
52
24.631
20.386
1.495
1.00
31.19
6
C

ATOM
243
CB
CYS
A
52
25.420
20.413
2.799
1.00
31.18
6
C

ATOM
244
SG
CYS
A
52
26.601
21.780
2.860
1.00
36.09
16
S

ATOM
245
C
CYS
A
52
23.861
19.074
1.371
1.00
29.52
6
C

ATOM
246
O
CYS
A
52
24.444
18.009
1.518
1.00
29.12
8
O

ATOM
247
N
ILE
A
53
22.562
19.150
1.107
1.00
28.43
7
N

ATOM
248
CA
ILE
A
53
21.753
17.939
0.942
1.00
28.06
6
C

ATOM
249
CB
ILE
A
53
20.289
18.316
0.567
1.00
28.23
6
C

ATOM
250
CG1
ILE
A
53
19.661
19.129
1.708
1.00
30.03
6
C

ATOM
251
CD1
ILE
A
53
18.283
19.739
1.391
1.00
32.54
6
C

ATOM
252
CG2
ILE
A
53
19.448
17.078
0.345
1.00
29.37
6
C

ATOM
253
C
ILE
A
53
22.429
17.048
−0.109
1.00
27.44
6
C

ATOM
254
O
ILE
A
53
22.935
17.550
−1.114
1.00
26.15
8
O

ATOM
255
N
PRO
A
54
22.469
15.740
0.133
1.00
27.31
7
N

ATOM
256
CA
PRO
A
54
23.141
14.815
−0.784
1.00
27.58
6
C

ATOM
257
CB
PRO
A
54
23.057
13.458
−0.065
1.00
27.83
6
C

ATOM
258
CG
PRO
A
54
22.513
13.719
1.293
1.00
28.07
6
C

ATOM
259
CD
PRO
A
54
21.853
15.052
1.281
1.00
27.24
6
C

ATOM
260
C
PRO
A
54
22.413
14.683
−2.117
1.00
27.78
6
C

ATOM
261
O
PRO
A
54
21.196
14.891
−2.189
1.00
27.38
8
O

ATOM
262
N
ILE
A
55
23.163
14.332
−3.154
1.00
27.67
7
N

ATOM
263
CA
ILE
A
55
22.585
14.048
−4.454
1.00
28.32
6
C

ATOM
264
CB
ILE
A
55
23.666
14.179
−5.548
1.00
28.85
6
C

ATOM
265
CG1
ILE
A
55
24.293
15.579
−5.494
1.00
30.95
6
C

ATOM
266
CD1
ILE
A
55
25.740
15.648
−5.965
1.00
33.86
6
C

ATOM
267
CG2
ILE
A
55
23.054
13.925
−6.929
1.00
30.22
6
C

ATOM
268
C
ILE
A
55
21.983
12.635
−4.455
1.00
27.58
6
C

ATOM
269
O
ILE
A
55
20.922
12.400
−5.017
1.00
27.41
8
O

ATOM
270
N
PHE
A
56
22.660
11.702
−3.790
1.00
26.32
7
N

ATOM
271
CA
PHE
A
56
22.237
10.314
−3.766
1.00
25.72
6
C

ATOM
272
CB
PHE
A
56
23.218
9.453
−4.581
1.00
25.89
6
C

ATOM
273
CG
PHE
A
56
23.324
9.836
−6.034
1.00
27.67
6
C

ATOM
274
CD1
PHE
A
56
24.429
10.528
−6.498
1.00
27.36
6
C

ATOM
275
CE1
PHE
A
56
24.546
10.875
−7.834
1.00
28.92
6
C

ATOM
276
CZ
PHE
A
56
23.556
10.552
−8.719
1.00
29.32
6
C

ATOM
277
CE2
PHE
A
56
22.437
9.856
−8.280
1.00
30.40
6
C

ATOM
278
CD2
PHE
A
56
22.327
9.496
−6.934
1.00
29.35
6
C

ATOM
279
C
PHE
A
56
22.226
9.718
−2.361
1.00
24.91
6
C

ATOM
280
O
PHE
A
56
23.036
10.085
−1.513
1.00
23.97
8
O

ATOM
281
N
TRP
A
57
21.312
8.781
−2.142
1.00
24.36
7
N

ATOM
282
CA
TRP
A
57
21.297
7.945
−0.942
1.00
24.65
6
C

ATOM
283
CB
TRP
A
57
20.622
8.641
0.260
1.00
24.01
6
C

ATOM
284
CG
TRP
A
57
19.175
9.036
0.010
1.00
25.03
6
C

ATOM
285
CD1
TRP
A
57
18.053
8.270
0.217
1.00
24.50
6
C

ATOM
286
NE1
TRP
A
57
16.924
8.976
−0.142
1.00
23.27
7
N

ATOM
287
CE2
TRP
A
57
17.298
10.223
−0.578
1.00
25.29
6
C

ATOM
288
CD2
TRP
A
57
18.705
10.298
−0.491
1.00
24.40
6
C

ATOM
289
CE3
TRP
A
57
19.337
11.486
−0.885
1.00
26.85
6
C

ATOM
290
CZ3
TRP
A
57
18.551
12.544
−1.342
1.00
28.68
6
C

ATOM
291
CH2
TRP
A
57
17.162
12.434
−1.409
1.00
27.81
6
C

ATOM
292
CZ2
TRP
A
57
16.516
11.289
−1.029
1.00
26.93
6
C

ATOM
293
C
TRP
A
57
20.572
6.649
−1.319
1.00
24.73
6
C

ATOM
294
O
TRP
A
57
19.945
6.577
−2.386
1.00
24.59
8
O

ATOM
295
N
VAL
A
58
20.684
5.630
−0.476
1.00
24.55
7
N

ATOM
296
CA
VAL
A
58
19.994
4.364
−0.702
1.00
24.83
6
C

ATOM
297
CB
VAL
A
58
20.741
3.191
−0.036
1.00
24.93
6
C

ATOM
298
CG1
VAL
A
58
19.939
1.887
−0.162
1.00
24.14
6
C

ATOM
299
CG2
VAL
A
58
22.109
3.016
−0.677
1.00
25.68
6
C

ATOM
300
C
VAL
A
58
18.544
4.447
−0.204
1.00
25.64
6
C

ATOM
301
O
VAL
A
58
18.296
4.723
0.976
1.00
25.76
8
O

ATOM
302
N
SER
A
59
17.597
4.220
−1.119
1.00
25.84
7
N

ATOM
303
CA
SER
A
59
16.170
4.341
−0.834
1.00
26.79
6
C

ATOM
304
CB
SER
A
59
15.449
4.907
−2.064
1.00
27.22
6
C

ATOM
305
OG
SER
A
59
15.274
6.298
−1.930
1.00
32.34
8
O

ATOM
306
C
SER
A
59
15.513
3.028
−0.437
1.00
26.12
6
C

ATOM
307
O
SER
A
59
14.527
3.018
0.314
1.00
25.45
8
O

ATOM
308
N
LYS
A
60
16.042
1.924
−0.965
1.00
25.71
7
N

ATOM
309
CA
LYS
A
60
15.524
0.592
−0.674
1.00
25.00
6
C

ATOM
310
CB
LYS
A
60
14.420
0.183
−1.665
1.00
25.95
6
C

ATOM
311
CG
LYS
A
60
13.282
1.185
−1.857
1.00
25.89
6
C

ATOM
312
CD
LYS
A
60
12.358
0.774
−3.030
1.00
27.60
6
C

ATOM
313
CE
LYS
A
60
11.199
1.774
−3.198
1.00
27.10
6
C

ATOM
314
NZ
LYS
A
60
10.221
1.300
−4.234
1.00
27.31
7
N

ATOM
315
C
LYS
A
60
16.697
−0.374
−0.821
1.00
24.95
6
C

ATOM
316
O
LYS
A
60
17.665
−0.065
−1.513
1.00
24.15
8
O

ATOM

317

N

TRP

A

61

16.625

−1.510

−0.148

1.00

24.47

7

N

ATOM

318

CA

TRP

A

61

17.656

−2.541

−0.280

1.00

25.47

6

C

ATOM

319

CB

TRP

A

61

18.899

−2.210

0.566

1.00

25.28

6

C

ATOM

320

CG

TRP

A

61

18.610

−2.062

2.003

1.00

25.61

6

C

ATOM

321

CD1

TRP

A

61

18.356

−0.900

2.677

1.00

25.43

6

C

ATOM

322

NE1

TRP

A

61

18.136

−1.169

4.008

1.00

25.56

7

N

ATOM

323

CE2

TRP

A

61

18.229

−2.520

4.213

1.00

26.39

6

C

ATOM

324

CD2

TRP

A

61

18.538

−3.113

2.974

1.00

26.25

6

C

ATOM

325

CE3

TRP

A

61

18.698

−4.504

2.918

1.00

26.30

6

C

ATOM

326

CZ3

TRP

A

61

18.543

−5.245

4.080

1.00

27.94

6

C

ATOM

327

CH2

TRP

A

61

18.229

−4.624

5.299

1.00

27.03

6

C

ATOM

328

CZ2

TRP

A

61

18.077

−3.267

5.388

1.00

26.38

6

C

ATOM

329

C

TRP

A

61

17.102

−3.920

0.083

1.00

26.16

6

C

ATOM

330

O

TRP

A

61

16.158

−4.037

0.871

1.00

25.82

8

O

ATOM
331
N
VAL
A
62
17.709
−4.958
−0.487
1.00
27.04
7
N

ATOM
332
CA
VAL
A
62
17.326
−6.346
−0.236
1.00
28.50
6
C

ATOM
333
CB
VAL
A
62
16.530
−6.937
−1.428
1.00
28.90
6
C

ATOM
334
CG1
VAL
A
62
16.036
−8.339
−1.100
1.00
30.44
6
C

ATOM
335
CG2
VAL
A
62
15.361
−6.042
−1.808
1.00
29.34
6
C

ATOM
336
C
VAL
A
62
18.600
−7.169
−0.076
1.00
29.14
6
C

ATOM
337
O
VAL
A
62
19.449
−7.171
−0.962
1.00
27.94
8
O

ATOM
338
N
ASP
A
63
18.726
−7.869
1.048
1.00
30.63
7
N

ATOM
339
CA
ASP
A
63
19.911
−8.672
1.335
1.00
31.95
6
C

ATOM
340
CB
ASP
A
63
20.241
−8.584
2.824
1.00
31.90
6
C

ATOM
341
CG
ASP
A
63
21.484
−9.378
3.215
1.00
32.72
6
C

ATOM
342
OD1
ASP
A
63
22.047
−10.133
2.383
1.00
33.46
8
O

ATOM
343
OD2
ASP
A
63
21.962
−9.306
4.361
1.00
31.63
8
O

ATOM
344
C
ASP
A
63
19.736
−10.130
0.898
1.00
33.18
6
C

ATOM
345
O
ASP
A
63
19.187
−10.958
1.632
1.00
33.29
8
O

ATOM
346
N
TYR
A
64
20.206
−10.435
−0.302
1.00
34.24
7
N

ATOM
347
CA
TYR
A
64
20.151
−11.797
−0.822
1.00
36.03
6
C

ATOM
348
CB
TYR
A
64
19.507
−11.794
−2.203
1.00
36.45
6
C

ATOM
349
CG
TYR
A
64
18.589
−12.965
−2.465
1.00
41.00
6
C

ATOM
350
CD1
TYR
A
64
17.298
−12.767
−2.940
1.00
44.13
6
C

ATOM
351
CE1
TYR
A
64
16.452
−13.837
−3.179
1.00
46.58
6
C

ATOM
352
CZ
TYR
A
64
16.898
−15.125
−2.943
1.00
47.54
6
C

ATOM
353
OH
TYR
A
64
16.068
−16.200
−3.177
1.00
50.35
8
O

ATOM
354
CE2
TYR
A
64
18.175
−15.346
−2.471
1.00
46.59
6
C

ATOM
355
CD2
TYR
A
64
19.011
−14.270
−2.233
1.00
44.35
6
C

ATOM
356
C
TYR
A
64
21.575
−12.330
−0.902
1.00
35.84
6
C

ATOM
357
O
TYR
A
64
21.925
−13.065
−1.823
1.00
35.73
8
O

ATOM
358
N
SER
A
65
22.398
−11.950
0.070
1.00
36.70
7
N

ATOM
359
CA
SER
A
65
23.818
−12.299
0.047
1.00
37.40
6
C

ATOM
360
CB
SER
A
65
24.627
−11.399
0.979
1.00
37.31
6
C

ATOM
361
OG
SER
A
65
24.385
−11.718
2.333
1.00
37.43
8
O

ATOM
362
C
SER
A
65
24.063
−13.766
0.369
1.00
38.51
6
C

ATOM
363
O
SER
A
65
25.198
−14.229
0.368
1.00
38.36
8
O

ATOM
364
N
ASP
A
66
22.979
−14.478
0.649
1.00
39.68
7
N

ATOM
365
CA
ASP
A
66
23.006
−15.906
0.892
1.00
40.94
6
C

ATOM
366
CB
ASP
A
66
21.603
−16.348
1.317
1.00
41.90
6
C

ATOM
367
CG
ASP
A
66
21.621
−17.525
2.252
1.00
45.04
6
C

ATOM
368
OD1
ASP
A
66
22.727
−17.914
2.693
1.00
49.21
8
O

ATOM
369
OD2
ASP
A
66
20.575
−18.128
2.603
1.00
48.82
8
O

ATOM
370
C
ASP
A
66
23.349
−16.621
−0.403
1.00
40.56
6
C

ATOM
371
O
ASP
A
66
23.967
−17.700
−0.396
1.00
40.67
8
O

ATOM
372
N
LYS
A
67
22.945
−16.018
−1.518
1.00
39.66
7
N

ATOM
373
CA
LYS
A
67
23.078
−16.670
−2.819
1.00
38.94
6
C

ATOM
374
CB
LYS
A
67
21.744
−17.344
−3.183
1.00
39.57
6
C

ATOM
375
CG
LYS
A
67
21.368
−18.486
−2.245
1.00
41.78
6
C

ATOM
376
CD
LYS
A
67
19.921
−18.941
−2.419
1.00
45.87
6
C

ATOM
377
CE
LYS
A
67
19.497
−19.841
−1.248
1.00
48.20
6
C

ATOM
378
NZ
LYS
A
67
18.171
−20.498
−1.466
1.00
49.72
7
N

ATOM
379
C
LYS
A
67
23.540
−15.783
−3.979
1.00
37.58
6
C

ATOM
380
O
LYS
A
67
24.265
−16.250
−4.857
1.00
37.13
8
O

ATOM
381
N
TYR
A
68
23.132
−14.514
−3.984
1.00
35.65
7
N

ATOM
382
CA
TYR
A
68
23.457
−13.624
−5.097
1.00
34.39
6
C

ATOM
383
CB
TYR
A
68
22.183
−13.231
−5.841
1.00
34.80
6
C

ATOM
384
CG
TYR
A
68
21.317
−14.414
−6.216
1.00
37.04
6
C

ATOM
385
CD1
TYR
A
68
20.096
−14.625
−5.593
1.00
39.02
6
C

ATOM
386
CE1
TYR
A
68
19.295
−15.708
−5.926
1.00
40.75
6
C

ATOM
387
CZ
TYR
A
68
19.710
−16.596
−6.895
1.00
41.85
6
C

ATOM
388
OH
TYR
A
68
18.897
−17.663
−7.223
1.00
44.07
8
O

ATOM
389
CE2
TYR
A
68
20.922
−16.411
−7.538
1.00
40.85
6
C

ATOM
390
CD2
TYR
A
68
21.723
−15.322
−7.192
1.00
38.99
6
C

ATOM
391
C
TYR
A
68
24.236
−12.365
−4.720
1.00
32.93
6
C

ATOM
392
O
TYR
A
68
25.242
−12.032
−5.354
1.00
31.97
8
O

ATOM
393
N
GLY
A
69
23.761
−11.655
−3.705
1.00
31.18
7
N

ATOM
394
CA
GLY
A
69
24.428
−10.437
−3.289
1.00
29.97
6
C

ATOM
395
C
GLY
A
69
23.447
−9.452
−2.675
1.00
29.19
6
C

ATOM
396
O
GLY
A
69
22.369
−9.840
−2.229
1.00
28.66
8
O

ATOM
397
N
LEU
A
70
23.831
−8.182
−2.652
1.00
28.13
7
N

ATOM
398
CA
LEU
A
70
22.985
−7.148
−2.089
1.00
27.85
6
C

ATOM
399
CB
LEU
A
70
23.752
−6.354
−1.044
1.00
27.96
6
C

ATOM
400
CG
LEU
A
70
22.788
−5.400
−0.333
1.00
29.94
6
C

ATOM
401
CD1
LEU
A
70
22.772
−5.653
1.159
1.00
29.95
6
C

ATOM
402
CD2
LEU
A
70
23.013
−3.946
−0.700
1.00
30.21
6
C

ATOM
403
C
LEU
A
70
22.467
−6.215
−3.163
1.00
27.20
6
C

ATOM
404
O
LEU
A
70
23.244
−5.529
−3.811
1.00
27.61
8
O

ATOM
405
N
GLY
A
71
21.148
−6.185
−3.337
1.00
26.88
7
N

ATOM
406
CA
GLY
A
71
20.513
−5.309
−4.312
1.00
26.41
6
C

ATOM
407
C
GLY
A
71
19.989
−4.043
−3.641
1.00
25.86
6
C

ATOM
408
O
GLY
A
71
19.567
−4.078
−2.488
1.00
25.29
8
O

ATOM
409
N
TYR
A
72
20.013
−2.924
−4.350
1.00
25.62
7
N

ATOM
410
CA
TYR
A
72
19.566
−1.666
−3.745
1.00
25.82
6
C

ATOM
411
CB
TYR
A
72
20.731
−1.032
−2.949
1.00
25.61
6
C

ATOM
412
CG
TYR
A
72
21.915
−0.698
−3.831
1.00
25.97
6
C

ATOM
413
CD1
TYR
A
72
21.987
0.524
−4.483
1.00
26.37
6
C

ATOM
414
CE1
TYR
A
72
23.041
0.830
−5.313
1.00
26.23
6
C

ATOM
415
CZ
TYR
A
72
24.049
−0.107
−5.506
1.00
26.67
6
C

ATOM
416
OH
TYR
A
72
25.095
0.210
−6.327
1.00
27.29
8
O

ATOM
417
CE2
TYR
A
72
24.005
−1.330
−4.868
1.00
24.54
6
C

ATOM
418
CD2
TYR
A
72
22.941
−1.626
−4.044
1.00
24.92
6
C

ATOM
419
C
TYR
A
72
19.026
−0.684
−4.790
1.00
25.98
6
C

ATOM
420
O
TYR
A
72
19.272
−0.829
−5.989
1.00
25.98
8
O

ATOM
421
N
GLN
A
73
18.243
0.287
−4.325
1.00
25.67
7
N

ATOM
422
CA
GLN
A
73
17.746
1.349
−5.172
1.00
26.04
6
C

ATOM
423
CB
GLN
A
73
16.218
1.439
−5.100
1.00
26.48
6
C

ATOM
424
CG
GLN
A
73
15.642
2.564
−5.972
1.00
27.53
6
C

ATOM
425
CD
GLN
A
73
14.208
2.947
−5.601
1.00
30.17
6
C

ATOM
426
OE1
GLN
A
73
13.944
3.366
−4.474
1.00
28.93
8
O

ATOM
427
NE2
GLN
A
73
13.288
2.825
−6.560
1.00
29.51
7
N

ATOM
428
C
GLN
A
73
18.329
2.675
−4.660
1.00
25.84
6
C

ATOM
429
O
GLN
A
73
18.397
2.899
−3.447
1.00
25.54
8
O

ATOM
430
N
LEU
A
74
18.779
3.528
−5.566
1.00
25.94
7
N

ATOM
431
CA
LEU
A
74
19.165
4.877
−5.161
1.00
25.91
6
C

ATOM
432
CB
LEU
A
74
20.345
5.427
−5.966
1.00
25.32
6
C

ATOM
433
CG
LEU
A
74
21.679
4.666
−5.878
1.00
26.95
6
C

ATOM
434
CD1
LEU
A
74
22.775
5.388
−6.660
1.00
26.40
6
C

ATOM
435
CD2
LEU
A
74
22.115
4.443
−4.424
1.00
27.89
6
C

ATOM
436
C
LEU
A
74
17.936
5.775
−5.292
1.00
26.16
6
C

ATOM
437
O
LEU
A
74
16.957
5.420
−5.958
1.00
25.70
8
O

ATOM
438
N
CYS
A
75
17.994
6.937
−4.656
1.00
25.94
7
N

ATOM
439
CA
CYS
A
75
16.862
7.872
−4.631
1.00
27.22
6
C

ATOM
440
CB
CYS
A
75
17.205
9.034
−3.697
1.00
26.98
6
C

ATOM
441
SG
CYS
A
75
18.557
10.044
−4.311
1.00
28.44
16
S

ATOM
442
C
CYS
A
75
16.402
8.414
−5.998
1.00
27.82
6
C

ATOM
443
O
CYS
A
75
15.287
8.945
−6.120
1.00
28.35
8
O

ATOM
444
N
ASP
A
76
17.253
8.294
−7.014
1.00
28.30
7
N

ATOM
445
CA
ASP
A
76
16.927
8.745
−8.375
1.00
29.16
6
C

ATOM
446
CB
ASP
A
76
18.199
9.183
−9.100
1.00
29.04
6
C

ATOM
447
CG
ASP
A
76
19.061
7.999
−9.514
1.00
30.05
6
C

ATOM
448
OD1
ASP
A
76
19.836
8.131
−10.488
1.00
31.11
8
O

ATOM
449
OD2
ASP
A
76
19.018
6.897
−8.925
1.00
28.83
8
O

ATOM
450
C
ASP
A
76
16.240
7.651
−9.197
1.00
29.24
6
C

ATOM
451
O
ASP
A
76
16.018
7.813
−10.403
1.00
29.76
8
O

ATOM
452
N
ASN
A
77
15.929
6.541
−8.534
1.00
29.36
7
N

ATOM
453
CA
ASN
A
77
15.269
5.367
−9.117
1.00
29.49
6
C

ATOM
454
CB
ASN
A
77
14.035
5.757
−9.938
1.00
30.31
6
C

ATOM
455
CG
ASN
A
77
13.039
6.559
−9.121
1.00
32.03
6
C

ATOM
456
OD1
ASN
A
77
12.765
6.237
−7.960
1.00
32.48
8
O

ATOM
457
ND2
ASN
A
77
12.510
7.618
−9.713
1.00
34.72
7
N

ATOM
458
C
ASN
A
77
16.169
4.355
−9.860
1.00
28.92
6
C

ATOM
459
O
ASN
A
77
15.682
3.323
−10.343
1.00
28.69
8
O

ATOM
460
N
SER
A
78
17.465
4.652
−9.943
1.00
28.22
7
N

ATOM
461
CA
SER
A
78
18.426
3.689
−10.479
1.00
27.62
6
C

ATOM
462
CB
SER
A
78
19.815
4.313
−10.669
1.00
27.53
6
C

ATOM
463
OG
SER
A
78
20.396
4.720
−9.427
1.00
28.06
8
O

ATOM
464
C
SER
A
78
18.517
2.540
−9.485
1.00
27.25
6
C

ATOM
465
O
SER
A
78
18.196
2.705
−8.312
1.00
26.90
8
O

ATOM
466
N
VAL
A
79
18.946
1.370
−9.947
1.00
27.24
7
N

ATOM
467
CA
VAL
A
79
19.108
0.237
−9.054
1.00
27.17
6
C

ATOM
468
CB
VAL
A
79
18.043
−0.866
−9.292
1.00
27.70
6
C

ATOM
469
CG1
VAL
A
79
16.627
−0.363
−8.933
1.00
27.98
6
C

ATOM
470
CG2
VAL
A
79
18.100
−1.369
−10.720
1.00
28.75
6
C

ATOM
471
C
VAL
A
79
20.516
−0.323
−9.224
1.00
27.02
6
C

ATOM
472
O
VAL
A
79
21.173
−0.094
−10.238
1.00
26.92
8
O

ATOM
473
N
GLY
A
80
20.993
−1.038
−8.220
1.00
26.41
7
N

ATOM
474
CA
GLY
A
80
22.324
−1.591
−8.302
1.00
26.60
6
C

ATOM
475
C
GLY
A
80
22.419
−2.870
−7.513
1.00
26.72
6
C

ATOM
476
O
GLY
A
80
21.537
−3.203
−6.730
1.00
26.68
8
O

ATOM
477
N
VAL
A
81
23.496
−3.602
−7.732
1.00
27.22
7
N

ATOM
478
CA
VAL
A
81
23.731
−4.813
−6.976
1.00
27.84
6
C

ATOM
479
CB
VAL
A
81
23.230
−6.069
−7.704
1.00
27.76
6
C

ATOM
480
CG1
VAL
A
81
23.847
−6.152
−9.075
1.00
30.13
6
C

ATOM
481
CG2
VAL
A
81
23.561
−7.335
−6.893
1.00
27.79
6
C

ATOM
482
C
VAL
A
81
25.218
−4.962
−6.729
1.00
27.52
6
C

ATOM
483
O
VAL
A
81
26.046
−4.662
−7.594
1.00
27.69
8
O

ATOM
484
N
LEU
A
82
25.551
−5.387
−5.524
1.00
26.88
7
N

ATOM
485
CA
LEU
A
82
26.921
−5.726
−5.211
1.00
27.04
6
C

ATOM
486
CB
LEU
A
82
27.370
−5.067
−3.907
1.00
26.63
6
C

ATOM
487
CG
LEU
A
82
28.677
−5.575
−3.286
1.00
29.59
6
C

ATOM
488
CD1
LEU
A
82
29.730
−5.961
−4.330
1.00
31.06
6
C

ATOM
489
CD2
LEU
A
82
29.224
−4.575
−2.259
1.00
29.35
6
C

ATOM
490
C
LEU
A
82
26.851
−7.239
−5.115
1.00
26.34
6
C

ATOM
491
O
LEU
A
82
26.353
−7.799
−4.127
1.00
26.20
8
O

ATOM
492
N
PHE
A
83
27.311
−7.898
−6.177
1.00
26.15
7
N

ATOM
493
CA
PHE
A
83
27.266
−9.350
−6.261
1.00
26.17
6
C

ATOM
494
CB
PHE
A
83
27.536
−9.809
−7.699
1.00
25.60
6
C

ATOM
495
CG
PHE
A
83
26.433
−9.467
−8.678
1.00
26.75
6
C

ATOM
496
CD1
PHE
A
83
26.644
−8.533
−9.676
1.00
26.59
6
C

ATOM
497
CE1
PHE
A
83
25.652
−8.235
−10.592
1.00
27.88
6
C

ATOM
498
CZ
PHE
A
83
24.432
−8.871
−10.510
1.00
28.25
6
C

ATOM
499
CE2
PHE
A
83
24.211
−9.810
−9.527
1.00
28.69
6
C

ATOM
500
CD2
PHE
A
83
25.209
−10.097
−8.607
1.00
26.35
6
C

ATOM
501
C
PHE
A
83
28.275
−10.036
−5.338
1.00
26.24
6
C

ATOM
502
O
PHE
A
83
29.308
−9.478
−4.993
1.00
25.94
8
O

ATOM
503
N
ASN
A
84
27.981
−11.277
−4.982
1.00
26.65
7
N

ATOM
504
CA
ASN
A
84
28.866
−12.062
−4.122
1.00
27.40
6
C

ATOM
505
CB
ASN
A
84
28.232
−13.424
−3.830
1.00
27.75
6
C

ATOM
506
CG
ASN
A
84
27.168
−13.348
−2.769
1.00
30.05
6
C

ATOM
507
OD1
ASN
A
84
26.839
−12.261
−2.277
1.00
29.88
8
O

ATOM
508
ND2
ASN
A
84
26.623
−14.504
−2.394
1.00
29.43
7
N

ATOM
509
C
ASN
A
84
30.275
−12.275
−4.676
1.00
27.09
6
C

ATOM
510
O
ASN
A
84
31.189
−12.621
−3.929
1.00
27.36
8
O

ATOM
511
N
ASN
A
85
30.449
−12.093
−5.979
1.00
26.58
7
N

ATOM
512
CA
ASN
A
85
31.763
−12.223
−6.588
1.00
26.96
6
C

ATOM
513
CB
ASN
A
85
31.644
−12.772
−8.009
1.00
27.42
6
C

ATOM
514
CG
ASN
A
85
30.946
−11.795
−8.950
1.00
26.92
6
C

ATOM
515
OD1
ASN
A
85
30.513
−10.720
−8.539
1.00
29.27
8
O

ATOM
516
ND2
ASN
A
85
30.851
−12.158
−10.211
1.00
26.62
7
N

ATOM
517
C
ASN
A
85
32.534
−10.898
−6.618
1.00
26.95
6
C

ATOM
518
O
ASN
A
85
33.563
−10.794
−7.273
1.00
26.21
8
O

ATOM
519
N
SER
A
86
32.010
−9.893
−5.919
1.00
27.52
7
N

ATOM
520
CA
SER
A
86
32.627
−8.563
−5.827
1.00
27.99
6
C

ATOM
521
CB
SER
A
86
34.076
−8.638
−5.354
1.00
28.25
6
C

ATOM
522
OG
SER
A
86
34.114
−9.058
−4.012
1.00
31.12
8
O

ATOM
523
C
SER
A
86
32.544
−7.699
−7.079
1.00
27.67
6
C

ATOM
524
O
SER
A
86
33.267
−6.704
−7.197
1.00
28.16
8
O

ATOM
525
N
THR
A
87
31.690
−8.067
−8.018
1.00
26.97
7
N

ATOM
526
CA
THR
A
87
31.472
−7.178
−9.157
1.00
26.07
6
C

ATOM
527
CB
THR
A
87
31.330
−7.954
−10.466
1.00
25.82
6
C

ATOM
528
OG1
THR
A
87
30.155
−8.782
−10.416
1.00
23.37
8
O

ATOM
529
CG2
THR
A
87
32.496
−8.954
−10.633
1.00
24.35
6
C

ATOM
530
C
THR
A
87
30.203
−6.416
−8.844
1.00
26.76
6
C

ATOM
531
O
THR
A
87
29.409
−6.851
−8.002
1.00
26.49
8
O

ATOM
532
N
ARG
A
88
30.007
−5.282
−9.504
1.00
26.58
7
N

ATOM
533
CA
ARG
A
88
28.807
−4.486
−9.277
1.00
27.64
6
C

ATOM
534
CB
ARG
A
88
29.136
−3.257
−8.418
1.00
28.83
6
C

ATOM
535
CG
ARG
A
88
30.493
−3.375
−7.687
1.00
32.71
6
C

ATOM
536
CD
ARG
A
88
30.554
−2.751
−6.330
1.00
38.97
6
C

ATOM
537
NE
ARG
A
88
31.926
−2.457
−5.938
1.00
42.42
7
N

ATOM
538
CZ
ARG
A
88
32.537
−2.966
−4.878
1.00
44.46
6
C

ATOM
539
NH1
ARG
A
88
31.909
−3.806
−4.081
1.00
47.01
7
N

ATOM
540
NH2
ARG
A
88
33.789
−2.628
−4.607
1.00
46.70
7
N

ATOM
541
C
ARG
A
88
28.184
−4.088
−10.610
1.00
27.27
6
C

ATOM
542
O
ARG
A
88
28.891
−3.834
−11.582
1.00
26.57
8
O

ATOM
543
N
LEU
A
89
26.859
−4.039
−10.647
1.00
27.53
7
N

ATOM
544
CA
LEU
A
89
26.136
−3.680
−11.859
1.00
27.62
6
C

ATOM
545
CB
LEU
A
89
25.461
−4.914
−12.447
1.00
27.92
6
C

ATOM
546
CG
LEU
A
89
24.688
−4.784
−13.759
1.00
28.76
6
C

ATOM
547
CD1
LEU
A
89
25.579
−4.250
−14.882
1.00
29.33
6
C

ATOM
548
CD2
LEU
A
89
24.090
−6.152
−14.140
1.00
30.55
6
C

ATOM
549
C
LEU
A
89
25.083
−2.647
−11.492
1.00
27.77
6
C

ATOM
550
O
LEU
A
89
24.386
−2.790
−10.482
1.00
27.57
8
O

ATOM
551
N
ILE
A
90
24.982
−1.604
−12.308
1.00
27.83
7
N

ATOM
552
CA
ILE
A
90
24.023
−0.532
−12.076
1.00
28.43
6
C

ATOM
553
CB
ILE
A
90
24.763
0.807
−11.845
1.00
28.80
6
C

ATOM
554
CG1
ILE
A
90
25.556
0.776
−10.538
1.00
30.29
6
C

ATOM
555
CD1
ILE
A
90
26.784
−0.098
−10.613
1.00
35.17
6
C

ATOM
556
CG2
ILE
A
90
23.783
1.978
−11.861
1.00
29.24
6
C

ATOM
557
C
ILE
A
90
23.133
−0.383
−13.296
1.00
28.55
6
C

ATOM
558
O
ILE
A
90
23.617
−0.415
−14.422
1.00
28.28
8
O

ATOM
559
N
LEU
A
91
21.836
−0.227
−13.065
1.00
28.68
7
N

ATOM
560
CA
LEU
A
91
20.892
0.034
−14.139
1.00
29.39
6
C

ATOM
561
CB
LEU
A
91
19.790
−1.021
−14.138
1.00
29.32
6
C

ATOM
562
CG
LEU
A
91
18.627
−0.740
−15.096
1.00
30.30
6
C

ATOM
563
CD1
LEU
A
91
19.064
−0.984
−16.540
1.00
30.54
6
C

ATOM
564
CD2
LEU
A
91
17.408
−1.595
−14.738
1.00
30.62
6
C

ATOM
565
C
LEU
A
91
20.329
1.441
−13.885
1.00
29.83
6
C

ATOM
566
O
LEU
A
91
19.727
1.695
−12.830
1.00
29.50
8
O

ATOM
567
N
TYR
A
92
20.579
2.358
−14.821
1.00
30.23
7
N

ATOM
568
CA
TYR
A
92
20.127
3.751
−14.697
1.00
30.91
6
C

ATOM
569
CB
TYR
A
92
20.768
4.626
−15.772
1.00
30.86
6
C

ATOM
570
CG
TYR
A
92
22.249
4.831
−15.576
1.00
32.76
6
C

ATOM
571
CD1
TYR
A
92
23.140
3.777
−15.715
1.00
33.72
6
C

ATOM
572
CE1
TYR
A
92
24.493
3.958
−15.528
1.00
35.16
6
C

ATOM
573
CZ
TYR
A
92
24.973
5.204
−15.199
1.00
34.76
6
C

ATOM
574
OH
TYR
A
92
26.318
5.388
−15.021
1.00
35.17
8
O

ATOM
575
CE2
TYR
A
92
24.115
6.268
−15.056
1.00
34.84
6
C

ATOM
576
CD2
TYR
A
92
22.758
6.078
−15.238
1.00
34.70
6
C

ATOM
577
C
TYR
A
92
18.610
3.886
−14.743
1.00
31.31
6
C

ATOM
578
O
TYR
A
92
17.918
2.977
−15.190
1.00
31.18
8
O

ATOM
579
N
ASN
A
93
18.096
5.028
−14.283
1.00
32.12
7
N

ATOM
580
CA
ASN
A
93
16.650
5.235
−14.234
1.00
33.32
6
C

ATOM
581
CB
ASN
A
93
16.261
6.432
−13.344
1.00
33.25
6
C

ATOM
582
CG
ASN
A
93
16.786
7.768
−13.866
1.00
33.58
6
C

ATOM
583
OD1
ASN
A
93
17.268
7.874
−14.998
1.00
32.74
8
O

ATOM
584
ND2
ASN
A
93
16.697
8.802
−13.025
1.00
33.00
7
N

ATOM
585
C
ASN
A
93
15.951
5.319
−15.598
1.00
34.12
6
C

ATOM
586
O
ASN
A
93
14.728
5.469
−15.661
1.00
34.09
8
O

ATOM
587
N
ASP
A
94
16.708
5.244
−16.687
1.00
34.86
7
N

ATOM
588
CA
ASP
A
94
16.053
5.211
−17.998
1.00
35.81
6
C

ATOM
589
CB
ASP
A
94
16.846
5.942
−19.087
1.00
36.25
6
C

ATOM
590
CG
ASP
A
94
18.336
5.762
−18.951
1.00
38.64
6
C

ATOM
591
OD1
ASP
A
94
18.966
5.230
−19.897
1.00
36.80
8
O

ATOM
592
OD2
ASP
A
94
18.964
6.124
−17.927
1.00
44.80
8
O

ATOM
593
C
ASP
A
94
15.721
3.777
−18.384
1.00
35.67
6
C

ATOM
594
O
ASP
A
94
15.157
3.522
−19.453
1.00
35.81
8
O

ATOM
595
N
GLY
A
95
16.071
2.846
−17.498
1.00
35.30
7
N

ATOM
596
CA
GLY
A
95
15.730
1.441
−17.658
1.00
34.65
6
C

ATOM
597
C
GLY
A
95
16.512
0.649
−18.693
1.00
34.46
6
C

ATOM
598
O
GLY
A
95
16.176
−0.506
−18.967
1.00
34.40
8
O

ATOM
599
N
ASP
A
96
17.563
1.242
−19.250
1.00
34.02
7
N

ATOM
600
CA
ASP
A
96
18.339
0.568
−20.293
1.00
33.87
6
C

ATOM
601
CB
ASP
A
96
17.887
1.059
−21.675
1.00
33.75
6
C

ATOM
602
CG
ASP
A
96
18.427
0.203
−22.811
1.00
34.99
6
C

ATOM
603
OD1
ASP
A
96
18.663
−1.003
−22.608
1.00
34.90
8
O

ATOM
604
OD2
ASP
A
96
18.647
0.659
−23.949
1.00
36.72
8
O

ATOM
605
C
ASP
A
96
19.852
0.741
−20.129
1.00
33.04
6
C

ATOM
606
O
ASP
A
96
20.624
−0.175
−20.402
1.00
33.28
8
O

ATOM
607
N
SER
A
97
20.280
1.911
−19.671
1.00
32.38
7
N

ATOM
608
CA
SER
A
97
21.709
2.180
−19.519
1.00
31.14
6
C

ATOM
609
CB
SER
A
97
21.951
3.681
−19.327
1.00
31.84
6
C

ATOM
610
OG
SER
A
97
21.665
4.395
−20.523
1.00
31.58
8
O

ATOM
611
C
SER
A
97
22.330
1.387
−18.364
1.00
30.82
6
C

ATOM
612
O
SER
A
97
21.708
1.223
−17.307
1.00
29.82
8
O

ATOM
613
N
LEU
A
98
23.545
0.892
−18.575
1.00
29.55
7
N

ATOM
614
CA
LEU
A
98
24.223
0.093
−17.559
1.00
29.94
6
C

ATOM
615
CB
LEU
A
98
24.370
−1.359
−18.022
1.00
29.99
6
C

ATOM
616
CG
LEU
A
98
23.131
−2.222
−18.248
1.00
29.63
6
C

ATOM
617
CD1
LEU
A
98
23.554
−3.504
−18.966
1.00
30.60
6
C

ATOM
618
CD2
LEU
A
98
22.433
−2.544
−16.933
1.00
29.49
6
C

ATOM
619
C
LEU
A
98
25.612
0.614
−17.275
1.00
29.90
6
C

ATOM
620
O
LEU
A
98
26.267
1.186
−18.152
1.00
29.73
8
O

ATOM
621
N
GLN
A
99
26.055
0.397
−16.040
1.00
29.63
7
N

ATOM
622
CA
GLN
A
99
27.436
0.632
−15.656
1.00
29.72
6
C

ATOM
623
CB
GLN
A
99
27.580
1.834
−14.710
1.00
29.87
6
C

ATOM
624
CG
GLN
A
99
29.006
2.022
−14.179
1.00
31.65
6
C

ATOM
625
CD
GLN
A
99
29.154
3.179
−13.176
1.00
34.86
6
C

ATOM
626
OE1
GLN
A
99
28.225
3.975
−12.969
1.00
38.05
8
O

ATOM
627
NE2
GLN
A
99
30.320
3.268
−12.558
1.00
35.00
7
N

ATOM
628
C
GLN
A
99
27.878
−0.649
−14.948
1.00
29.09
6
C

ATOM
629
O
GLN
A
99
27.285
−1.027
−13.939
1.00
28.62
8
O

ATOM
630
N
TYR
A
100
28.885
−1.326
−15.496
1.00
28.74
7
N

ATOM
631
CA
TYR
A
100
29.428
−2.553
−14.902
1.00
28.36
6
C

ATOM
632
CB
TYR
A
100
29.588
−3.661
−15.952
1.00
28.26
6
C

ATOM
633
CG
TYR
A
100
29.947
−5.033
−15.395
1.00
28.17
6
C

ATOM
634
CD1
TYR
A
100
29.237
−5.587
−14.329
1.00
28.77
6
C

ATOM
635
CE1
TYR
A
100
29.544
−6.851
−13.835
1.00
28.30
6
C

ATOM
636
CZ
TYR
A
100
30.580
−7.566
−14.398
1.00
27.40
6
C

ATOM
637
OH
TYR
A
100
30.886
−8.818
−13.917
1.00
27.32
8
O

ATOM
638
CE2
TYR
A
100
31.303
−7.029
−15.446
1.00
27.53
6
C

ATOM
639
CD2
TYR
A
100
30.981
−5.782
−15.941
1.00
27.24
6
C

ATOM
640
C
TYR
A
100
30.782
−2.244
−14.300
1.00
28.71
6
C

ATOM
641
O
TYR
A
100
31.638
−1.639
−14.963
1.00
29.05
8
O

ATOM
642
N
ILE
A
101
30.973
−2.630
−13.040
1.00
28.17
7
N

ATOM
643
CA
ILE
A
101
32.246
−2.421
−12.363
1.00
28.15
6
C

ATOM
644
CB
ILE
A
101
32.094
−1.589
−11.065
1.00
28.44
6
C

ATOM
645
CG1
ILE
A
101
31.429
−0.235
−11.339
1.00
29.47
6
C

ATOM
646
CD1
ILE
A
101
29.939
−0.276
−11.252
1.00
32.69
6
C

ATOM
647
CG2
ILE
A
101
33.444
−1.322
−10.461
1.00
28.54
6
C

ATOM
648
C
ILE
A
101
32.816
−3.778
−12.014
1.00
28.30
6
C

ATOM
649
O
ILE
A
101
32.228
−4.518
−11.212
1.00
26.96
8
O

ATOM
650
N
GLU
A
102
33.951
−4.099
−12.625
1.00
28.36
7
N

ATOM
651
CA
GLU
A
102
34.618
−5.373
−12.391
1.00
29.48
6
C

ATOM
652
CB
GLU
A
102
35.545
−5.713
−13.558
1.00
29.40
6
C

ATOM
653
CG
GLU
A
102
34.783
−6.010
−14.839
1.00
29.63
6
C

ATOM
654
CD
GLU
A
102
35.686
−6.094
−16.049
1.00
31.22
6
C

ATOM
655
OE1
GLU
A
102
36.394
−7.111
−16.206
1.00
31.47
8
O

ATOM
656
OE2
GLU
A
102
35.691
−5.132
−16.841
1.00
34.12
8
O

ATOM
657
C
GLU
A
102
35.380
−5.349
−11.072
1.00
30.52
6
C

ATOM
658
O
GLU
A
102
35.519
−4.299
−10.446
1.00
29.92
8
O

ATOM
659
N
ARG
A
103
35.854
−6.518
−10.647
1.00
31.46
7
N

ATOM
660
CA
ARG
A
103
36.540
−6.644
−9.365
1.00
33.21
6
C

ATOM
661
CB
ARG
A
103
37.104
−8.056
−9.195
1.00
33.45
6
C

ATOM
662
CG
ARG
A
103
36.036
−9.120
−9.017
1.00
35.12
6
C

ATOM
663
CD
ARG
A
103
36.541
−10.534
−9.294
1.00
39.05
6
C

ATOM
664
NE
ARG
A
103
37.045
−11.209
−8.112
1.00
42.34
7
N

ATOM
665
CZ
ARG
A
103
38.106
−12.010
−8.100
1.00
43.44
6
C

ATOM
666
NH1
ARG
A
103
38.817
−12.210
−9.204
1.00
44.15
7
N

ATOM
667
NH2
ARG
A
103
38.474
−12.594
−6.972
1.00
44.98
7
N

ATOM
668
C
ARG
A
103
37.659
−5.641
−9.179
1.00
33.82
6
C

ATOM
669
O
ARG
A
103
37.835
−5.100
−8.101
1.00
34.04
8
O

ATOM
670
N
ASP
A
104
38.415
−5.400
−10.236
1.00
35.17
7
N

ATOM
671
CA
ASP
A
104
39.541
−4.483
−10.173
1.00
36.39
6
C

ATOM
672
CB
ASP
A
104
40.521
−4.855
−11.266
1.00
37.19
6
C

ATOM
673
CG
ASP
A
104
39.825
−5.106
−12.580
1.00
40.35
6
C

ATOM
674
OD1
ASP
A
104
39.635
−6.296
−12.946
1.00
45.85
8
O

ATOM
675
OD2
ASP
A
104
39.375
−4.177
−13.279
1.00
39.52
8
O

ATOM
676
C
ASP
A
104
39.114
−3.023
−10.344
1.00
36.08
6
C

ATOM
677
O
ASP
A
104
39.958
−2.139
−10.469
1.00
36.56
8
O

ATOM
678
N
GLY
A
105
37.812
−2.768
−10.359
1.00
35.42
7
N

ATOM
679
CA
GLY
A
105
37.328
−1.407
−10.502
1.00
35.09
6
C

ATOM
680
C
GLY
A
105
37.112
−0.922
−11.930
1.00
34.77
6
C

ATOM
681
O
GLY
A
105
36.659
0.201
−12.136
1.00
34.65
8
O

ATOM
682
N
THR
A
106
37.413
−1.754
−12.922
1.00
34.37
7
N

ATOM
683
CA
THR
A
106
37.198
−1.344
−14.315
1.00
34.21
6
C

ATOM
684
CB
THR
A
106
37.736
−2.400
−15.295
1.00
34.25
6
C

ATOM
685
OG1
THR
A
106
39.147
−2.568
−15.095
1.00
33.88
8
O

ATOM
686
CG2
THR
A
106
37.638
−1.888
−16.738
1.00
34.18
6
C

ATOM
687
C
THR
A
106
35.713
−1.081
−14.577
1.00
34.43
6
C

ATOM
688
O
THR
A
106
34.864
−1.919
−14.263
1.00
33.73
8
O

ATOM
689
N
GLU
A
107
35.401
0.080
−15.154
1.00
34.65
7
N

ATOM
690
CA
GLU
A
107
34.009
0.456
−15.410
1.00
35.83
6
C

ATOM
691
CB
GLU
A
107
33.721
1.882
−14.917
1.00
35.89
6
C

ATOM
692
CG
GLU
A
107
33.975
2.149
−13.440
1.00
38.03
6
C

ATOM
693
CD
GLU
A
107
33.784
3.616
−13.066
1.00
40.69
6
C

ATOM
694
OE1
GLU
A
107
33.359
4.413
−13.938
1.00
42.02
8
O

ATOM
695
OE2
GLU
A
107
34.062
3.982
−11.902
1.00
42.05
8
O

ATOM
696
C
GLU
A
107
33.649
0.369
−16.890
1.00
35.91
6
C

ATOM
697
O
GLU
A
107
34.394
0.848
−17.750
1.00
36.37
8
O

ATOM
698
N
SER
A
108
32.508
−0.244
−17.182
1.00
35.87
7
N

ATOM
699
CA
SER
A
108
32.015
−0.356
−18.551
1.00
36.36
6
C

ATOM
700
CB
SER
A
108
31.931
−1.820
−18.984
1.00
36.01
6
C

ATOM
701
OG
SER
A
108
33.217
−2.393
−19.154
1.00
36.34
8
O

ATOM
702
C
SER
A
108
30.624
0.261
−18.612
1.00
36.72
6
C

ATOM
703
O
SER
A
108
29.822
0.043
−17.708
1.00
36.15
8
O

ATOM
704
N
TYR
A
109
30.353
1.044
−19.658
1.00
36.80
7
N

ATOM
705
CA
TYR
A
109
29.036
1.654
−19.838
1.00
37.56
6
C

ATOM
706
CB
TYR
A
109
29.135
3.179
−19.970
1.00
37.52
6
C

ATOM
707
CG
TYR
A
109
29.685
3.801
−18.705
1.00
38.15
6
C

ATOM
708
CD1
TYR
A
109
31.044
3.771
−18.435
1.00
39.00
6
C

ATOM
709
CE1
TYR
A
109
31.560
4.308
−17.266
1.00
38.75
6
C

ATOM
710
CZ
TYR
A
109
30.710
4.878
−16.347
1.00
39.50
6
C

ATOM
711
OH
TYR
A
109
31.236
5.409
−15.186
1.00
39.40
8
O

ATOM
712
CE2
TYR
A
109
29.346
4.909
−16.586
1.00
39.18
6
C

ATOM
713
CD2
TYR
A
109
28.842
4.366
−17.759
1.00
38.80
6
C

ATOM
714
C
TYR
A
109
28.365
1.011
−21.036
1.00
37.99
6
C

ATOM
715
O
TYR
A
109
28.862
1.095
−22.159
1.00
38.19
8
O

ATOM
716
N
LEU
A
110
27.251
0.338
−20.777
1.00
38.47
7
N

ATOM
717
CA
LEU
A
110
26.557
−0.425
−21.803
1.00
39.08
6
C

ATOM
718
CB
LEU
A
110
26.799
−1.926
−21.594
1.00
39.57
6
C

ATOM
719
CG
LEU
A
110
28.213
−2.444
−21.365
1.00
40.82
6
C

ATOM
720
CD1
LEU
A
110
28.195
−3.950
−21.138
1.00
41.92
6
C

ATOM
721
CD2
LEU
A
110
29.094
−2.098
−22.558
1.00
42.45
6
C

ATOM
722
C
LEU
A
110
25.067
−0.202
−21.738
1.00
38.83
6
C

ATOM
723
O
LEU
A
110
24.565
0.584
−20.932
1.00
38.56
8
O

ATOM
724
N
THR
A
111
24.358
−0.912
−22.605
1.00
38.56
7
N

ATOM
725
CA
THR
A
111
22.911
−0.869
−22.607
1.00
38.47
6
C

ATOM
726
CB
THR
A
111
22.403
−0.170
−23.884
1.00
38.99
6
C

ATOM
727
OG1
THR
A
111
22.813
1.205
−23.879
1.00
40.54
8
O

ATOM
728
CG2
THR
A
111
20.922
−0.042
−23.840
1.00
40.63
6
C

ATOM
729
C
THR
A
111
22.389
−2.300
−22.523
1.00
37.37
6
C

ATOM
730
O
THR
A
111
23.043
−3.234
−23.004
1.00
37.13
8
O

ATOM
731
N
VAL
A
112
21.225
−2.480
−21.908
1.00
36.49
7
N

ATOM
732
CA
VAL
A
112
20.606
−3.797
−21.842
1.00
36.02
6
C

ATOM
733
CB
VAL
A
112
19.387
−3.814
−20.899
1.00
36.12
6
C

ATOM
734
CG1
VAL
A
112
18.649
−5.153
−20.973
1.00
35.72
6
C

ATOM
735
CG2
VAL
A
112
19.819
−3.524
−19.464
1.00
35.10
6
C

ATOM
736
C
VAL
A
112
20.179
−4.189
−23.260
1.00
36.45
6
C

ATOM
737
O
VAL
A
112
20.398
−5.317
−23.696
1.00
35.73
8
O

ATOM
738
N
SER
A
113
19.607
−3.229
−23.980
1.00
36.64
7
N

ATOM
739
CA
SER
A
113
19.122
−3.462
−25.338
1.00
37.54
6
C

ATOM
740
CB
SER
A
113
18.484
−2.190
−25.912
1.00
37.50
6
C

ATOM
741
OG
SER
A
113
19.411
−1.120
−25.988
1.00
38.07
8
O

ATOM
742
C
SER
A
113
20.203
−3.999
−26.273
1.00
37.96
6
C

ATOM
743
O
SER
A
113
19.897
−4.727
−27.224
1.00
38.50
8
O

ATOM
744
N
SER
A
114
21.459
−3.648
−26.005
1.00
38.01
7
N

ATOM
745
CA
SER
A
114
22.583
−4.107
−26.826
1.00
38.47
6
C

ATOM
746
CB
SER
A
114
23.839
−3.268
−26.562
1.00
38.34
6
C

ATOM
747
OG
SER
A
114
24.459
−3.627
−25.338
1.00
38.19
8
O

ATOM
748
C
SER
A
114
22.910
−5.588
−26.628
1.00
38.68
6
C

ATOM
749
O
SER
A
114
23.719
−6.155
−27.370
1.00
38.77
8
O

ATOM
750
N
HIS
A
115
22.296
−6.199
−25.619
1.00
38.57
7
N

ATOM
751
CA
HIS
A
115
22.502
−7.616
−25.308
1.00
38.72
6
C

ATOM
752
CB
HIS
A
115
21.892
−8.516
−26.398
1.00
39.09
6
C

ATOM
753
CG
HIS
A
115
21.849
−9.966
−26.026
1.00
39.56
6
C

ATOM
754
ND1
HIS
A
115
20.675
−10.623
−25.724
1.00
40.68
7
N

ATOM
755
CE1
HIS
A
115
20.941
−11.882
−25.428
1.00
40.16
6
C

ATOM
756
NE2
HIS
A
115
22.246
−12.066
−25.525
1.00
40.59
7
N

ATOM
757
CD2
HIS
A
115
22.836
−10.884
−25.899
1.00
39.60
6
C

ATOM
758
C
HIS
A
115
23.969
−7.997
−25.067
1.00
38.66
6
C

ATOM
759
O
HIS
A
115
24.575
−8.716
−25.871
1.00
38.76
8
O

ATOM
760
N
PRO
A
116
24.538
−7.509
−23.969
1.00
38.46
7
N

ATOM
761
CA
PRO
A
116
25.911
−7.855
−23.578
1.00
38.23
6
C

ATOM
762
CB
PRO
A
116
26.177
−6.905
−22.412
1.00
38.56
6
C

ATOM
763
CG
PRO
A
116
24.818
−6.660
−21.847
1.00
38.47
6
C

ATOM
764
CD
PRO
A
116
23.924
−6.544
−23.039
1.00
38.20
6
C

ATOM
765
C
PRO
A
116
25.963
−9.307
−23.103
1.00
37.95
6
C

ATOM
766
O
PRO
A
116
25.567
−9.620
−21.977
1.00
37.64
8
O

ATOM
767
N
ASN
A
117
26.460
−10.188
−23.966
1.00
37.62
7
N

ATOM
768
CA
ASN
A
117
26.448
−11.631
−23.712
1.00
37.09
6
C

ATOM
769
CB
ASN
A
117
27.238
−12.351
−24.816
1.00
37.51
6
C

ATOM
770
CG
ASN
A
117
26.686
−12.059
−26.199
1.00
38.58
6
C

ATOM
771
OD1
ASN
A
117
25.471
−12.017
−26.389
1.00
40.21
8
O

ATOM
772
ND2
ASN
A
117
27.571
−11.839
−27.169
1.00
40.37
7
N

ATOM
773
C
ASN
A
117
26.895
−12.111
−22.320
1.00
36.55
6
C

ATOM
774
O
ASN
A
117
26.206
−12.907
−21.671
1.00
36.33
8
O

ATOM
775
N
ALA
A
118
28.046
−11.630
−21.865
1.00
35.80
7
N

ATOM
776
CA
ALA
A
118
28.604
−12.066
−20.591
1.00
35.11
6
C

ATOM
777
CB
ALA
A
118
30.063
−11.596
−20.465
1.00
35.22
6
C

ATOM
778
C
ALA
A
118
27.786
−11.594
−19.385
1.00
34.02
6
C

ATOM
779
O
ALA
A
118
27.867
−12.181
−18.303
1.00
33.55
8
O

ATOM
780
N
LEU
A
119
26.972
−10.565
−19.587
1.00
33.40
7
N

ATOM
781
CA
LEU
A
119
26.227
−9.942
−18.491
1.00
33.05
6
C

ATOM
782
CB
LEU
A
119
26.328
−8.416
−18.609
1.00
33.25
6
C

ATOM
783
CG
LEU
A
119
27.690
−7.786
−18.323
1.00
34.22
6
C

ATOM
784
CD1
LEU
A
119
27.621
−6.275
−18.502
1.00
35.25
6
C

ATOM
785
CD2
LEU
A
119
28.119
−8.137
−16.917
1.00
36.37
6
C

ATOM
786
C
LEU
A
119
24.745
−10.322
−18.391
1.00
32.71
6
C

ATOM
787
O
LEU
A
119
24.071
−9.939
−17.436
1.00
31.84
8
O

ATOM
788
N
MET
A
120
24.234
−11.080
−19.356
1.00
32.22
7
N

ATOM
789
CA
MET
A
120
22.806
−11.400
−19.362
1.00
31.92
6
C

ATOM
790
CB
MET
A
120
22.444
−12.265
−20.567
1.00
32.59
6
C

ATOM
791
CG
MET
A
120
22.717
−11.571
−21.905
1.00
34.09
6
C

ATOM
792
SD
MET
A
120
22.013
−9.907
−22.079
1.00
38.77
16
S

ATOM
793
CE
MET
A
120
20.285
−10.247
−21.682
1.00
38.80
6
C

ATOM
794
C
MET
A
120
22.259
−11.979
−18.042
1.00
31.30
6
C

ATOM
795
O
MET
A
120
21.224
−11.531
−17.558
1.00
30.96
8
O

ATOM
796
N
LYS
A
121
22.960
−12.934
−17.441
1.00
30.40
7
N

ATOM
797
CA
LYS
A
121
22.510
−13.503
−16.167
1.00
30.26
6
C

ATOM
798
CB
LYS
A
121
23.373
−14.696
−15.761
1.00
30.27
6
C

ATOM
799
CG
LYS
A
121
23.150
−15.967
−16.604
1.00
33.46
6
C

ATOM
800
CD
LYS
A
121
23.970
−17.127
−16.066
1.00
35.55
6
C

ATOM
801
CE
LYS
A
121
23.753
−18.400
−16.889
1.00
38.65
6
C

ATOM
802
NZ
LYS
A
121
23.433
−18.087
−18.308
1.00
39.64
7
N

ATOM
803
C
LYS
A
121
22.513
−12.456
−15.025
1.00
29.34
6
C

ATOM
804
O
LYS
A
121
21.611
−12.425
−14.180
1.00
28.21
8
O

ATOM
805
N
LYS
A
122
23.540
−11.623
−14.989
1.00
28.61
7
N

ATOM
806
CA
LYS
A
122
23.610
−10.593
−13.951
1.00
28.77
6
C

ATOM
807
CB
LYS
A
122
25.008
−9.966
−13.893
1.00
28.40
6
C

ATOM
808
CG
LYS
A
122
26.040
−10.890
−13.202
1.00
28.80
6
C

ATOM
809
CD
LYS
A
122
27.469
−10.367
−13.341
1.00
27.89
6
C

ATOM
810
CE
LYS
A
122
28.417
−11.049
−12.351
1.00
29.28
6
C

ATOM
811
NZ
LYS
A
122
29.821
−10.547
−12.489
1.00
27.94
7
N

ATOM
812
C
LYS
A
122
22.502
−9.558
−14.155
1.00
28.77
6
C

ATOM
813
O
LYS
A
122
21.872
−9.110
−13.192
1.00
28.75
8
O

ATOM
814
N
ILE
A
123
22.247
−9.199
−15.408
1.00
28.79
7
N

ATOM
815
CA
ILE
A
123
21.157
−8.279
−15.725
1.00
29.61
6
C

ATOM
816
CB
ILE
A
123
21.107
−8.009
−17.237
1.00
29.77
6
C

ATOM
817
CG1
ILE
A
123
22.237
−7.059
−17.646
1.00
30.44
6
C

ATOM
818
CD1
ILE
A
123
22.525
−7.043
−19.147
1.00
30.78
6
C

ATOM
819
CG2
ILE
A
123
19.744
−7.442
−17.629
1.00
30.39
6
C

ATOM
820
C
ILE
A
123
19.807
−8.833
−15.256
1.00
29.86
6
C

ATOM
821
O
ILE
A
123
18.965
−8.097
−14.733
1.00
29.63
8
O

ATOM
822
N
THR
A
124
19.603
−10.135
−15.442
1.00
30.05
7
N

ATOM
823
CA
THR
A
124
18.347
−10.774
−15.045
1.00
30.32
6
C

ATOM
824
CB
THR
A
124
18.306
−12.235
−15.529
1.00
30.81
6
C

ATOM
825
OG1
THR
A
124
18.216
−12.258
−16.963
1.00
31.11
8
O

ATOM
826
CG2
THR
A
124
17.012
−12.909
−15.086
1.00
31.08
6
C

ATOM
827
C
THR
A
124
18.141
−10.705
−13.544
1.00
30.49
6
C

ATOM
828
O
THR
A
124
17.042
−10.417
−13.062
1.00
29.67
8
O

ATOM
829
N
LEU
A
125
19.212
−10.964
−12.807
1.00
30.74
7
N

ATOM
830
CA
LEU
A
125
19.172
−10.897
−11.356
1.00
31.48
6
C

ATOM
831
CB
LEU
A
125
20.505
−11.357
−10.780
1.00
31.93
6
C

ATOM
832
CG
LEU
A
125
20.591
−12.868
−10.563
1.00
34.56
6
C

ATOM
833
CD1
LEU
A
125
22.040
−13.324
−10.481
1.00
37.59
6
C

ATOM
834
CD2
LEU
A
125
19.841
−13.214
−9.287
1.00
36.58
6
C

ATOM
835
C
LEU
A
125
18.872
−9.475
−10.893
1.00
31.05
6
C

ATOM
836
O
LEU
A
125
18.117
−9.273
−9.945
1.00
31.71
8
O

ATOM
837
N
LEU
A
126
19.472
−8.494
−11.554
1.00
30.76
7
N

ATOM
838
CA
LEU
A
126
19.265
−7.098
−11.173
1.00
30.69
6
C

ATOM
839
CB
LEU
A
126
20.215
−6.172
−11.936
1.00
30.65
6
C

ATOM
840
CG
LEU
A
126
20.182
−4.691
−11.530
1.00
30.81
6
C

ATOM
841
CD1
LEU
A
126
20.007
−4.521
−10.014
1.00
31.78
6
C

ATOM
842
CD2
LEU
A
126
21.431
−3.961
−12.006
1.00
30.87
6
C

ATOM
843
C
LEU
A
126
17.815
−6.694
−11.397
1.00
31.15
6
C

ATOM
844
O
LEU
A
126
17.205
−6.038
−10.552
1.00
30.24
8
O

ATOM
845
N
LYS
A
127
17.256
−7.090
−12.538
1.00
31.51
7
N

ATOM
846
CA
LYS
A
127
15.858
−6.790
−12.814
1.00
32.55
6
C

ATOM
847
CB
LYS
A
127
15.478
−7.183
−14.238
1.00
32.83
6
C

ATOM
848
CG
LYS
A
127
15.983
−6.183
−15.249
1.00
35.21
6
C

ATOM
849
CD
LYS
A
127
15.786
−6.665
−16.663
1.00
38.59
6
C

ATOM
850
CE
LYS
A
127
16.504
−5.741
−17.630
1.00
40.76
6
C

ATOM
851
NZ
LYS
A
127
15.590
−4.779
−18.294
1.00
42.33
7
N

ATOM
852
C
LYS
A
127
14.944
−7.442
−11.786
1.00
32.75
6
C

ATOM
853
O
LYS
A
127
13.950
−6.842
−11.365
1.00
32.80
8
O

ATOM
854
N
TYR
A
128
15.268
−8.662
−11.372
1.00
33.10
7
N

ATOM
855
CA
TYR
A
128
14.495
−9.284
−10.298
1.00
33.78
6
C

ATOM
856
CB
TYR
A
128
15.046
−10.647
−9.904
1.00
34.29
6
C

ATOM
857
CG
TYR
A
128
14.322
−11.233
−8.714
1.00
37.18
6
C

ATOM
858
CD1
TYR
A
128
14.931
−11.307
−7.474
1.00
40.84
6
C

ATOM
859
CE1
TYR
A
128
14.270
−11.835
−6.382
1.00
42.52
6
C

ATOM
860
CZ
TYR
A
128
12.984
−12.297
−6.518
1.00
43.44
6
C

ATOM
861
OH
TYR
A
128
12.340
−12.821
−5.419
1.00
46.68
8
O

ATOM
862
CE2
TYR
A
128
12.347
−12.235
−7.736
1.00
42.86
6
C

ATOM
863
CD2
TYR
A
128
13.016
−11.700
−8.829
1.00
40.68
6
C

ATOM
864
C
TYR
A
128
14.497
−8.385
−9.059
1.00
33.36
6
C

ATOM
865
O
TYR
A
128
13.445
−8.136
−8.469
1.00
32.70
8
O

ATOM
866
N
PHE
A
129
15.683
−7.922
−8.659
1.00
33.07
7
N

ATOM
867
CA
PHE
A
129
15.797
−7.020
−7.501
1.00
33.01
6
C

ATOM
868
CB
PHE
A
129
17.255
−6.609
−7.244
1.00
33.14
6
C

ATOM
869
CG
PHE
A
129
18.074
−7.648
−6.523
1.00
34.24
6
C

ATOM
870
CD1
PHE
A
129
19.076
−8.325
−7.178
1.00
35.93
6
C

ATOM
871
CE1
PHE
A
129
19.839
−9.288
−6.523
1.00
36.77
6
C

ATOM
872
CZ
PHE
A
129
19.611
−9.552
−5.183
1.00
37.12
6
C

ATOM
873
CE2
PHE
A
129
18.624
−8.859
−4.505
1.00
36.61
6
C

ATOM
874
CD2
PHE
A
129
17.862
−7.914
−5.177
1.00
36.22
6
C

ATOM
875
C
PHE
A
129
14.956
−5.762
−7.707
1.00
32.81
6
C

ATOM
876
O
PHE
A
129
14.213
−5.347
−6.812
1.00
32.43
8
O

ATOM
877
N
ARG
A
130
15.090
−5.144
−8.876
1.00
32.83
7
N

ATOM
878
CA
ARG
A
130
14.347
−3.922
−9.173
1.00
33.47
6
C

ATOM
879
CB
ARG
A
130
14.625
−3.434
−10.595
1.00
33.52
6
C

ATOM
880
CG
ARG
A
130
13.696
−2.287
−11.010
1.00
34.77
6
C

ATOM
881
CD
ARG
A
130
13.624
−2.022
−12.500
1.00
36.82
6
C

ATOM
882
NE
ARG
A
130
13.117
−3.171
−13.245
1.00
37.03
7
N

ATOM
883
CZ
ARG
A
130
13.233
−3.298
−14.557
1.00
38.07
6
C

ATOM
884
NH1
ARG
A
130
13.833
−2.344
−15.253
1.00
39.49
7
N

ATOM
885
NH2
ARG
A
130
12.755
−4.370
−15.174
1.00
37.52
7
N

ATOM
886
C
ARG
A
130
12.845
−4.140
−9.000
1.00
33.31
6
C

ATOM
887
O
ARG
A
130
12.151
−3.328
−8.386
1.00
32.73
8
O

ATOM
888
N
ASN
A
131
12.352
−5.245
−9.548
1.00
33.24
7
N

ATOM
889
CA
ASN
A
131
10.933
−5.561
−9.480
1.00
33.80
6
C

ATOM
890
CB
ASN
A
131
10.596
−6.732
−10.406
1.00
33.96
6
C

ATOM
891
CG
ASN
A
131
10.819
−6.399
−11.867
1.00
35.72
6
C

ATOM
892
OD1
ASN
A
131
10.921
−5.229
−12.244
1.00
38.11
8
O

ATOM
893
ND2
ASN
A
131
10.893
−7.428
−12.702
1.00
36.73
7
N

ATOM
894
C
ASN
A
131
10.486
−5.870
−8.061
1.00
33.52
6
C

ATOM
895
O
ASN
A
131
9.419
−5.433
−7.640
1.00
33.38
8
O

ATOM
896
N
TYR
A
132
11.305
−6.617
−7.321
1.00
33.15
7
N

ATOM
897
CA
TYR
A
132
10.978
−6.933
−5.938
1.00
33.32
6
C

ATOM
898
CB
TYR
A
132
12.040
−7.850
−5.323
1.00
33.61
6
C

ATOM
899
CG
TYR
A
132
11.686
−8.294
−3.923
1.00
35.78
6
C

ATOM
900
CD1
TYR
A
132
11.066
−9.515
−3.700
1.00
36.19
6
C

ATOM
901
CE1
TYR
A
132
10.727
−9.920
−2.422
1.00
38.23
6
C

ATOM
902
CZ
TYR
A
132
11.009
−9.103
−1.352
1.00
38.11
6
C

ATOM
903
OH
TYR
A
132
10.674
−9.504
−0.077
1.00
39.25
8
O

ATOM
904
CE2
TYR
A
132
11.623
−7.886
−1.548
1.00
37.39
6
C

ATOM
905
CD2
TYR
A
132
11.958
−7.487
−2.825
1.00
36.22
6
C

ATOM
906
C
TYR
A
132
10.849
−5.658
−5.094
1.00
33.10
6
C

ATOM
907
O
TYR
A
132
9.887
−5.483
−4.336
1.00
32.61
8
O

ATOM
908
N
MET
A
133
11.821
−4.766
−5.236
1.00
32.43
7
N

ATOM
909
CA
MET
A
133
11.833
−3.533
−4.457
1.00
32.77
6
C

ATOM
910
CB
MET
A
133
13.163
−2.802
−4.644
1.00
32.05
6
C

ATOM
911
CG
MET
A
133
14.345
−3.557
−4.063
1.00
30.87
6
C

ATOM
912
SD
MET
A
133
15.894
−2.591
−4.095
1.00
29.32
16
S

ATOM
913
CE
MET
A
133
16.221
−2.588
−5.846
1.00
28.38
6
C

ATOM
914
C
MET
A
133
10.659
−2.621
−4.802
1.00
33.03
6
C

ATOM
915
O
MET
A
133
10.058
−2.009
−3.921
1.00
32.77
8
O

ATOM
916
N
SER
A
134
10.334
−2.538
−6.086
1.00
33.87
7
N

ATOM
917
CA
SER
A
134
9.218
−1.717
−6.533
1.00
35.17
6
C

ATOM
918
CB
SER
A
134
9.214
−1.615
−8.059
1.00
35.68
6
C

ATOM
919
OG
SER
A
134
7.952
−1.180
−8.534
1.00
37.31
8
O

ATOM
920
C
SER
A
134
7.867
−2.237
−6.021
1.00
35.37
6
C

ATOM
921
O
SER
A
134
6.973
−1.453
−5.694
1.00
35.81
8
O

ATOM
922
N
GLU
A
135
7.719
−3.553
−5.934
1.00
35.53
7
N

ATOM
923
CA
GLU
A
135
6.452
−4.133
−5.486
1.00
35.74
6
C

ATOM
924
CB
GLU
A
135
6.284
−5.545
−6.052
1.00
36.44
6
C

ATOM
925
CG
GLU
A
135
6.145
−5.599
−7.567
1.00
39.80
6
C

ATOM
926
CD
GLU
A
135
4.701
−5.532
−8.032
1.00
44.09
6
C

ATOM
927
OE1
GLU
A
135
3.819
−5.185
−7.213
1.00
45.82
8
O

ATOM
928
OE2
GLU
A
135
4.444
−5.840
−9.221
1.00
46.81
8
O

ATOM
929
C
GLU
A
135
6.251
−4.171
−3.967
1.00
34.96
6
C

ATOM
930
O
GLU
A
135
5.125
−4.011
−3.482
1.00
34.64
8
O

ATOM
931
N
HIS
A
136
7.333
−4.358
−3.216
1.00
33.49
7
N

ATOM
932
CA
HIS
A
136
7.220
−4.574
−1.777
1.00
32.95
6
C

ATOM
933
CB
HIS
A
136
7.933
−5.875
−1.405
1.00
33.31
6
C

ATOM
934
CG
HIS
A
136
7.430
−7.075
−2.142
1.00
35.42
6
C

ATOM
935
ND1
HIS
A
136
6.323
−7.787
−1.735
1.00
36.88
7
N

ATOM
936
CE1
HIS
A
136
6.118
−8.791
−2.570
1.00
38.14
6
C

ATOM
937
NE2
HIS
A
136
7.050
−8.752
−3.506
1.00
38.14
7
N

ATOM
938
CD2
HIS
A
136
7.884
−7.688
−3.261
1.00
37.14
6
C

ATOM
939
C
HIS
A
136
7.757
−3.503
−0.827
1.00
31.87
6
C

ATOM
940
O
HIS
A
136
7.369
−3.482
0.332
1.00
31.61
8
O

ATOM
941
N
LEU
A
137
8.643
−2.630
−1.297
1.00
30.88
7
N

ATOM
942
CA
LEU
A
137
9.362
−1.751
−0.361
1.00
30.11
6
C

ATOM
943
CB
LEU
A
137
10.871
−2.021
−0.444
1.00
29.63
6
C

ATOM
944
CG
LEU
A
137
11.300
−3.497
−0.339
1.00
29.18
6
C

ATOM
945
CD1
LEU
A
137
12.823
−3.621
−0.382
1.00
27.36
6
C

ATOM
946
CD2
LEU
A
137
10.755
−4.166
0.918
1.00
27.32
6
C

ATOM
947
C
LEU
A
137
9.108
−0.254
−0.468
1.00
29.92
6
C

ATOM
948
O
LEU
A
137
8.901
0.286
−1.545
1.00
29.22
8
O

ATOM

949

N

LEU

A

138

9.165

0.401

0.687

1.00

29.80

7

N

ATOM

950

CA

LEU

A

138

8.936

1.834

0.809

1.00

30.37

6

C

ATOM

951

CB

LEU

A

138

8.575

2.142

2.255

1.00

30.84

6

C

ATOM

952

CG

LEU

A

138

8.219

3.594

2.534

1.00

31.89

6

C

ATOM

953

CD1

LEU

A

138

6.971

3.986

1.732

1.00

34.04

6

C

ATOM

954

CD2

LEU

A

138

7.996

3.742

4.020

1.00

35.04

6

C

ATOM

955

C

LEU

A

138

10.181

2.633

0.423

1.00

30.37

6

C

ATOM

956

O

LEU

A

138

11.286

2.272

0.805

1.00

30.44

8

O

ATOM
957
N
LYS
A
139
10.008
3.703
−0.346
1.00
30.35
7
N

ATOM
958
CA
LYS
A
139
11.150
4.525
−0.761
1.00
30.71
6
C

ATOM
959
CB
LYS
A
139
10.823
5.264
−2.061
1.00
30.82
6
C

ATOM
960
CG
LYS
A
139
11.970
6.066
−2.618
1.00
31.83
6
C

ATOM
961
CD
LYS
A
139
11.733
6.531
−4.057
1.00
32.82
6
C

ATOM
962
CE
LYS
A
139
12.878
7.451
−4.495
1.00
32.63
6
C

ATOM
963
NZ
LYS
A
139
12.797
7.904
−5.908
1.00
32.89
7
N

ATOM
964
C
LYS
A
139
11.585
5.513
0.326
1.00
30.83
6
C

ATOM
965
O
LYS
A
139
10.830
6.420
0.692
1.00
30.70
8
O

ATOM
966
N
ALA
A
140
12.797
5.333
0.846
1.00
30.65
7
N

ATOM
967
CA
ALA
A
140
13.313
6.226
1.877
1.00
30.81
6
C

ATOM
968
CB
ALA
A
140
14.529
5.619
2.574
1.00
30.74
6
C

ATOM
969
C
ALA
A
140
13.667
7.586
1.288
1.00
31.03
6
C

ATOM
970
O
ALA
A
140
14.249
7.678
0.208
1.00
29.95
8
O

ATOM
971
N
GLY
A
141
13.302
8.642
2.010
1.00
31.87
7
N

ATOM
972
CA
GLY
A
141
13.600
9.989
1.568
1.00
33.39
6
C

ATOM
973
C
GLY
A
141
12.687
10.419
0.443
1.00
34.35
6
C

ATOM
974
O
GLY
A
141
13.056
11.249
−0.386
1.00
34.20
8
O

ATOM
975
N
ALA
A
142
11.490
9.852
0.421
1.00
35.76
7
N

ATOM
976
CA
ALA
A
142
10.509
10.176
−0.606
1.00
37.44
6
C

ATOM
977
CB
ALA
A
142
9.254
9.343
−0.410
1.00
37.52
6
C

ATOM
978
C
ALA
A
142
10.162
11.666
−0.588
1.00
38.51
6
C

ATOM
979
O
ALA
A
142
9.806
12.243
−1.618
1.00
38.73
8
O

ATOM
980
N
ASN
A
143
10.265
12.282
0.585
1.00
39.78
7
N

ATOM
981
CA
ASN
A
143
9.947
13.702
0.729
1.00
41.20
6
C

ATOM
982
CB
ASN
A
143
9.266
13.962
2.076
1.00
41.20
6
C

ATOM
983
CG
ASN
A
143
10.130
13.547
3.257
1.00
41.87
6
C

ATOM
984
OD1
ASN
A
143
11.169
12.898
3.087
1.00
42.25
8
O

ATOM
985
ND2
ASN
A
143
9.704
13.919
4.463
1.00
40.49
7
N

ATOM
986
C
ASN
A
143
11.163
14.611
0.581
1.00
42.02
6
C

ATOM
987
O
ASN
A
143
11.069
15.825
0.781
1.00
42.10
8
O

ATOM
988
N
ILE
A
144
12.307
14.031
0.230
1.00
42.79
7
N

ATOM
989
CA
ILE
A
144
13.530
14.817
0.099
1.00
43.76
6
C

ATOM
990
CB
ILE
A
144
14.733
14.093
0.739
1.00
43.56
6
C

ATOM
991
CG1
ILE
A
144
14.392
13.605
2.150
1.00
43.51
6
C

ATOM
992
CD1
ILE
A
144
15.560
12.895
2.865
1.00
44.14
6
C

ATOM
993
CG2
ILE
A
144
15.941
15.012
0.751
1.00
43.45
6
C

ATOM
994
C
ILE
A
144
13.858
15.124
−1.351
1.00
44.78
6
C

ATOM
995
O
ILE
A
144
13.718
14.266
−2.231
1.00
44.82
8
O

ATOM
996
N
THR
A
145
14.306
16.352
−1.588
1.00
46.00
7
N

ATOM
997
CA
THR
A
145
14.717
16.781
−2.914
1.00
47.32
6
C

ATOM
998
CB
THR
A
145
14.106
18.153
−3.251
1.00
47.48
6
C

ATOM
999
OG1
THR
A
145
12.681
18.032
−3.379
1.00
47.75
8
O

ATOM
1000
CG2
THR
A
145
14.555
18.603
−4.637
1.00
47.41
6
C

ATOM
1001
C
THR
A
145
16.236
16.870
−2.948
1.00
48.35
6
C

ATOM
1002
O
THR
A
145
16.824
17.752
−2.327
1.00
47.79
8
O

ATOM
1003
N
PRO
A
146
16.871
15.965
−3.687
1.00
49.56
7
N

ATOM
1004
CA
PRO
A
146
18.334
15.916
−3.748
1.00
50.70
6
C

ATOM
1005
CB
PRO
A
146
18.610
14.687
−4.623
1.00
50.57
6
C

ATOM
1006
CG
PRO
A
146
17.377
14.486
−5.410
1.00
50.15
6
C

ATOM
1007
CD
PRO
A
146
16.236
14.963
−4.561
1.00
49.76
6
C

ATOM
1008
C
PRO
A
146
18.870
17.157
−4.433
1.00
52.00
6
C

ATOM
1009
O
PRO
A
146
18.140
17.784
−5.203
1.00
52.03
8
O

ATOM
1010
N
ARG
A
147
20.114
17.525
−4.155
1.00
53.52
7
N

ATOM
1011
CA
ARG
A
147
20.694
18.665
−4.843
1.00
55.35
6
C

ATOM
1012
CB
ARG
A
147
21.863
19.266
−4.053
1.00
55.28
6
C

ATOM
1013
CG
ARG
A
147
23.214
18.590
−4.231
1.00
55.08
6
C

ATOM
1014
CD
ARG
A
147
24.306
19.208
−3.360
1.00
54.57
6
C

ATOM
1015
NE
ARG
A
147
25.650
18.751
−3.702
1.00
54.12
7
N

ATOM
1016
CZ
ARG
A
147
26.310
17.808
−3.044
1.00
54.07
6
C

ATOM
1017
NH1
ARG
A
147
25.750
17.199
−2.008
1.00
53.40
7
N

ATOM
1018
NH2
ARG
A
147
27.533
17.464
−3.424
1.00
54.71
7
N

ATOM
1019
C
ARG
A
147
21.114
18.207
−6.235
1.00
56.73
6
C

ATOM
1020
O
ARG
A
147
20.917
17.047
−6.597
1.00
56.81
8
O

ATOM
1021
N
GLU
A
148
21.667
19.114
−7.028
1.00
58.51
7
N

ATOM
1022
CA
GLU
A
148
22.093
18.751
−8.371
1.00
60.30
6
C

ATOM
1023
CB
GLU
A
148
21.094
19.278
−9.409
1.00
60.58
6
C

ATOM
1024
CG
GLU
A
148
19.741
18.573
−9.355
1.00
61.94
6
C

ATOM
1025
CD
GLU
A
148
18.802
18.971
−10.481
1.00
63.38
6
C

ATOM
1026
OE1
GLU
A
148
19.203
19.779
−11.350
1.00
64.12
8
O

ATOM
1027
OE2
GLU
A
148
17.655
18.470
−10.498
1.00
63.83
8
O

ATOM
1028
C
GLU
A
148
23.503
19.255
−8.653
1.00
61.21
6
C

ATOM
1029
O
GLU
A
148
23.875
20.351
−8.223
1.00
61.66
8
O

ATOM
1030
N
GLY
A
149
24.286
18.446
−9.360
1.00
62.09
7
N

ATOM
1031
CA
GLY
A
149
25.647
18.812
−9.718
1.00
62.88
6
C

ATOM
1032
C
GLY
A
149
26.148
18.045
−10.929
1.00
63.42
6
C

ATOM
1033
O
GLY
A
149
26.292
16.822
−10.881
1.00
63.58
8
O

ATOM
1034
N
ASP
A
150
26.421
18.765
−12.015
1.00
63.96
7
N

ATOM
1035
CA
ASP
A
150
26.880
18.152
−13.261
1.00
64.35
6
C

ATOM
1036
CB
ASP
A
150
28.190
17.388
−13.049
1.00
64.61
6
C

ATOM
1037
CG
ASP
A
150
29.268
18.247
−12.419
1.00
65.54
6
C

ATOM
1038
OD1
ASP
A
150
29.231
19.484
−12.603
1.00
66.88
8
O

ATOM
1039
OD2
ASP
A
150
30.189
17.775
−11.719
1.00
66.50
8
O

ATOM
1040
C
ASP
A
150
25.809
17.226
−13.838
1.00
64.29
6
C

ATOM
1041
O
ASP
A
150
24.663
17.639
−14.015
1.00
64.48
8
O

ATOM
1042
N
GLU
A
151
26.192
15.982
−14.122
1.00
64.02
7
N

ATOM
1043
CA
GLU
A
151
25.292
14.969
−14.684
1.00
63.67
6
C

ATOM
1044
CB
GLU
A
151
24.029
15.603
−15.288
1.00
63.88
6
C

ATOM
1045
CG
GLU
A
151
22.862
15.768
−14.322
1.00
64.91
6
C

ATOM
1046
CD
GLU
A
151
21.638
16.391
−14.979
1.00
66.57
6
C

ATOM
1047
OE1
GLU
A
151
21.428
16.155
−16.190
1.00
66.99
8
O

ATOM
1048
OE2
GLU
A
151
20.882
17.113
−14.288
1.00
67.29
8
O

ATOM
1049
C
GLU
A
151
26.001
14.137
−15.752
1.00
63.01
6
C

ATOM
1050
O
GLU
A
151
25.558
13.040
−16.094
1.00
63.25
8
O

ATOM
1051
N
LEU
A
152
27.110
14.662
−16.267
1.00
61.93
7
N

ATOM
1052
CA
LEU
A
152
27.850
14.017
−17.354
1.00
60.75
6
C

ATOM
1053
CB
LEU
A
152
28.805
15.023
−18.002
1.00
61.03
6
C

ATOM
1054
CG
LEU
A
152
28.167
16.369
−18.359
1.00
61.49
6
C

ATOM
1055
CD1
LEU
A
152
29.199
17.326
−18.939
1.00
62.25
6
C

ATOM
1056
CD2
LEU
A
152
27.006
16.180
−19.326
1.00
62.22
6
C

ATOM
1057
C
LEU
A
152
28.604
12.737
−16.961
1.00
59.54
6
C

ATOM
1058
O
LEU
A
152
28.966
11.939
−17.826
1.00
59.82
8
O

ATOM
1059
N
ALA
A
153
28.842
12.554
−15.664
1.00
57.73
7
N

ATOM
1060
CA
ALA
A
153
29.528
11.370
−15.140
1.00
55.74
6
C

ATOM
1061
CB
ALA
A
153
31.002
11.395
−15.533
1.00
55.94
6
C

ATOM
1062
C
ALA
A
153
29.389
11.389
−13.624
1.00
54.05
6
C

ATOM
1063
O
ALA
A
153
30.163
12.071
−12.951
1.00
54.29
8
O

ATOM
1064
N
ARG
A
154
28.433
10.642
−13.065
1.00
51.55
7
N

ATOM
1065
CA
ARG
A
154
28.196
10.803
−11.632
1.00
48.72
6
C

ATOM
1066
CB
ARG
A
154
27.585
12.188
−11.413
1.00
48.98
6
C

ATOM
1067
CG
ARG
A
154
26.314
12.428
−12.218
1.00
49.65
6
C

ATOM
1068
CD
ARG
A
154
25.262
11.353
−12.036
1.00
50.74
6
C

ATOM
1069
NE
ARG
A
154
23.946
11.781
−12.481
1.00
52.59
7
N

ATOM
1070
CZ
ARG
A
154
22.901
10.977
−12.581
1.00
53.47
6
C

ATOM
1071
NH1
ARG
A
154
23.016
9.695
−12.263
1.00
55.62
7
N

ATOM
1072
NH2
ARG
A
154
21.737
11.452
−12.993
1.00
53.86
7
N

ATOM
1073
C
ARG
A
154
27.355
9.811
−10.815
1.00
46.34
6
C

ATOM
1074
O
ARG
A
154
26.956
10.148
−9.704
1.00
46.32
8
O

ATOM
1075
N
LEU
A
155
27.064
8.619
−11.318
1.00
42.78
7
N

ATOM
1076
CA
LEU
A
155
26.300
7.677
−10.496
1.00
39.40
6
C

ATOM
1077
CB
LEU
A
155
25.388
6.807
−11.360
1.00
39.89
6
C

ATOM
1078
CG
LEU
A
155
24.122
6.271
−10.690
1.00
40.23
6
C

ATOM
1079
CD1
LEU
A
155
23.327
7.409
−10.101
1.00
41.05
6
C

ATOM
1080
CD2
LEU
A
155
23.259
5.508
−11.679
1.00
40.15
6
C

ATOM
1081
C
LEU
A
155
27.262
6.804
−9.683
1.00
36.59
6
C

ATOM
1082
O
LEU
A
155
28.112
6.130
−10.249
1.00
35.75
8
O

ATOM
1083
N
PRO
A
156
27.140
6.825
−8.358
1.00
33.97
7
N

ATOM
1084
CA
PRO
A
156
28.030
6.030
−7.504
1.00
32.21
6
C

ATOM
1085
CB
PRO
A
156
27.895
6.711
−6.137
1.00
32.09
6
C

ATOM
1086
CG
PRO
A
156
26.505
7.245
−6.128
1.00
33.10
6
C

ATOM
1087
CD
PRO
A
156
26.175
7.614
−7.569
1.00
33.75
6
C

ATOM
1088
C
PRO
A
156
27.543
4.590
−7.382
1.00
30.42
6
C

ATOM
1089
O
PRO
A
156
26.371
4.322
−7.627
1.00
29.50
8
O

ATOM
1090
N
TYR
A
157
28.435
3.671
−7.017
1.00
28.48
7
N

ATOM
1091
CA
TYR
A
157
28.012
2.297
−6.793
1.00
27.62
6
C

ATOM
1092
CB
TYR
A
157
28.701
1.325
−7.750
1.00
27.64
6
C

ATOM
1093
CG
TYR
A
157
30.199
1.396
−7.711
1.00
28.30
6
C

ATOM
1094
CD1
TYR
A
157
30.927
0.684
−6.762
1.00
28.82
6
C

ATOM
1095
CE1
TYR
A
157
32.308
0.759
−6.724
1.00
30.62
6
C

ATOM
1096
CZ
TYR
A
157
32.966
1.538
−7.664
1.00
31.02
6
C

ATOM
1097
OH
TYR
A
157
34.336
1.621
−7.646
1.00
33.38
8
O

ATOM
1098
CE2
TYR
A
157
32.262
2.261
−8.600
1.00
29.83
6
C

ATOM
1099
CD2
TYR
A
157
30.895
2.184
−8.626
1.00
30.03
6
C

ATOM
1100
C
TYR
A
157
28.328
1.930
−5.354
1.00
26.56
6
C

ATOM
1101
O
TYR
A
157
29.028
2.657
−4.655
1.00
25.59
8
O

ATOM
1102
N
LEU
A
158
27.818
0.795
−4.917
1.00
26.50
7
N

ATOM
1103
CA
LEU
A
158
28.028
0.361
−3.549
1.00
25.95
6
C

ATOM
1104
CB
LEU
A
158
26.927
−0.627
−3.159
1.00
26.13
6
C

ATOM
1105
CG
LEU
A
158
26.975
−1.109
−1.717
1.00
24.82
6
C

ATOM
1106
CD1
LEU
A
158
26.752
0.064
−0.751
1.00
25.10
6
C

ATOM
1107
CD2
LEU
A
158
25.919
−2.197
−1.532
1.00
24.37
6
C

ATOM
1108
C
LEU
A
158
29.413
−0.286
−3.418
1.00
26.52
6
C

ATOM
1109
O
LEU
A
158
29.679
−1.318
−4.025
1.00
26.29
8
O

ATOM
1110
N
ARG
A
159
30.298
0.339
−2.648
1.00
26.73
7
N

ATOM
1111
CA
ARG
A
159
31.658
−0.176
−2.451
1.00
27.63
6
C

ATOM
1112
CB
ARG
A
159
32.561
0.902
−1.854
1.00
28.30
6
C

ATOM
1113
CG
ARG
A
159
33.108
1.914
−2.848
1.00
31.65
6
C

ATOM
1114
CD
ARG
A
159
34.205
2.815
−2.253
1.00
36.75
6
C

ATOM
1115
NE
ARG
A
159
34.803
3.710
−3.249
1.00
40.48
7
N

ATOM
1116
CZ
ARG
A
159
35.301
4.920
−2.979
1.00
42.21
6
C

ATOM
1117
NH1
ARG
A
159
35.282
5.406
−1.737
1.00
41.23
7
N

ATOM
1118
NH2
ARG
A
159
35.818
5.649
−3.960
1.00
44.20
7
N

ATOM
1119
C
ARG
A
159
31.672
−1.372
−1.513
1.00
27.88
6
C

ATOM
1120
O
ARG
A
159
32.331
−2.383
−1.771
1.00
27.60
8
O

ATOM
1121
N
THR
A
160
30.967
−1.234
−0.396
1.00
27.42
7
N

ATOM
1122
CA
THR
A
160
30.840
−2.330
0.557
1.00
28.18
6
C

ATOM
1123
CB
THR
A
160
32.181
−2.595
1.292
1.00
28.55
6
C

ATOM
1124
OG1
THR
A
160
32.102
−3.825
2.033
1.00
31.68
8
O

ATOM
1125
CG2
THR
A
160
32.441
−1.542
2.352
1.00
30.45
6
C

ATOM
1126
C
THR
A
160
29.721
−1.994
1.535
1.00
27.01
6
C

ATOM
1127
O
THR
A
160
29.190
−0.872
1.542
1.00
26.02
8
O

ATOM
1128
N
TRP
A
161
29.360
−2.974
2.345
1.00
26.52
7
N

ATOM
1129
CA
TRP
A
161
28.300
−2.817
3.325
1.00
25.88
6
C

ATOM
1130
CB
TRP
A
161
26.932
−2.972
2.657
1.00
25.89
6
C

ATOM
1131
CG
TRP
A
161
26.714
−4.349
2.082
1.00
26.34
6
C

ATOM
1132
CD1
TRP
A
161
27.141
−4.805
0.865
1.00
27.76
6
C

ATOM
1133
NE1
TRP
A
161
26.775
−6.119
0.694
1.00
27.89
7
N

ATOM
1134
CE2
TRP
A
161
26.093
−6.542
1.803
1.00
29.07
6
C

ATOM
1135
CD2
TRP
A
161
26.036
−5.453
2.702
1.00
28.34
6
C

ATOM
1136
CE3
TRP
A
161
25.390
−5.635
3.928
1.00
29.01
6
C

ATOM
1137
CZ3
TRP
A
161
24.820
−6.868
4.209
1.00
30.77
6
C

ATOM
1138
CH2
TRP
A
161
24.888
−7.928
3.292
1.00
30.47
6
C

ATOM
1139
CZ2
TRP
A
161
25.520
−7.787
2.088
1.00
30.10
6
C

ATOM
1140
C
TRP
A
161
28.462
−3.936
4.329
1.00
25.71
6
C

ATOM
1141
O
TRP
A
161
29.123
−4.933
4.046
1.00
25.32
8
O

ATOM
1142
N
PHE
A
162
27.851
−3.766
5.492
1.00
25.51
7
N

ATOM
1143
CA
PHE
A
162
27.813
−4.820
6.492
1.00
25.62
6
C

ATOM
1144
CB
PHE
A
162
29.146
−4.947
7.250
1.00
25.67
6
C

ATOM
1145
CG
PHE
A
162
29.507
−3.752
8.100
1.00
25.29
6
C

ATOM
1146
CD1
PHE
A
162
29.037
−3.645
9.408
1.00
25.53
6
C

ATOM
1147
CE1
PHE
A
162
29.370
−2.568
10.197
1.00
24.54
6
C

ATOM
1148
CZ
PHE
A
162
30.196
−1.566
9.693
1.00
25.18
6
C

ATOM
1149
CE2
PHE
A
162
30.673
−1.654
8.398
1.00
26.15
6
C

ATOM
1150
CD2
PHE
A
162
30.336
−2.758
7.610
1.00
26.22
6
C

ATOM
1151
C
PHE
A
162
26.622
−4.541
7.401
1.00
26.20
6
C

ATOM
1152
O
PHE
A
162
26.070
−3.432
7.386
1.00
25.37
8
O

ATOM
1153
N
ARG
A
163
26.202
−5.541
8.166
1.00
26.36
7
N

ATOM
1154
CA
ARG
A
163
25.066
−5.356
9.064
1.00
27.40
6
C

ATOM
1155
CB
ARG
A
163
23.899
−6.261
8.644
1.00
28.06
6
C

ATOM
1156
CG
ARG
A
163
24.291
−7.736
8.578
1.00
32.20
6
C

ATOM
1157
CD
ARG
A
163
23.123
−8.716
8.483
1.00
36.60
6
C

ATOM
1158
NE
ARG
A
163
22.269
−8.429
7.336
1.00
38.18
7
N

ATOM
1159
CZ
ARG
A
163
21.027
−7.987
7.435
1.00
39.16
6
C

ATOM
1160
NH1
ARG
A
163
20.488
−7.791
8.633
1.00
40.03
7
N

ATOM
1161
NH2
ARG
A
163
20.315
−7.755
6.343
1.00
39.07
7
N

ATOM
1162
C
ARG
A
163
25.463
−5.684
10.492
1.00
26.54
6
C

ATOM
1163
O
ARG
A
163
26.263
−6.594
10.717
1.00
26.51
8
O

ATOM
1164
N
THR
A
164
24.947
−4.906
11.442
1.00
26.05
7
N

ATOM
1165
CA
THR
A
164
25.115
−5.214
12.867
1.00
26.45
6
C

ATOM
1166
CB
THR
A
164
25.641
−4.012
13.660
1.00
26.07
6
C

ATOM
1167
OG1
THR
A
164
24.684
−2.945
13.600
1.00
25.53
8
O

ATOM
1168
CG2
THR
A
164
26.901
−3.434
13.016
1.00
26.06
6
C

ATOM
1169
C
THR
A
164
23.731
−5.605
13.385
1.00
26.76
6
C

ATOM
1170
O
THR
A
164
22.777
−5.676
12.613
1.00
26.66
8
O

ATOM
1171
N
ARG
A
165
23.602
−5.828
14.684
1.00
27.66
7
N

ATOM
1172
CA
ARG
A
165
22.295
−6.172
15.225
1.00
28.33
6
C

ATOM
1173
CB
ARG
A
165
22.429
−6.707
16.661
1.00
28.64
6
C

ATOM
1174
CG
ARG
A
165
22.973
−5.690
17.667
1.00
29.97
6
C

ATOM
1175
CD
ARG
A
165
23.038
−6.213
19.116
1.00
32.01
6
C

ATOM
1176
NE
ARG
A
165
23.350
−5.147
20.065
1.00
33.50
7
N

ATOM
1177
CZ
ARG
A
165
23.006
−5.159
21.346
1.00
34.29
6
C

ATOM
1178
NH1
ARG
A
165
22.335
−6.191
21.846
1.00
35.50
7
N

ATOM
1179
NH2
ARG
A
165
23.337
−4.140
22.132
1.00
35.43
7
N

ATOM
1180
C
ARG
A
165
21.381
−4.942
15.189
1.00
28.74
6
C

ATOM
1181
O
ARG
A
165
20.151
−5.064
15.256
1.00
29.22
8
O

ATOM
1182
N
SER
A
166
21.982
−3.759
15.061
1.00
27.82
7
N

ATOM
1183
CA
SER
A
166
21.228
−2.508
15.132
1.00
27.68
6
C

ATOM
1184
CB
SER
A
166
21.906
−1.526
16.103
1.00
27.82
6
C

ATOM
1185
OG
SER
A
166
22.192
−2.112
17.363
1.00
30.66
8
O

ATOM
1186
C
SER
A
166
21.036
−1.773
13.803
1.00
26.93
6
C

ATOM
1187
O
SER
A
166
20.139
−0.936
13.688
1.00
26.34
8
O

ATOM
1188
N
ALA
A
167
21.871
−2.066
12.811
1.00
25.82
7
N

ATOM
1189
CA
ALA
A
167
21.812
−1.282
11.584
1.00
25.43
6
C

ATOM
1190
CB
ALA
A
167
22.478
0.087
11.839
1.00
24.92
6
C

ATOM
1191
C
ALA
A
167
22.485
−1.926
10.390
1.00
24.77
6
C

ATOM
1192
O
ALA
A
167
23.257
−2.884
10.538
1.00
24.32
8
O

ATOM
1193
N
ILE
A
168
22.197
−1.371
9.210
1.00
24.07
7
N

ATOM
1194
CA
ILE
A
168
22.915
−1.733
7.997
1.00
24.20
6
C

ATOM
1195
CB
ILE
A
168
21.979
−2.205
6.840
1.00
24.66
6
C

ATOM
1196
CG1
ILE
A
168
22.816
−2.618
5.625
1.00
24.70
6
C

ATOM
1197
CD1
ILE
A
168
22.053
−3.476
4.600
1.00
26.32
6
C

ATOM
1198
CG2
ILE
A
168
20.959
−1.138
6.458
1.00
24.77
6
C

ATOM
1199
C
ILE
A
168
23.746
−0.507
7.618
1.00
23.55
6
C

ATOM
1200
O
ILE
A
168
23.278
0.636
7.720
1.00
23.26
8
O

ATOM
1201
N
ILE
A
169
25.000
−0.747
7.251
1.00
23.24
7
N

ATOM
1202
CA
ILE
A
169
25.950
0.326
6.933
1.00
22.80
6
C

ATOM
1203
CB
ILE
A
169
27.248
0.165
7.804
1.00
23.33
6
C

ATOM
1204
CG1
ILE
A
169
26.972
0.486
9.276
1.00
22.62
6
C

ATOM
1205
CD1
ILE
A
169
26.159
−0.586
10.034
1.00
23.51
6
C

ATOM
1206
CG2
ILE
A
169
28.389
1.042
7.284
1.00
22.78
6
C

ATOM
1207
C
ILE
A
169
26.269
0.178
5.461
1.00
22.89
6
C

ATOM
1208
O
ILE
A
169
26.661
−0.898
5.037
1.00
22.95
8
O

ATOM
1209
N
LEU
A
170
26.081
1.253
4.694
1.00
22.59
7
N

ATOM
1210
CA
LEU
A
170
26.267
1.254
3.253
1.00
23.18
6
C

ATOM
1211
CB
LEU
A
170
24.942
1.584
2.565
1.00
23.34
6
C

ATOM
1212
CG
LEU
A
170
23.794
0.623
2.903
1.00
23.78
6
C

ATOM
1213
CD1
LEU
A
170
22.445
1.335
2.810
1.00
24.60
6
C

ATOM
1214
CD2
LEU
A
170
23.847
−0.553
1.958
1.00
24.60
6
C

ATOM
1215
C
LEU
A
170
27.315
2.296
2.879
1.00
23.39
6
C

ATOM
1216
O
LEU
A
170
27.197
3.464
3.241
1.00
23.48
8
O

ATOM
1217
N
HIS
A
171
28.331
1.870
2.137
1.00
23.66
7
N

ATOM
1218
CA
HIS
A
171
29.408
2.766
1.751
1.00
24.20
6
C

ATOM
1219
CB
HIS
A
171
30.751
2.196
2.220
1.00
24.28
6
C

ATOM
1220
CG
HIS
A
171
31.930
3.061
1.885
1.00
26.53
6
C

ATOM
1221
ND1
HIS
A
171
31.832
4.424
1.718
1.00
27.97
7
N

ATOM
1222
CE1
HIS
A
171
33.029
4.922
1.446
1.00
29.66
6
C

ATOM
1223
NE2
HIS
A
171
33.901
3.931
1.443
1.00
31.07
7
N

ATOM
1224
CD2
HIS
A
171
33.239
2.755
1.712
1.00
27.93
6
C

ATOM
1225
C
HIS
A
171
29.408
2.911
0.238
1.00
24.50
6
C

ATOM
1226
O
HIS
A
171
29.715
1.950
−0.481
1.00
24.62
8
O

ATOM
1227
N
LEU
A
172
29.065
4.107
−0.229
1.00
24.25
7
N

ATOM
1228
CA
LEU
A
172
28.988
4.403
−1.660
1.00
25.47
6
C

ATOM
1229
CB
LEU
A
172
27.897
5.462
−1.915
1.00
25.00
6
C

ATOM
1230
CG
LEU
A
172
26.453
5.059
−1.582
1.00
27.92
6
C

ATOM
1231
CD1
LEU
A
172
25.466
6.107
−2.079
1.00
28.86
6
C

ATOM
1232
CD2
LEU
A
172
26.112
3.706
−2.191
1.00
28.96
6
C

ATOM
1233
C
LEU
A
172
30.340
4.863
−2.227
1.00
25.07
6
C

ATOM
1234
O
LEU
A
172
31.170
5.427
−1.499
1.00
24.85
8
O

ATOM
1235
N
SER
A
173
30.537
4.664
−3.528
1.00
25.35
7
N

ATOM
1236
CA
SER
A
173
31.809
5.003
−4.182
1.00
25.98
6
C

ATOM
1237
CB
SER
A
173
31.879
4.396
−5.590
1.00
26.00
6
C

ATOM
1238
OG
SER
A
173
30.838
4.902
−6.402
1.00
24.57
8
O

ATOM
1239
C
SER
A
173
32.102
6.507
−4.241
1.00
26.40
6
C

ATOM
1240
O
SER
A
173
33.217
6.912
−4.578
1.00
27.27
8
O

ATOM
1241
N
ASN
A
174
31.106
7.336
−3.938
1.00
26.37
7
N

ATOM
1242
CA
ASN
A
174
31.326
8.786
−3.902
1.00
26.18
6
C

ATOM
1243
CB
ASN
A
174
30.071
9.556
−4.355
1.00
26.32
6
C

ATOM
1244
CG
ASN
A
174
28.887
9.340
−3.444
1.00
26.00
6
C

ATOM
1245
OD1
ASN
A
174
28.971
8.617
−2.442
1.00
24.32
8
O

ATOM
1246
ND2
ASN
A
174
27.767
9.982
−3.778
1.00
26.41
7
N

ATOM
1247
C
ASN
A
174
31.806
9.248
−2.515
1.00
26.35
6
C

ATOM
1248
O
ASN
A
174
31.957
10.456
−2.262
1.00
26.26
8
O

ATOM
1249
N
GLY
A
175
32.054
8.275
−1.636
1.00
25.33
7
N

ATOM
1250
CA
GLY
A
175
32.550
8.526
−0.291
1.00
25.96
6
C

ATOM
1251
C
GLY
A
175
31.475
8.588
0.787
1.00
25.46
6
C

ATOM
1252
O
GLY
A
175
31.776
8.570
1.980
1.00
25.69
8
O

ATOM
1253
N
SER
A
176
30.216
8.675
0.374
1.00
25.33
7
N

ATOM
1254
CA
SER
A
176
29.115
8.757
1.335
1.00
25.17
6
C

ATOM
1255
CB
SER
A
176
27.797
9.051
0.608
1.00
25.53
6
C

ATOM
1256
OG
SER
A
176
27.809
10.376
0.083
1.00
28.20
8
O

ATOM
1257
C
SER
A
176
28.961
7.466
2.134
1.00
24.26
6
C

ATOM
1258
O
SER
A
176
29.149
6.373
1.600
1.00
23.81
8
O

ATOM
1259
N
VAL
A
177
28.617
7.608
3.411
1.00
23.56
7
N

ATOM
1260
CA
VAL
A
177
28.325
6.464
4.267
1.00
23.31
6
C

ATOM
1261
CB
VAL
A
177
29.328
6.306
5.428
1.00
23.77
6
C

ATOM
1262
CG1
VAL
A
177
28.852
5.217
6.392
1.00
24.27
6
C

ATOM
1263
CG2
VAL
A
177
30.722
5.959
4.891
1.00
23.70
6
C

ATOM
1264
C
VAL
A
177
26.907
6.634
4.813
1.00
23.11
6
C

ATOM
1265
O
VAL
A
177
26.578
7.683
5.388
1.00
22.34
8
O

ATOM
1266
N
GLN
A
178
26.067
5.619
4.603
1.00
22.44
7
N

ATOM
1267
CA
GLN
A
178
24.684
5.670
5.074
1.00
22.30
6
C

ATOM
1268
CB
GLN
A
178
23.712
5.489
3.901
1.00
22.62
6
C

ATOM
1269
CG
GLN
A
178
22.230
5.506
4.290
1.00
23.11
6
C

ATOM
1270
CD
GLN
A
178
21.321
5.526
3.078
1.00
24.18
6
C

ATOM
1271
OE1
GLN
A
178
21.713
5.999
2.004
1.00
24.06
8
O

ATOM
1272
NE2
GLN
A
178
20.114
4.996
3.235
1.00
23.29
7
N

ATOM
1273
C
GLN
A
178
24.459
4.594
6.122
1.00
21.55
6
C

ATOM
1274
O
GLN
A
178
24.934
3.473
5.977
1.00
21.29
8
O

ATOM
1275
N
ILE
A
179
23.776
4.955
7.202
1.00
21.05
7
N

ATOM
1276
CA
ILE
A
179
23.473
3.995
8.264
1.00
21.59
6
C

ATOM
1277
CB
ILE
A
179
24.273
4.340
9.570
1.00
21.66
6
C

ATOM
1278
CG1
ILE
A
179
25.773
4.433
9.280
1.00
21.63
6
C

ATOM
1279
CD1
ILE
A
179
26.620
4.908
10.469
1.00
22.78
6
C

ATOM
1280
CG2
ILE
A
179
24.021
3.268
10.644
1.00
22.47
6
C

ATOM
1281
C
ILE
A
179
21.976
4.045
8.532
1.00
21.86
6
C

ATOM
1282
O
ILE
A
179
21.451
5.108
8.902
1.00
21.55
8
O

ATOM
1283
N
ASN
A
180
21.304
2.910
8.335
1.00
22.26
7
N

ATOM
1284
CA
ASN
A
180
19.870
2.769
8.567
1.00
23.11
6
C

ATOM
1285
CB
ASN
A
180
19.183
2.006
7.422
1.00
22.91
6
C

ATOM
1286
CG
ASN
A
180
19.032
2.825
6.152
1.00
23.65
6
C

ATOM
1287
OD1
ASN
A
180
19.694
3.852
5.955
1.00
23.20
8
O

ATOM
1288
ND2
ASN
A
180
18.151
2.362
5.267
1.00
24.48
7
N

ATOM
1289
C
ASN
A
180
19.679
1.948
9.823
1.00
23.44
6
C

ATOM
1290
O
ASN
A
180
20.101
0.791
9.868
1.00
23.66
8
O

ATOM
1291
N
PHE
A
181
19.049
2.537
10.837
1.00
24.40
7
N

ATOM
1292
CA
PHE
A
181
18.801
1.842
12.097
1.00
25.35
6
C

ATOM
1293
CB
PHE
A
181
18.794
2.827
13.277
1.00
24.90
6
C

ATOM
1294
CG
PHE
A
181
20.128
3.511
13.500
1.00
25.75
6
C

ATOM
1295
CD1
PHE
A
181
20.368
4.774
12.996
1.00
25.28
6
C

ATOM
1296
CE1
PHE
A
181
21.604
5.392
13.186
1.00
26.59
6
C

ATOM
1297
CZ
PHE
A
181
22.597
4.744
13.886
1.00
26.44
6
C

ATOM
1298
CE2
PHE
A
181
22.372
3.494
14.388
1.00
27.49
6
C

ATOM
1299
CD2
PHE
A
181
21.139
2.873
14.190
1.00
25.89
6
C

ATOM
1300
C
PHE
A
181
17.498
1.049
12.007
1.00
26.42
6
C

ATOM
1301
O
PHE
A
181
16.460
1.585
11.601
1.00
26.61
8
O

ATOM
1302
N
PHE
A
182
17.564
−0.229
12.369
1.00
27.90
7
N

ATOM
1303
CA
PHE
A
182
16.421
−1.137
12.216
1.00
29.59
6
C

ATOM
1304
CB
PHE
A
182
16.851
−2.602
12.398
1.00
29.28
6
C

ATOM
1305
CG
PHE
A
182
17.902
−3.069
11.423
1.00
28.88
6
C

ATOM
1306
CD1
PHE
A
182
19.009
−3.762
11.875
1.00
28.98
6
C

ATOM
1307
CE1
PHE
A
182
19.982
−4.199
10.992
1.00
29.40
6
C

ATOM
1308
CZ
PHE
A
182
19.848
−3.955
9.638
1.00
29.35
6
C

ATOM
1309
CE2
PHE
A
182
18.743
−3.263
9.172
1.00
29.15
6
C

ATOM
1310
CD2
PHE
A
182
17.776
−2.831
10.067
1.00
29.92
6
C

ATOM
1311
C
PHE
A
182
15.264
−0.852
13.173
1.00
30.82
6
C

ATOM
1312
O
PHE
A
182
14.102
−0.862
12.772
1.00
32.18
8
O

ATOM
1313
N
GLN
A
183
15.591
−0.583
14.428
1.00
32.18
7
N

ATOM
1314
CA
GLN
A
183
14.591
−0.443
15.491
1.00
33.29
6
C

ATOM
1315
CB
GLN
A
183
15.304
−0.334
16.839
1.00
33.98
6
C

ATOM
1316
CG
GLN
A
183
14.415
−0.518
18.052
1.00
37.89
6
C

ATOM
1317
CD
GLN
A
183
15.216
−0.852
19.295
1.00
41.93
6
C

ATOM
1318
OE1
GLN
A
183
16.179
−0.149
19.629
1.00
44.34
8
O

ATOM
1319
NE2
GLN
A
183
14.829
−1.921
19.982
1.00
43.28
7
N

ATOM
1320
C
GLN
A
183
13.601
0.713
15.328
1.00
32.74
6
C

ATOM
1321
O
GLN
A
183
12.392
0.534
15.504
1.00
33.69
8
O

ATOM
1322
N
ASP
A
184
14.093
1.891
14.973
1.00
31.46
7
N

ATOM
1323
CA
ASP
A
184
13.210
3.048
14.889
1.00
30.53
6
C

ATOM
1324
CB
ASP
A
184
13.603
4.079
15.944
1.00
30.82
6
C

ATOM
1325
CG
ASP
A
184
15.022
4.596
15.763
1.00
31.38
6
C

ATOM
1326
OD1
ASP
A
184
15.690
4.271
14.741
1.00
30.84
8
O

ATOM
1327
OD2
ASP
A
184
15.547
5.350
16.601
1.00
31.85
8
O

ATOM
1328
C
ASP
A
184
13.142
3.688
13.510
1.00
29.56
6
C

ATOM
1329
O
ASP
A
184
12.552
4.752
13.340
1.00
28.90
8
O

ATOM

1330

N

HIS

A

185

13.761

3.036

12.527

1.00

28.47

7

N

ATOM

1331

CA

HIS

A

185

13.753

3.525

11.155

1.00

28.01

6

C

ATOM

1332

CB

HIS

A

185

12.316

3.630

10.643

1.00

28.20

6

C

ATOM

1333

CG

HIS

A

185

11.530

2.365

10.792

1.00

30.16

6

C

ATOM

1334

ND1

HIS

A

185

11.815

1.223

10.075

1.00

30.90

7

N

ATOM

1335

CE1

HIS

A

185

10.958

0.271

10.405

1.00

33.28

6

C

ATOM

1336

NE2

HIS

A

185

10.126

0.756

11.310

1.00

32.23

7

N

ATOM

1337

CD2

HIS

A

185

10.464

2.063

11.574

1.00

32.17

6

C

ATOM

1338

C

HIS

A

185

14.480

4.858

10.936

1.00

26.92

6

C

ATOM

1339

O

HIS

A

185

14.302

5.486

9.886

1.00

26.86

8

O

ATOM
1340
N
THR
A
186
15.279
5.303
11.905
1.00
26.53
7
N

ATOM
1341
CA
THR
A
186
16.040
6.552
11.714
1.00
25.66
6
C

ATOM
1342
CB
THR
A
186
16.544
7.190
13.041
1.00
25.96
6
C

ATOM
1343
OG1
THR
A
186
17.269
6.228
13.806
1.00
25.64
8
O

ATOM
1344
CG2
THR
A
186
15.389
7.622
13.962
1.00
26.85
6
C

ATOM
1345
C
THR
A
186
17.237
6.255
10.810
1.00
25.19
6
C

ATOM
1346
O
THR
A
186
17.713
5.121
10.769
1.00
24.35
8
O

ATOM

1347

N

LYS

A

187

17.753

7.278

10.133

1.00

24.27

7

N

ATOM

1348

CA

LYS

A

187

18.831

7.065

9.158

1.00

24.03

6

C

ATOM

1349

CB

LYS

A

187

18.240

6.886

7.748

1.00

24.02

6

C

ATOM

1350

CG

LYS

A

187

17.219

5.759

7.624

1.00

23.58

6

C

ATOM

1351

CD

LYS

A

187

16.588

5.693

6.221

1.00

25.16

6

C

ATOM

1352

CE

LYS

A

187

15.579

4.537

6.132

1.00

24.31

6

C

ATOM

1353

NZ

LYS

A

187

14.412

4.734

7.055

1.00

24.25

7

N

ATOM

1354

C

LYS

A

187

19.776

8.251

9.120

1.00

24.10

6

C

ATOM

1355

O

LYS

A

187

19.354

9.391

9.325

1.00

24.85

8

O

ATOM
1356
N
LEU
A
188
21.047
7.975
8.851
1.00
23.49
7
N

ATOM
1357
CA
LEU
A
188
22.055
9.014
8.674
1.00
23.57
6
C

ATOM
1358
CB
LEU
A
188
23.187
8.823
9.673
1.00
24.22
6
C

ATOM
1359
CG
LEU
A
188
22.913
8.997
11.160
1.00
25.94
6
C

ATOM
1360
CD1
LEU
A
188
24.207
8.835
11.924
1.00
26.57
6
C

ATOM
1361
CD2
LEU
A
188
22.320
10.366
11.419
1.00
28.31
6
C

ATOM
1362
C
LEU
A
188
22.677
8.861
7.297
1.00
23.47
6
C

ATOM
1363
O
LEU
A
188
22.961
7.735
6.877
1.00
22.53
8
O

ATOM
1364
N
ILE
A
189
22.901
9.978
6.605
1.00
23.57
7
N

ATOM
1365
CA
ILE
A
189
23.648
9.965
5.348
1.00
24.10
6
C

ATOM
1366
CB
ILE
A
189
22.783
10.443
4.181
1.00
24.59
6
C

ATOM
1367
CG1
ILE
A
189
21.523
9.575
4.044
1.00
24.75
6
C

ATOM
1368
CD1
ILE
A
189
20.426
10.284
3.221
1.00
25.98
6
C

ATOM
1369
CG2
ILE
A
189
23.592
10.403
2.890
1.00
25.55
6
C

ATOM
1370
C
ILE
A
189
24.822
10.929
5.555
1.00
24.28
6
C

ATOM
1371
O
ILE
A
189
24.613
12.129
5.705
1.00
23.81
8
O

ATOM
1372
N
LEU
A
190
26.034
10.383
5.611
1.00
24.25
7
N

ATOM
1373
CA
LEU
A
190
27.240
11.151
5.873
1.00
25.06
6
C

ATOM
1374
CB
LEU
A
190
28.137
10.388
6.850
1.00
25.79
6
C

ATOM
1375
CG
LEU
A
190
27.580
10.239
8.263
1.00
27.20
6
C

ATOM
1376
CD1
LEU
A
190
28.056
8.935
8.903
1.00
28.40
6
C

ATOM
1377
CD2
LEU
A
190
28.010
11.468
9.085
1.00
30.40
6
C

ATOM
1378
C
LEU
A
190
28.020
11.363
4.595
1.00
25.17
6
C

ATOM
1379
O
LEU
A
190
28.271
10.409
3.869
1.00
24.80
8
O

ATOM
1380
N
CYS
A
191
28.422
12.608
4.345
1.00
25.78
7
N

ATOM
1381
CA
CYS
A
191
29.239
12.926
3.178
1.00
26.60
6
C

ATOM
1382
CB
CYS
A
191
28.477
13.798
2.184
1.00
26.74
6
C

ATOM
1383
SG
CYS
A
191
29.522
14.300
0.771
1.00
29.77
16
S

ATOM
1384
C
CYS
A
191
30.512
13.648
3.604
1.00
26.28
6
C

ATOM
1385
O
CYS
A
191
30.464
14.735
4.154
1.00
25.81
8
O

ATOM
1386
N
PRO
A
192
31.655
13.046
3.319
1.00
26.92
7
N

ATOM
1387
CA
PRO
A
192
32.937
13.622
3.713
1.00
27.35
6
C

ATOM
1388
CB
PRO
A
192
33.899
12.459
3.531
1.00
27.11
6
C

ATOM
1389
CG
PRO
A
192
33.290
11.633
2.441
1.00
27.39
6
C

ATOM
1390
CD
PRO
A
192
31.805
11.796
2.556
1.00
26.86
6
C

ATOM
1391
C
PRO
A
192
33.352
14.774
2.789
1.00
27.90
6
C

ATOM
1392
O
PRO
A
192
34.255
15.530
3.138
1.00
28.13
8
O

ATOM
1393
N
LEU
A
193
32.717
14.895
1.630
1.00
28.55
7
N

ATOM
1394
CA
LEU
A
193
33.070
15.966
0.689
1.00
29.46
6
C

ATOM
1395
CB
LEU
A
193
32.635
15.614
−0.738
1.00
30.04
6
C

ATOM
1396
CG
LEU
A
193
33.225
14.307
−1.290
1.00
30.91
6
C

ATOM
1397
CD1
LEU
A
193
32.849
14.105
−2.754
1.00
34.39
6
C

ATOM
1398
CD2
LEU
A
193
34.738
14.306
−1.131
1.00
33.74
6
C

ATOM
1399
C
LEU
A
193
32.420
17.257
1.163
1.00
29.81
6
C

ATOM
1400
O
LEU
A
193
33.048
18.321
1.164
1.00
30.32
8
O

ATOM
1401
N
MET
A
194
31.164
17.158
1.590
1.00
29.55
7
N

ATOM
1402
CA
MET
A
194
30.470
18.308
2.158
1.00
30.51
6
C

ATOM
1403
CB
MET
A
194
28.949
18.191
1.951
1.00
30.64
6
C

ATOM
1404
CG
MET
A
194
28.497
18.114
0.492
1.00
33.89
6
C

ATOM
1405
SD
MET
A
194
28.350
19.743
−0.282
1.00
40.49
16
S

ATOM
1406
CE
MET
A
194
29.874
20.489
0.168
1.00
38.93
6
C

ATOM
1407
C
MET
A
194
30.772
18.452
3.657
1.00
29.73
6
C

ATOM
1408
O
MET
A
194
30.501
19.497
4.240
1.00
30.14
8
O

ATOM
1409
N
ALA
A
195
31.326
17.404
4.268
1.00
29.26
7
N

ATOM
1410
CA
ALA
A
195
31.543
17.367
5.722
1.00
28.29
6
C

ATOM
1411
CB
ALA
A
195
32.578
18.420
6.168
1.00
28.53
6
C

ATOM
1412
C
ALA
A
195
30.201
17.599
6.395
1.00
27.41
6
C

ATOM
1413
O
ALA
A
195
30.058
18.437
7.284
1.00
27.21
8
O

ATOM
1414
N
ALA
A
196
29.209
16.834
5.962
1.00
26.60
7
N

ATOM
1415
CA
ALA
A
196
27.846
17.048
6.403
1.00
25.81
6
C

ATOM
1416
CB
ALA
A
196
27.066
17.729
5.308
1.00
26.13
6
C

ATOM
1417
C
ALA
A
196
27.163
15.744
6.769
1.00
25.38
6
C

ATOM
1418
O
ALA
A
196
27.605
14.665
6.377
1.00
25.32
8
O

ATOM
1419
N
VAL
A
197
26.093
15.853
7.543
1.00
25.22
7
N

ATOM
1420
CA
VAL
A
197
25.289
14.691
7.888
1.00
24.87
6
C

ATOM
1421
CB
VAL
A
197
25.497
14.215
9.353
1.00
25.21
6
C

ATOM
1422
CG1
VAL
A
197
25.102
15.298
10.359
1.00
24.80
6
C

ATOM
1423
CG2
VAL
A
197
24.701
12.929
9.618
1.00
26.76
6
C

ATOM
1424
C
VAL
A
197
23.828
15.008
7.663
1.00
25.04
6
C

ATOM
1425
O
VAL
A
197
23.346
16.069
8.053
1.00
24.74
8
O

ATOM
1426
N
THR
A
198
23.120
14.077
7.027
1.00
24.63
7
N

ATOM
1427
CA
THR
A
198
21.687
14.218
6.852
1.00
25.28
6
C

ATOM
1428
CB
THR
A
198
21.309
13.955
5.385
1.00
25.27
6
C

ATOM
1429
OG1
THR
A
198
21.801
15.031
4.591
1.00
24.40
8
O

ATOM
1430
CG2
THR
A
198
19.779
14.020
5.178
1.00
25.08
6
C

ATOM
1431
C
THR
A
198
21.026
13.216
7.786
1.00
25.74
6
C

ATOM
1432
O
THR
A
198
21.331
12.032
7.743
1.00
25.91
8
O

ATOM
1433
N
TYR
A
199
20.161
13.709
8.666
1.00
26.25
7
N

ATOM
1434
CA
TYR
A
199
19.473
12.877
9.635
1.00
26.86
6
C

ATOM
1435
CB
TYR
A
199
19.636
13.490
11.034
1.00
26.82
6
C

ATOM
1436
CG
TYR
A
199
18.974
12.708
12.139
1.00
28.67
6
C

ATOM
1437
CD1
TYR
A
199
19.057
11.328
12.182
1.00
29.47
6
C

ATOM
1438
CE1
TYR
A
199
18.455
10.606
13.196
1.00
32.81
6
C

ATOM
1439
CZ
TYR
A
199
17.764
11.272
14.188
1.00
34.25
6
C

ATOM
1440
OH
TYR
A
199
17.163
10.561
15.205
1.00
38.37
8
O

ATOM
1441
CE2
TYR
A
199
17.671
12.643
14.174
1.00
33.45
6
C

ATOM
1442
CD2
TYR
A
199
18.277
13.357
13.151
1.00
30.73
6
C

ATOM
1443
C
TYR
A
199
18.000
12.780
9.268
1.00
26.79
6
C

ATOM
1444
O
TYR
A
199
17.345
13.803
9.042
1.00
26.93
8
O

ATOM
1445
N
ILE
A
200
17.502
11.550
9.168
1.00
26.74
7
N

ATOM
1446
CA
ILE
A
200
16.097
11.284
8.868
1.00
27.16
6
C

ATOM
1447
CB
ILE
A
200
15.948
10.266
7.714
1.00
26.97
6
C

ATOM
1448
CG1
ILE
A
200
16.456
10.861
6.398
1.00
26.38
6
C

ATOM
1449
CD1
ILE
A
200
16.462
9.862
5.225
1.00
26.08
6
C

ATOM
1450
CG2
ILE
A
200
14.482
9.858
7.550
1.00
26.89
6
C

ATOM
1451
C
ILE
A
200
15.545
10.696
10.157
1.00
27.75
6
C

ATOM
1452
O
ILE
A
200
15.995
9.639
10.595
1.00
27.06
8
O

ATOM
1453
N
ASP
A
201
14.605
11.393
10.790
1.00
29.03
7
N

ATOM
1454
CA
ASP
A
201
14.122
10.955
12.099
1.00
30.64
6
C

ATOM
1455
CB
ASP
A
201
13.854
12.161
13.017
1.00
31.21
6
C

ATOM
1456
CG
ASP
A
201
12.624
12.960
12.611
1.00
32.40
6
C

ATOM
1457
OD1
ASP
A
201
11.793
12.475
11.812
1.00
33.54
8
O

ATOM
1458
OD2
ASP
A
201
12.397
14.096
13.071
1.00
35.66
8
O

ATOM
1459
C
ASP
A
201
12.921
10.002
12.011
1.00
31.64
6
C

ATOM
1460
O
ASP
A
201
12.446
9.696
10.916
1.00
31.11
8
O

ATOM
1461
N
GLU
A
202
12.443
9.525
13.160
1.00
33.23
7
N

ATOM
1462
CA
GLU
A
202
11.361
8.542
13.173
1.00
34.74
6
C

ATOM
1463
CB
GLU
A
202
11.138
7.940
14.571
1.00
35.27
6
C

ATOM
1464
CG
GLU
A
202
11.226
8.923
15.722
1.00
38.23
6
C

ATOM
1465
CD
GLU
A
202
12.657
9.148
16.179
1.00
41.73
6
C

ATOM
1466
OE1
GLU
A
202
13.209
8.265
16.888
1.00
43.97
8
O

ATOM
1467
OE2
GLU
A
202
13.230
10.204
15.827
1.00
41.84
8
O

ATOM
1468
C
GLU
A
202
10.050
9.052
12.583
1.00
35.39
6
C

ATOM
1469
O
GLU
A
202
9.156
8.265
12.282
1.00
35.88
8
O

ATOM
1470
N
LYS
A
203
9.938
10.360
12.398
1.00
35.90
7
N

ATOM
1471
CA
LYS
A
203
8.740
10.919
11.790
1.00
36.58
6
C

ATOM
1472
CB
LYS
A
203
8.337
12.212
12.508
1.00
37.33
6
C

ATOM
1473
CG
LYS
A
203
8.233
12.040
14.025
1.00
38.95
6
C

ATOM
1474
CD
LYS
A
203
7.774
13.318
14.718
1.00
42.84
6
C

ATOM
1475
CE
LYS
A
203
7.529
13.084
16.207
1.00
44.43
6
C

ATOM
1476
NZ
LYS
A
203
6.740
14.186
16.831
1.00
46.60
7
N

ATOM
1477
C
LYS
A
203
8.957
11.146
10.295
1.00
36.53
6
C

ATOM
1478
O
LYS
A
203
8.072
11.626
9.594
1.00
36.51
8
O

ATOM
1479
N
ARG
A
204
10.139
10.765
9.814
1.00
36.40
7
N

ATOM
1480
CA
ARG
A
204
10.523
10.908
8.405
1.00
36.41
6
C

ATOM
1481
CB
ARG
A
204
9.467
10.330
7.468
1.00
36.80
6
C

ATOM
1482
CG
ARG
A
204
9.260
8.842
7.667
1.00
38.81
6
C

ATOM
1483
CD
ARG
A
204
8.316
8.209
6.664
1.00
42.51
6
C

ATOM
1484
NE
ARG
A
204
7.496
7.175
7.291
1.00
47.42
7
N

ATOM
1485
CZ
ARG
A
204
7.602
5.882
7.028
1.00
48.72
6
C

ATOM
1486
NH1
ARG
A
204
8.500
5.465
6.151
1.00
50.90
7
N

ATOM
1487
NH2
ARG
A
204
6.819
5.003
7.640
1.00
49.75
7
N

ATOM
1488
C
ARG
A
204
10.872
12.344
8.053
1.00
36.27
6
C

ATOM
1489
O
ARG
A
204
11.058
12.702
6.886
1.00
35.30
8
O

ATOM
1490
N
ASP
A
205
10.958
13.163
9.091
1.00
36.42
7
N

ATOM
1491
CA
ASP
A
205
11.386
14.534
8.939
1.00
37.02
6
C

ATOM
1492
CB
ASP
A
205
11.021
15.342
10.176
1.00
37.71
6
C

ATOM
1493
CG
ASP
A
205
10.499
16.710
9.831
1.00
40.93
6
C

ATOM
1494
OD1
ASP
A
205
11.285
17.681
9.909
1.00
42.87
8
O

ATOM
1495
OD2
ASP
A
205
9.316
16.901
9.460
1.00
44.32
8
O

ATOM
1496
C
ASP
A
205
12.895
14.482
8.761
1.00
36.46
6
C

ATOM
1497
O
ASP
A
205
13.564
13.562
9.247
1.00
36.31
8
O

ATOM
1498
N
PHE
A
206
13.446
15.458
8.060
1.00
35.73
7
N

ATOM
1499
CA
PHE
A
206
14.866
15.414
7.794
1.00
34.97
6
C

ATOM
1500
CB
PHE
A
206
15.096
14.828
6.403
1.00
35.12
6
C

ATOM
1501
CG
PHE
A
206
14.542
15.677
5.298
1.00
35.80
6
C

ATOM
1502
CD1
PHE
A
206
15.337
16.622
4.663
1.00
36.66
6
C

ATOM
1503
CE1
PHE
A
206
14.831
17.407
3.647
1.00
36.61
6
C

ATOM
1504
CZ
PHE
A
206
13.516
17.260
3.255
1.00
37.26
6
C

ATOM
1505
CE2
PHE
A
206
12.711
16.328
3.881
1.00
37.55
6
C

ATOM
1506
CD2
PHE
A
206
13.222
15.546
4.899
1.00
37.21
6
C

ATOM
1507
C
PHE
A
206
15.514
16.787
7.870
1.00
34.23
6
C

ATOM
1508
O
PHE
A
206
14.846
17.813
7.736
1.00
34.04
8
O

ATOM
1509
N
ARG
A
207
16.829
16.775
8.059
1.00
32.42
7
N

ATOM
1510
CA
ARG
A
207
17.639
17.973
8.080
1.00
31.15
6
C

ATOM
1511
CB
ARG
A
207
17.721
18.530
9.504
1.00
31.65
6
C

ATOM
1512
CG
ARG
A
207
17.048
19.867
9.750
1.00
34.21
6
C

ATOM
1513
CD
ARG
A
207
15.763
20.088
9.013
1.00
37.48
6
C

ATOM
1514
NE
ARG
A
207
14.911
21.073
9.676
1.00
39.43
7
N

ATOM
1515
CZ
ARG
A
207
13.596
20.943
9.761
1.00
40.61
6
C

ATOM
1516
NH1
ARG
A
207
13.009
19.882
9.221
1.00
40.43
7
N

ATOM
1517
NH2
ARG
A
207
12.865
21.865
10.375
1.00
41.27
7
N

ATOM
1518
C
ARG
A
207
19.044
17.589
7.646
1.00
29.46
6
C

ATOM
1519
O
ARG
A
207
19.516
16.488
7.939
1.00
28.39
8
O

ATOM
1520
N
THR
A
208
19.717
18.509
6.973
1.00
27.70
7
N

ATOM
1521
CA
THR
A
208
21.117
18.324
6.635
1.00
26.90
6
C

ATOM
1522
CB
THR
A
208
21.340
18.595
5.155
1.00
27.18
6
C

ATOM
1523
OG1
THR
A
208
20.704
17.563
4.396
1.00
28.05
8
O

ATOM
1524
CG2
THR
A
208
22.824
18.467
4.810
1.00
26.41
6
C

ATOM
1525
C
THR
A
208
21.914
19.317
7.477
1.00
26.37
6
C

ATOM
1526
O
THR
A
208
21.589
20.491
7.498
1.00
26.33
8
O

ATOM
1527
N
TYR
A
209
22.938
18.837
8.179
1.00
25.78
7
N

ATOM
1528
CA
TYR
A
209
23.731
19.689
9.074
1.00
25.54
6
C

ATOM
1529
CB
TYR
A
209
23.648
19.141
10.505
1.00
25.31
6
C

ATOM
1530
CG
TYR
A
209
22.274
19.111
11.095
1.00
25.56
6
C

ATOM
1531
CD1
TYR
A
209
21.540
17.936
11.127
1.00
27.19
6
C

ATOM
1532
CE1
TYR
A
209
20.278
17.897
11.678
1.00
27.25
6
C

ATOM
1533
CZ
TYR
A
209
19.731
19.048
12.198
1.00
27.58
6
C

ATOM
1534
OH
TYR
A
209
18.471
19.004
12.741
1.00
28.08
8
O

ATOM
1535
CE2
TYR
A
209
20.439
20.235
12.180
1.00
27.33
6
C

ATOM
1536
CD2
TYR
A
209
21.705
20.259
11.632
1.00
27.03
6
C

ATOM
1537
C
TYR
A
209
25.199
19.673
8.713
1.00
25.06
6
C

ATOM
1538
O
TYR
A
209
25.746
18.617
8.387
1.00
24.78
8
O

ATOM
1539
N
ARG
A
210
25.856
20.828
8.799
1.00
24.92
7
N

ATOM
1540
CA
ARG
A
210
27.298
20.864
8.640
1.00
24.96
6
C

ATOM
1541
CB
ARG
A
210
27.773
22.300
8.432
1.00
25.52
6
C

ATOM
1542
CG
ARG
A
210
28.709
22.478
7.288
1.00
29.28
6
C

ATOM
1543
CD
ARG
A
210
28.950
23.949
6.924
1.00
31.30
6
C

ATOM
1544
NE
ARG
A
210
28.560
24.209
5.547
1.00
36.97
7
N

ATOM
1545
CZ
ARG
A
210
29.356
24.017
4.512
1.00
37.48
6
C

ATOM
1546
NH1
ARG
A
210
30.595
23.578
4.709
1.00
38.89
7
N

ATOM
1547
NH2
ARG
A
210
28.925
24.276
3.294
1.00
37.04
7
N

ATOM
1548
C
ARG
A
210
27.877
20.350
9.951
1.00
24.74
6
C

ATOM
1549
O
ARG
A
210
27.537
20.862
11.030
1.00
24.12
8
O

ATOM
1550
N
LEU
A
211
28.762
19.362
9.867
1.00
23.95
7
N

ATOM
1551
CA
LEU
A
211
29.324
18.756
11.076
1.00
24.48
6
C

ATOM
1552
CB
LEU
A
211
30.228
17.573
10.714
1.00
24.81
6
C

ATOM
1553
CG
LEU
A
211
29.472
16.344
10.216
1.00
25.82
6
C

ATOM
1554
CD1
LEU
A
211
30.420
15.356
9.535
1.00
28.04
6
C

ATOM
1555
CD2
LEU
A
211
28.746
15.673
11.405
1.00
27.62
6
C

ATOM
1556
C
LEU
A
211
30.085
19.742
11.950
1.00
24.79
6
C

ATOM
1557
O
LEU
A
211
29.979
19.701
13.183
1.00
24.62
8
O

ATOM
1558
N
SER
A
212
30.859
20.623
11.323
1.00
24.24
7
N

ATOM
1559
CA
SER
A
212
31.621
21.621
12.080
1.00
24.90
6
C

ATOM
1560
CB
SER
A
212
32.659
22.353
11.195
1.00
24.80
6
C

ATOM
1561
OG
SER
A
212
32.048
23.064
10.141
1.00
25.93
8
O

ATOM
1562
C
SER
A
212
30.704
22.602
12.816
1.00
24.67
6
C

ATOM
1563
O
SER
A
212
31.068
23.094
13.880
1.00
24.77
8
O

ATOM
1564
N
LEU
A
213
29.507
22.855
12.286
1.00
24.57
7
N

ATOM
1565
CA
LEU
A
213
28.556
23.740
12.974
1.00
24.86
6
C

ATOM
1566
CB
LEU
A
213
27.526
24.316
12.002
1.00
24.39
6
C

ATOM
1567
CG
LEU
A
213
28.111
25.341
11.026
1.00
24.18
6
C

ATOM
1568
CD1
LEU
A
213
27.047
25.832
10.040
1.00
23.86
6
C

ATOM
1569
CD2
LEU
A
213
28.745
26.527
11.790
1.00
24.15
6
C

ATOM
1570
C
LEU
A
213
27.855
23.053
14.151
1.00
25.20
6
C

ATOM
1571
O
LEU
A
213
27.463
23.714
15.123
1.00
24.93
8
O

ATOM
1572
N
LEU
A
214
27.672
21.737
14.059
1.00
25.82
7
N

ATOM
1573
CA
LEU
A
214
27.120
20.975
15.197
1.00
26.69
6
C

ATOM
1574
CB
LEU
A
214
26.877
19.513
14.812
1.00
26.57
6
C

ATOM
1575
CG
LEU
A
214
25.718
19.265
13.847
1.00
26.82
6
C

ATOM
1576
CD1
LEU
A
214
25.672
17.794
13.400
1.00
26.32
6
C

ATOM
1577
CD2
LEU
A
214
24.401
19.674
14.490
1.00
26.19
6
C

ATOM
1578
C
LEU
A
214
28.132
21.043
16.335
1.00
27.38
6
C

ATOM
1579
O
LEU
A
214
27.778
21.077
17.525
1.00
27.59
8
O

ATOM
1580
N
GLU
A
215
29.405
21.044
15.961
1.00
27.94
7
N

ATOM
1581
CA
GLU
A
215
30.480
21.129
16.938
1.00
29.14
6
C

ATOM
1582
CB
GLU
A
215
31.832
20.999
16.233
1.00
29.52
6
C

ATOM
1583
CG
GLU
A
215
33.024
21.144
17.151
1.00
31.92
6
C

ATOM
1584
CD
GLU
A
215
34.330
20.820
16.457
1.00
35.42
6
C

ATOM
1585
OE1
GLU
A
215
34.317
20.077
15.442
1.00
37.83
8
O

ATOM
1586
OE2
GLU
A
215
35.371
21.311
16.932
1.00
38.01
8
O

ATOM
1587
C
GLU
A
215
30.398
22.459
17.681
1.00
29.04
6
C

ATOM
1588
O
GLU
A
215
30.588
22.537
18.901
1.00
28.72
8
O

ATOM
1589
N
GLU
A
216
30.074
23.512
16.939
1.00
28.90
7
N

ATOM
1590
CA
GLU
A
216
30.031
24.846
17.518
1.00
28.96
6
C

ATOM
1591
CB
GLU
A
216
30.291
25.894
16.435
1.00
29.27
6
C

ATOM
1592
CG
GLU
A
216
31.704
25.831
15.898
1.00
32.49
6
C

ATOM
1593
CD
GLU
A
216
31.881
26.639
14.634
1.00
35.60
6
C

ATOM
1594
OE1
GLU
A
216
31.369
27.788
14.599
1.00
31.06
8
O

ATOM
1595
OE2
GLU
A
216
32.525
26.100
13.693
1.00
36.77
8
O

ATOM
1596
C
GLU
A
216
28.728
25.158
18.229
1.00
28.41
6
C

ATOM
1597
O
GLU
A
216
28.737
25.791
19.282
1.00
27.84
8
O

ATOM
1598
N
TYR
A
217
27.611
24.686
17.675
1.00
28.11
7
N

ATOM
1599
CA
TYR
A
217
26.295
25.039
18.201
1.00
28.19
6
C

ATOM
1600
CB
TYR
A
217
25.402
25.548
17.070
1.00
28.42
6
C

ATOM
1601
CG
TYR
A
217
25.858
26.892
16.546
1.00
29.01
6
C

ATOM
1602
CD1
TYR
A
217
26.601
26.996
15.375
1.00
30.89
6
C

ATOM
1603
CE1
TYR
A
217
27.031
28.241
14.908
1.00
31.06
6
C

ATOM
1604
CZ
TYR
A
217
26.711
29.381
15.631
1.00
31.94
6
C

ATOM
1605
OH
TYR
A
217
27.125
30.617
15.199
1.00
32.46
8
O

ATOM
1606
CE2
TYR
A
217
25.980
29.288
16.794
1.00
30.74
6
C

ATOM
1607
CD2
TYR
A
217
25.557
28.057
17.240
1.00
30.04
6
C

ATOM
1608
C
TYR
A
217
25.578
23.951
19.007
1.00
28.54
6
C

ATOM
1609
O
TYR
A
217
24.570
24.239
19.662
1.00
28.35
8
O

ATOM
1610
N
GLY
A
218
26.092
22.723
18.947
1.00
28.00
7
N

ATOM
1611
CA
GLY
A
218
25.524
21.602
19.696
1.00
28.61
6
C

ATOM
1612
C
GLY
A
218
24.399
20.886
18.972
1.00
28.81
6
C

ATOM
1613
O
GLY
A
218
23.959
21.325
17.914
1.00
28.46
8
O

ATOM
1614
N
CYS
A
219
23.937
19.770
19.535
1.00
29.39
7
N

ATOM
1615
CA
CYS
A
219
22.804
19.047
18.974
1.00
30.37
6
C

ATOM
1616
CB
CYS
A
219
23.192
18.175
17.771
1.00
30.56
6
C

ATOM
1617
SG
CYS
A
219
24.248
16.767
18.144
1.00
32.81
16
S

ATOM
1618
C
CYS
A
219
22.139
18.210
20.045
1.00
30.63
6
C

ATOM
1619
O
CYS
A
219
22.665
18.061
21.146
1.00
30.45
8
O

ATOM
1620
N
CYS
A
220
20.978
17.661
19.719
1.00
31.60
7
N

ATOM
1621
CA
CYS
A
220
20.224
16.889
20.695
1.00
32.42
6
C

ATOM
1622
CB
CYS
A
220
18.800
16.657
20.189
1.00
32.94
6
C

ATOM
1623
SG
CYS
A
220
18.712
15.547
18.750
1.00
35.67
16
S

ATOM
1624
C
CYS
A
220
20.877
15.543
20.957
1.00
32.66
6
C

ATOM
1625
O
CYS
A
220
21.647
15.042
20.142
1.00
31.36
8
O

ATOM
1626
N
LYS
A
221
20.593
14.975
22.123
1.00
33.15
7
N

ATOM
1627
CA
LYS
A
221
20.997
13.620
22.378
1.00
34.52
6
C

ATOM
1628
CB
LYS
A
221
20.503
13.168
23.762
1.00
35.08
6
C

ATOM
1629
CG
LYS
A
221
20.396
11.664
23.931
1.00
37.45
6
C

ATOM
1630
CD
LYS
A
221
19.537
11.281
25.137
1.00
41.50
6
C

ATOM
1631
CE
LYS
A
221
19.220
9.793
25.114
1.00
43.16
6
C

ATOM
1632
NZ
LYS
A
221
18.022
9.437
25.937
1.00
44.32
7
N

ATOM
1633
C
LYS
A
221
20.200
12.966
21.261
1.00
34.63
6
C

ATOM
1634
O
LYS
A
221
19.268
13.553
20.750
1.00
36.08
8
O

ATOM
1635
N
GLU
A
222
20.543
11.777
20.837
1.00
34.66
7
N

ATOM
1636
CA
GLU
A
222
19.779
11.175
19.742
1.00
33.55
6
C

ATOM
1637
CB
GLU
A
222
18.390
11.802
19.530
1.00
34.71
6
C

ATOM
1638
CG
GLU
A
222
17.261
11.284
20.433
1.00
37.70
6
C

ATOM
1639
CD
GLU
A
222
16.841
12.297
21.487
1.00
41.31
6
C

ATOM
1640
OE1
GLU
A
222
17.183
13.496
21.338
1.00
40.54
8
O

ATOM
1641
OE2
GLU
A
222
16.163
11.901
22.471
1.00
44.12
8
O

ATOM
1642
C
GLU
A
222
20.601
11.365
18.494
1.00
31.77
6
C

ATOM
1643
O
GLU
A
222
21.102
10.399
17.961
1.00
31.65
8
O

ATOM
1644
N
LEU
A
223
20.737
12.600
18.011
1.00
30.02
7
N

ATOM
1645
CA
LEU
A
223
21.627
12.797
16.869
1.00
28.45
6
C

ATOM
1646
CB
LEU
A
223
21.482
14.186
16.236
1.00
28.53
6
C

ATOM
1647
CG
LEU
A
223
22.461
14.498
15.100
1.00
28.64
6
C

ATOM
1648
CD1
LEU
A
223
22.416
13.409
14.023
1.00
28.96
6
C

ATOM
1649
CD2
LEU
A
223
22.160
15.859
14.506
1.00
28.18
6
C

ATOM
1650
C
LEU
A
223
23.044
12.546
17.374
1.00
27.23
6
C

ATOM
1651
O
LEU
A
223
23.826
11.848
16.738
1.00
25.93
8
O

ATOM
1652
N
ALA
A
224
23.356
13.073
18.559
1.00
26.68
7
N

ATOM
1653
CA
ALA
A
224
24.683
12.874
19.127
1.00
26.35
6
C

ATOM
1654
CB
ALA
A
224
24.822
13.605
20.474
1.00
26.82
6
C

ATOM
1655
C
ALA
A
224
25.033
11.405
19.292
1.00
25.93
6
C

ATOM
1656
O
ALA
A
224
26.161
10.988
19.001
1.00
24.68
8
O

ATOM
1657
N
SER
A
225
24.076
10.618
19.772
1.00
25.86
7
N

ATOM
1658
CA
SER
A
225
24.341
9.209
20.004
1.00
26.40
6
C

ATOM
1659
CB
SER
A
225
23.220
8.566
20.831
1.00
26.51
6
C

ATOM
1660
OG
SER
A
225
21.999
8.630
20.130
1.00
31.54
8
O

ATOM
1661
C
SER
A
225
24.520
8.478
18.670
1.00
25.53
6
C

ATOM
1662
O
SER
A
225
25.331
7.550
18.559
1.00
25.79
8
O

ATOM
1663
N
ARG
A
226
23.759
8.893
17.668
1.00
24.77
7
N

ATOM
1664
CA
ARG
A
226
23.867
8.272
16.354
1.00
24.66
6
C

ATOM
1665
CB
ARG
A
226
22.660
8.614
15.485
1.00
24.86
6
C

ATOM
1666
CG
ARG
A
226
21.403
7.808
15.900
1.00
25.80
6
C

ATOM
1667
CD
ARG
A
226
20.076
8.383
15.422
1.00
27.28
6
C

ATOM
1668
NE
ARG
A
226
18.934
7.584
15.889
1.00
27.33
7
N

ATOM
1669
CZ
ARG
A
226
18.443
7.622
17.129
1.00
29.35
6
C

ATOM
1670
NH1
ARG
A
226
18.974
8.429
18.041
1.00
28.48
7
N

ATOM
1671
NH2
ARG
A
226
17.403
6.864
17.458
1.00
30.48
7
N

ATOM
1672
C
ARG
A
226
25.202
8.615
15.686
1.00
24.36
6
C

ATOM
1673
O
ARG
A
226
25.783
7.777
14.985
1.00
24.15
8
O

ATOM
1674
N
LEU
A
227
25.687
9.835
15.915
1.00
24.00
7
N

ATOM
1675
CA
LEU
A
227
26.993
10.243
15.385
1.00
24.49
6
C

ATOM
1676
CB
LEU
A
227
27.197
11.751
15.522
1.00
25.07
6
C

ATOM
1677
CG
LEU
A
227
26.409
12.631
14.546
1.00
25.52
6
C

ATOM
1678
CD1
LEU
A
227
26.575
14.116
14.901
1.00
25.92
6
C

ATOM
1679
CD2
LEU
A
227
26.832
12.394
13.087
1.00
27.53
6
C

ATOM
1680
C
LEU
A
227
28.139
9.470
16.066
1.00
24.12
6
C

ATOM
1681
O
LEU
A
227
29.154
9.164
15.436
1.00
23.19
8
O

ATOM
1682
N
ARG
A
228
27.981
9.151
17.351
1.00
23.81
7
N

ATOM
1683
CA
ARG
A
228
28.973
8.310
18.026
1.00
24.03
6
C

ATOM
1684
CB
ARG
A
228
28.654
8.178
19.526
1.00
23.73
6
C

ATOM
1685
CG
ARG
A
228
29.133
9.349
20.409
1.00
26.03
6
C

ATOM
1686
CD
ARG
A
228
28.765
9.173
21.909
1.00
31.16
6
C

ATOM
1687
NE
ARG
A
228
27.857
10.250
22.283
1.00
36.96
7
N

ATOM
1688
CZ
ARG
A
228
26.632
10.098
22.737
1.00
36.30
6
C

ATOM
1689
NH1
ARG
A
228
26.118
8.889
22.949
1.00
37.17
7
N

ATOM
1690
NH2
ARG
A
228
25.928
11.174
23.012
1.00
38.46
7
N

ATOM
1691
C
ARG
A
228
28.992
6.929
17.375
1.00
23.69
6
C

ATOM
1692
O
ARG
A
228
30.053
6.366
17.098
1.00
23.53
8
O

ATOM
1693
N
TYR
A
229
27.805
6.371
17.157
1.00
24.48
7
N

ATOM
1694
CA
TYR
A
229
27.673
5.069
16.509
1.00
24.19
6
C

ATOM
1695
CB
TYR
A
229
26.196
4.703
16.373
1.00
24.66
6
C

ATOM
1696
CG
TYR
A
229
25.971
3.265
15.968
1.00
25.51
6
C

ATOM
1697
CD1
TYR
A
229
25.931
2.264
16.930
1.00
26.27
6
C

ATOM
1698
CE1
TYR
A
229
25.730
0.944
16.580
1.00
28.27
6
C

ATOM
1699
CZ
TYR
A
229
25.563
0.598
15.258
1.00
26.61
6
C

ATOM
1700
OH
TYR
A
229
25.367
−0.732
14.947
1.00
27.01
8
O

ATOM
1701
CE2
TYR
A
229
25.601
1.561
14.274
1.00
27.00
6
C

ATOM
1702
CD2
TYR
A
229
25.807
2.902
14.628
1.00
25.01
6
C

ATOM
1703
C
TYR
A
229
28.312
5.117
15.110
1.00
23.94
6
C

ATOM
1704
O
TYR
A
229
29.032
4.211
14.713
1.00
23.19
8
O

ATOM
1705
N
ALA
A
230
28.044
6.190
14.376
1.00
23.63
7
N

ATOM
1706
CA
ALA
A
230
28.627
6.350
13.033
1.00
23.80
6
C

ATOM
1707
CB
ALA
A
230
28.128
7.634
12.391
1.00
23.61
6
C

ATOM
1708
C
ALA
A
230
30.165
6.297
13.042
1.00
23.70
6
C

ATOM
1709
O
ALA
A
230
30.790
5.651
12.186
1.00
23.89
8
O

ATOM
1710
N
ARG
A
231
30.780
6.956
14.016
1.00
23.74
7
N

ATOM
1711
CA
ARG
A
231
32.234
6.917
14.138
1.00
24.06
6
C

ATOM
1712
CB
ARG
A
231
32.710
7.811
15.283
1.00
24.34
6
C

ATOM
1713
CG
ARG
A
231
34.223
7.959
15.371
1.00
26.14
6
C

ATOM
1714
CD
ARG
A
231
34.902
7.010
16.354
1.00
28.93
6
C

ATOM
1715
NE
ARG
A
231
36.355
7.205
16.370
1.00
31.60
7
N

ATOM
1716
CZ
ARG
A
231
36.961
8.234
16.942
1.00
33.08
6
C

ATOM
1717
NH1
ARG
A
231
36.251
9.163
17.571
1.00
34.81
7
N

ATOM
1718
NH2
ARG
A
231
38.283
8.339
16.895
1.00
35.10
7
N

ATOM
1719
C
ARG
A
231
32.755
5.487
14.313
1.00
23.52
6
C

ATOM
1720
O
ARG
A
231
33.743
5.103
13.686
1.00
24.27
8
O

ATOM
1721
N
THR
A
232
32.084
4.707
15.155
1.00
23.50
7
N

ATOM
1722
CA
THR
A
232
32.442
3.312
15.370
1.00
23.31
6
C

ATOM
1723
CB
THR
A
232
31.491
2.700
16.424
1.00
24.05
6
C

ATOM
1724
OG1
THR
A
232
31.636
3.415
17.666
1.00
24.01
8
O

ATOM
1725
CG2
THR
A
232
31.905
1.260
16.755
1.00
24.30
6
C

ATOM
1726
C
THR
A
232
32.330
2.533
14.056
1.00
23.53
6
C

ATOM
1727
O
THR
A
232
33.188
1.705
13.718
1.00
22.83
8
O

ATOM
1728
N
MET
A
233
31.258
2.792
13.317
1.00
23.02
7
N

ATOM
1729
CA
MET
A
233
31.071
2.114
12.042
1.00
23.97
6
C

ATOM
1730
CB
MET
A
233
29.681
2.389
11.474
1.00
23.70
6
C

ATOM
1731
CG
MET
A
233
28.548
1.893
12.339
1.00
24.89
6
C

ATOM
1732
SD
MET
A
233
28.603
0.124
12.651
1.00
25.92
16
S

ATOM
1733
CE
MET
A
233
29.192
0.099
14.342
1.00
27.03
6
C

ATOM
1734
C
MET
A
233
32.141
2.509
11.032
1.00
23.90
6
C

ATOM
1735
O
MET
A
233
32.602
1.671
10.255
1.00
24.29
8
O

ATOM
1736
N
VAL
A
234
32.528
3.779
11.034
1.00
24.08
7
N

ATOM
1737
CA
VAL
A
234
33.561
4.232
10.099
1.00
25.69
6
C

ATOM
1738
CB
VAL
A
234
33.647
5.767
10.036
1.00
25.75
6
C

ATOM
1739
CG1
VAL
A
234
34.896
6.230
9.251
1.00
26.58
6
C

ATOM
1740
CG2
VAL
A
234
32.379
6.325
9.415
1.00
26.02
6
C

ATOM
1741
C
VAL
A
234
34.912
3.586
10.434
1.00
26.22
6
C

ATOM
1742
O
VAL
A
234
35.628
3.137
9.531
1.00
27.03
8
O

ATOM
1743
N
ASP
A
235
35.242
3.502
11.723
1.00
27.15
7
N

ATOM
1744
CA
ASP
A
235
36.470
2.813
12.153
1.00
27.94
6
C

ATOM
1745
CB
ASP
A
235
36.618
2.810
13.685
1.00
28.58
6
C

ATOM
1746
CG
ASP
A
235
37.272
4.061
14.226
1.00
30.71
6
C

ATOM
1747
OD1
ASP
A
235
38.060
4.718
13.498
1.00
31.20
8
O

ATOM
1748
OD2
ASP
A
235
37.073
4.460
15.397
1.00
33.27
8
O

ATOM
1749
C
ASP
A
235
36.458
1.372
11.655
1.00
27.90
6
C

ATOM
1750
O
ASP
A
235
37.482
0.848
11.198
1.00
27.86
8
O

ATOM
1751
N
LYS
A
236
35.301
0.718
11.758
1.00
28.17
7
N

ATOM
1752
CA
LYS
A
236
35.167
−0.661
11.292
1.00
29.21
6
C

ATOM
1753
CB
LYS
A
236
33.805
−1.255
11.651
1.00
28.80
6
C

ATOM
1754
CG
LYS
A
236
33.766
−2.755
11.453
1.00
31.44
6
C

ATOM
1755
CD
LYS
A
236
32.463
−3.365
11.880
1.00
33.65
6
C

ATOM
1756
CE
LYS
A
236
32.677
−4.762
12.424
1.00
35.73
6
C

ATOM
1757
NZ
LYS
A
236
33.676
−5.582
11.682
1.00
33.42
7
N

ATOM
1758
C
LYS
A
236
35.412
−0.779
9.781
1.00
29.51
6
C

ATOM
1759
O
LYS
A
236
36.136
−1.676
9.336
1.00
29.82
8
O

ATOM
1760
N
LEU
A
237
34.813
0.123
9.006
1.00
29.66
7
N

ATOM
1761
CA
LEU
A
237
35.000
0.144
7.556
1.00
30.62
6
C

ATOM
1762
CB
LEU
A
237
34.222
1.310
6.923
1.00
29.80
6
C

ATOM
1763
CG
LEU
A
237
32.701
1.153
6.793
1.00
28.82
6
C

ATOM
1764
CD1
LEU
A
237
32.026
2.447
6.386
1.00
29.45
6
C

ATOM
1765
CD2
LEU
A
237
32.362
0.031
5.797
1.00
28.42
6
C

ATOM
1766
C
LEU
A
237
36.488
0.282
7.234
1.00
31.81
6
C

ATOM
1767
O
LEU
A
237
37.002
−0.373
6.326
1.00
31.85
8
O

ATOM
1768
N
LEU
A
238
37.174
1.133
7.986
1.00
33.83
7
N

ATOM
1769
CA
LEU
A
238
38.610
1.323
7.794
1.00
36.16
6
C

ATOM
1770
CB
LEU
A
238
39.098
2.564
8.539
1.00
35.64
6
C

ATOM
1771
CG
LEU
A
238
38.733
3.870
7.834
1.00
35.77
6
C

ATOM
1772
CD1
LEU
A
238
38.772
5.047
8.792
1.00
35.86
6
C

ATOM
1773
CD2
LEU
A
238
39.644
4.104
6.622
1.00
35.95
6
C

ATOM
1774
C
LEU
A
238
39.427
0.110
8.219
1.00
37.98
6
C

ATOM
1775
O
LEU
A
238
40.483
−0.154
7.650
1.00
38.49
8
O

ATOM
1776
N
SER
A
239
38.939
−0.627
9.210
1.00
40.20
7
N

ATOM
1777
CA
SER
A
239
39.658
−1.793
9.715
1.00
42.61
6
C

ATOM
1778
CB
SER
A
239
39.069
−2.256
11.048
1.00
42.53
6
C

ATOM
1779
OG
SER
A
239
37.938
−3.081
10.828
1.00
41.85
8
O

ATOM
1780
C
SER
A
239
39.597
−2.942
8.723
1.00
44.55
6
C

ATOM
1781
O
SER
A
239
40.564
−3.680
8.551
1.00
45.33
8
O

ATOM
1782
N
SER
A
240
38.448
−3.099
8.076
1.00
46.75
7
N

ATOM
1783
CA
SER
A
240
38.266
−4.176
7.118
1.00
48.84
6
C

ATOM
1784
CB
SER
A
240
36.812
−4.656
7.104
1.00
48.94
6
C

ATOM
1785
OG
SER
A
240
35.913
−3.574
6.949
1.00
50.43
8
O

ATOM
1786
C
SER
A
240
38.705
−3.724
5.734
1.00
49.93
6
C

ATOM
1787
O
SER
A
240
38.692
−4.508
4.790
1.00
50.59
8
O

ATOM
1788
N
ALA
A
241
39.105
−2.458
5.635
1.00
51.23
7
N

ATOM
1789
CA
ALA
A
241
39.580
−1.867
4.382
1.00
52.19
6
C

ATOM
1790
CB
ALA
A
241
40.848
−1.074
4.625
1.00
52.24
6
C

ATOM
1791
C
ALA
A
241
39.819
−2.906
3.294
1.00
52.79
6
C

ATOM
1792
O
ALA
A
241
40.907
−3.488
3.238
1.00
53.19
8
O

ATOM
1793
OXT
ALA
A
241
38.934
−3.162
2.470
1.00
53.32
8
O

ATOM
1794
N
ALA
B
20
−18.462
10.374
−32.692
1.00
40.15
7
N

ATOM
1795
CA
ALA
B
20
−18.787
10.792
−31.295
1.00
39.44
6
C

ATOM
1796
CB
ALA
B
20
−19.597
12.064
−31.300
1.00
39.55
6
C

ATOM
1797
C
ALA
B
20
−19.538
9.676
−30.576
1.00
38.88
6
C

ATOM
1798
O
ALA
B
20
−20.122
8.802
−31.212
1.00
38.93
8
O

ATOM
1799
N
LEU
B
21
−19.515
9.710
−29.249
1.00
38.33
7
N

ATOM
1800
CA
LEU
B
21
−20.162
8.679
−28.452
1.00
37.95
6
C

ATOM
1801
CB
LEU
B
21
−19.852
8.865
−26.969
1.00
38.46
6
C

ATOM
1802
CG
LEU
B
21
−18.459
8.484
−26.486
1.00
39.45
6
C

ATOM
1803
CD1
LEU
B
21
−18.389
8.609
−24.970
1.00
40.66
6
C

ATOM
1804
CD2
LEU
B
21
−18.114
7.069
−26.923
1.00
40.86
6
C

ATOM
1805
C
LEU
B
21
−21.663
8.693
−28.674
1.00
37.29
6
C

ATOM
1806
O
LEU
B
21
−22.288
7.643
−28.802
1.00
36.75
8
O

ATOM
1807
N
SER
B
22
−22.235
9.894
−28.706
1.00
36.31
7
N

ATOM
1808
CA
SER
B
22
−23.662
10.057
−28.948
1.00
35.97
6
C

ATOM
1809
CB
SER
B
22
−24.027
11.551
−28.960
1.00
36.01
6
C

ATOM
1810
OG
SER
B
22
−25.386
11.740
−29.301
1.00
38.45
8
O

ATOM
1811
C
SER
B
22
−24.081
9.355
−30.248
1.00
34.55
6
C

ATOM
1812
O
SER
B
22
−25.046
8.597
−30.264
1.00
34.41
8
O

ATOM
1813
N
ASP
B
23
−23.346
9.583
−31.332
1.00
33.54
7
N

ATOM
1814
CA
ASP
B
23
−23.635
8.906
−32.596
1.00
32.88
6
C

ATOM
1815
CB
ASP
B
23
−22.679
9.371
−33.696
1.00
32.93
6
C

ATOM
1816
CG
ASP
B
23
−22.915
10.820
−34.120
1.00
34.64
6
C

ATOM
1817
OD1
ASP
B
23
−24.013
11.362
−33.878
1.00
34.23
8
O

ATOM
1818
OD2
ASP
B
23
−22.050
11.485
−34.718
1.00
35.74
8
O

ATOM
1819
C
ASP
B
23
−23.534
7.375
−32.453
1.00
32.47
6
C

ATOM
1820
O
ASP
B
23
−24.386
6.633
−32.945
1.00
31.81
8
O

ATOM
1821
N
MET
B
24
−22.490
6.906
−31.781
1.00
31.81
7
N

ATOM
1822
CA
MET
B
24
−22.307
5.459
−31.625
1.00
32.01
6
C

ATOM
1823
CB
MET
B
24
−20.997
5.143
−30.894
1.00
32.02
6
C

ATOM
1824
CG
MET
B
24
−20.641
3.656
−30.899
1.00
33.91
6
C

ATOM
1825
SD
MET
B
24
−19.040
3.300
−30.170
1.00
36.01
16
S

ATOM
1826
CE
MET
B
24
−17.947
3.957
−31.436
1.00
36.64
6
C

ATOM
1827
C
MET
B
24
−23.492
4.849
−30.882
1.00
31.30
6
C

ATOM
1828
O
MET
B
24
−23.984
3.784
−31.245
1.00
31.16
8
O

ATOM
1829
N
LEU
B
25
−23.956
5.539
−29.846
1.00
31.37
7
N

ATOM
1830
CA
LEU
B
25
−25.086
5.057
−29.060
1.00
31.48
6
C

ATOM
1831
CB
LEU
B
25
−25.356
5.979
−27.871
1.00
31.48
6
C

ATOM
1832
CG
LEU
B
25
−26.522
5.539
−26.982
1.00
31.97
6
C

ATOM
1833
CD1
LEU
B
25
−26.211
4.186
−26.338
1.00
32.19
6
C

ATOM
1834
CD2
LEU
B
25
−26.852
6.594
−25.919
1.00
34.03
6
C

ATOM
1835
C
LEU
B
25
−26.333
4.927
−29.928
1.00
31.62
6
C

ATOM
1836
O
LEU
B
25
−27.015
3.904
−29.895
1.00
31.80
8
O

ATOM
1837
N
GLN
B
26
−26.619
5.949
−30.726
1.00
31.03
7
N

ATOM
1838
CA
GLN
B
26
−27.785
5.895
−31.608
1.00
30.78
6
C

ATOM
1839
CB
GLN
B
26
−27.993
7.236
−32.330
1.00
31.06
6
C

ATOM
1840
CG
GLN
B
26
−28.570
8.351
−31.445
1.00
33.92
6
C

ATOM
1841
CD
GLN
B
26
−28.926
9.607
−32.236
1.00
38.19
6
C

ATOM
1842
OE1
GLN
B
26
−28.178
10.021
−33.114
1.00
39.61
8
O

ATOM
1843
NE2
GLN
B
26
−30.068
10.214
−31.919
1.00
40.60
7
N

ATOM
1844
C
GLN
B
26
−27.678
4.750
−32.620
1.00
29.63
6
C

ATOM
1845
O
GLN
B
26
−28.666
4.101
−32.938
1.00
29.49
8
O

ATOM
1846
N
GLN
B
27
−26.482
4.524
−33.146
1.00
29.11
7
N

ATOM
1847
CA
GLN
B
27
−26.258
3.449
−34.102
1.00
28.58
6
C

ATOM
1848
CB
GLN
B
27
−24.844
3.560
−34.694
1.00
28.95
6
C

ATOM
1849
CG
GLN
B
27
−24.627
4.822
−35.541
1.00
30.01
6
C

ATOM
1850
CD
GLN
B
27
−23.158
5.178
−35.722
1.00
31.97
6
C

ATOM
1851
OE1
GLN
B
27
−22.277
4.377
−35.412
1.00
30.66
8
O

ATOM
1852
NE2
GLN
B
27
−22.894
6.381
−36.235
1.00
30.20
7
N

ATOM
1853
C
GLN
B
27
−26.462
2.070
−33.454
1.00
28.10
6
C

ATOM
1854
O
GLN
B
27
−27.047
1.168
−34.050
1.00
27.55
8
O

ATOM
1855
N
LEU
B
28
−25.953
1.907
−32.239
1.00
27.60
7
N

ATOM
1856
CA
LEU
B
28
−26.105
0.640
−31.534
1.00
27.94
6
C

ATOM
1857
CB
LEU
B
28
−25.145
0.574
−30.344
1.00
27.52
6
C

ATOM
1858
CG
LEU
B
28
−23.674
0.414
−30.741
1.00
27.57
6
C

ATOM
1859
CD1
LEU
B
28
−22.758
0.627
−29.547
1.00
29.37
6
C

ATOM
1860
CD2
LEU
B
28
−23.410
−0.943
−31.367
1.00
28.78
6
C

ATOM
1861
C
LEU
B
28
−27.560
0.445
−31.114
1.00
28.02
6
C

ATOM
1862
O
LEU
B
28
−28.134
−0.632
−31.298
1.00
28.14
8
O

ATOM
1863
N
HIS
B
29
−28.172
1.498
−30.580
1.00
28.71
7
N

ATOM
1864
CA
HIS
B
29
−29.567
1.411
−30.178
1.00
29.41
6
C

ATOM
1865
CB
HIS
B
29
−30.088
2.755
−29.660
1.00
29.83
6
C

ATOM
1866
CG
HIS
B
29
−31.563
2.756
−29.388
1.00
31.05
6
C

ATOM
1867
ND1
HIS
B
29
−32.112
2.192
−28.256
1.00
33.08
7
N

ATOM
1868
CE1
HIS
B
29
−33.427
2.325
−28.291
1.00
33.88
6
C

ATOM
1869
NE2
HIS
B
29
−33.751
2.954
−29.408
1.00
33.28
7
N

ATOM
1870
CD2
HIS
B
29
−32.604
3.236
−30.111
1.00
33.23
6
C

ATOM
1871
C
HIS
B
29
−30.406
0.949
−31.363
1.00
29.61
6
C

ATOM
1872
O
HIS
B
29
−31.248
0.059
−31.243
1.00
29.22
8
O

ATOM
1873
N
SER
B
30
−30.169
1.556
−32.516
1.00
29.41
7
N

ATOM
1874
CA
SER
B
30
−30.928
1.205
−33.710
1.00
30.33
6
C

ATOM
1875
CB
SER
B
30
−30.558
2.150
−34.857
1.00
30.29
6
C

ATOM
1876
OG
SER
B
30
−31.372
1.911
−35.981
1.00
32.27
8
O

ATOM
1877
C
SER
B
30
−30.747
−0.256
−34.145
1.00
29.67
6
C

ATOM
1878
O
SER
B
30
−31.725
−0.971
−34.379
1.00
29.62
8
O

ATOM
1879
N
VAL
B
31
−29.508
−0.716
−34.264
1.00
29.60
7
N

ATOM
1880
CA
VAL
B
31
−29.321
−2.091
−34.727
1.00
29.79
6
C

ATOM
1881
CB
VAL
B
31
−27.859
−2.398
−35.181
1.00
29.90
6
C

ATOM
1882
CG1
VAL
B
31
−26.890
−2.255
−34.045
1.00
29.99
6
C

ATOM
1883
CG2
VAL
B
31
−27.780
−3.784
−35.806
1.00
30.78
6
C

ATOM
1884
C
VAL
B
31
−29.858
−3.109
−33.711
1.00
29.27
6
C

ATOM
1885
O
VAL
B
31
−30.505
−4.081
−34.086
1.00
29.15
8
O

ATOM
1886
N
ASN
B
32
−29.637
−2.859
−32.424
1.00
29.19
7
N

ATOM
1887
CA
ASN
B
32
−30.106
−3.792
−31.407
1.00
29.31
6
C

ATOM
1888
CB
ASN
B
32
−29.550
−3.434
−30.021
1.00
28.38
6
C

ATOM
1889
CG
ASN
B
32
−28.034
−3.544
−29.955
1.00
28.07
6
C

ATOM
1890
OD1
ASN
B
32
−27.414
−4.173
−30.811
1.00
27.63
8
O

ATOM
1891
ND2
ASN
B
32
−27.429
−2.936
−28.930
1.00
26.72
7
N

ATOM
1892
C
ASN
B
32
−31.629
−3.892
−31.401
1.00
29.61
6
C

ATOM
1893
O
ASN
B
32
−32.175
−4.979
−31.303
1.00
29.49
8
O

ATOM
1894
N
ALA
B
33
−32.305
−2.752
−31.535
1.00
30.63
7
N

ATOM
1895
CA
ALA
B
33
−33.768
−2.699
−31.534
1.00
31.08
6
C

ATOM
1896
CB
ALA
B
33
−34.242
−1.259
−31.578
1.00
31.16
6
C

ATOM
1897
C
ALA
B
33
−34.396
−3.513
−32.676
1.00
31.77
6
C

ATOM
1898
O
ALA
B
33
−35.542
−3.989
−32.564
1.00
31.28
8
O

ATOM
1899
N
SER
B
34
−33.642
−3.696
−33.759
1.00
31.76
7
N

ATOM
1900
CA
SER
B
34
−34.128
−4.482
−34.889
1.00
32.48
6
C

ATOM
1901
CB
SER
B
34
−33.389
−4.092
−36.177
1.00
32.31
6
C

ATOM
1902
OG
SER
B
34
−32.074
−4.628
−36.186
1.00
31.09
8
O

ATOM
1903
C
SER
B
34
−33.992
−5.993
−34.655
1.00
33.19
6
C

ATOM
1904
O
SER
B
34
−34.454
−6.791
−35.479
1.00
33.37
8
O

ATOM
1905
N
LYS
B
35
−33.370
−6.368
−33.535
1.00
33.91
7
N

ATOM
1906
CA
LYS
B
35
−33.127
−7.769
−33.169
1.00
34.68
6
C

ATOM
1907
CB
LYS
B
35
−34.403
−8.404
−32.615
1.00
35.29
6
C

ATOM
1908
CG
LYS
B
35
−35.058
−7.602
−31.485
1.00
36.72
6
C

ATOM
1909
CD
LYS
B
35
−34.692
−8.140
−30.122
1.00
40.57
6
C

ATOM
1910
CE
LYS
B
35
−35.496
−7.457
−29.015
1.00
40.76
6
C

ATOM
1911
NZ
LYS
B
35
−36.831
−8.105
−28.810
1.00
42.80
7
N

ATOM
1912
C
LYS
B
35
−32.630
−8.572
−34.366
1.00
34.80
6
C

ATOM
1913
O
LYS
B
35
−33.317
−9.475
−34.844
1.00
34.35
8
O

ATOM
1914
N
PRO
B
36
−31.430
−8.253
−34.837
1.00
35.09
7
N

ATOM
1915
CA
PRO
B
36
−30.903
−8.840
−36.077
1.00
35.28
6
C

ATOM
1916
CB
PRO
B
36
−29.565
−8.116
−36.256
1.00
35.30
6
C

ATOM
1917
CG
PRO
B
36
−29.192
−7.714
−34.847
1.00
35.62
6
C

ATOM
1918
CD
PRO
B
36
−30.496
−7.276
−34.249
1.00
34.89
6
C

ATOM
1919
C
PRO
B
36
−30.705
−10.360
−36.077
1.00
35.65
6
C

ATOM
1920
O
PRO
B
36
−30.595
−10.922
−37.167
1.00
35.42
8
O

ATOM
1921
N
SER
B
37
−30.662
−11.017
−34.916
1.00
35.73
7
N

ATOM
1922
CA
SER
B
37
−30.474
−12.469
−34.915
1.00
36.28
6
C

ATOM
1923
CB
SER
B
37
−29.538
−12.929
−33.789
1.00
36.41
6
C

ATOM
1924
OG
SER
B
37
−30.183
−12.868
−32.533
1.00
35.91
8
O

ATOM
1925
C
SER
B
37
−31.789
−13.239
−34.869
1.00
36.95
6
C

ATOM
1926
O
SER
B
37
−31.803
−14.464
−34.989
1.00
37.58
8
O

ATOM
1927
N
GLU
B
38
−32.893
−12.524
−34.699
1.00
37.36
7
N

ATOM
1928
CA
GLU
B
38
−34.199
−13.161
−34.657
1.00
38.54
6
C

ATOM
1929
CB
GLU
B
38
−35.069
−12.541
−33.555
1.00
38.20
6
C

ATOM
1930
CG
GLU
B
38
−34.497
−12.752
−32.162
1.00
39.65
6
C

ATOM
1931
CD
GLU
B
38
−35.307
−12.080
−31.061
1.00
41.33
6
C

ATOM
1932
OE1
GLU
B
38
−36.512
−11.811
−31.263
1.00
41.59
8
O

ATOM
1933
OE2
GLU
B
38
−34.733
−11.822
−29.983
1.00
42.51
8
O

ATOM
1934
C
GLU
B
38
−34.866
−13.032
−36.018
1.00
38.82
6
C

ATOM
1935
O
GLU
B
38
−35.934
−12.459
−36.143
1.00
39.40
8
O

ATOM
1936
N
ARG
B
39
−34.213
−13.558
−37.043
1.00
39.56
7
N

ATOM
1937
CA
ARG
B
39
−34.746
−13.508
−38.395
1.00
40.06
6
C

ATOM
1938
CB
ARG
B
39
−33.852
−12.652
−39.288
1.00
39.74
6
C

ATOM
1939
CG
ARG
B
39
−33.605
−11.249
−38.760
1.00
38.73
6
C

ATOM
1940
CD
ARG
B
39
−34.740
−10.274
−39.009
1.00
36.27
6
C

ATOM
1941
NE
ARG
B
39
−34.464
−8.983
−38.391
1.00
34.61
7
N

ATOM
1942
CZ
ARG
B
39
−33.754
−8.019
−38.963
1.00
35.62
6
C

ATOM
1943
NH1
ARG
B
39
−33.256
−8.188
−40.186
1.00
34.56
7
N

ATOM
1944
NH2
ARG
B
39
−33.550
−6.876
−38.318
1.00
34.83
7
N

ATOM
1945
C
ARG
B
39
−34.796
−14.920
−38.945
1.00
40.94
6
C

ATOM
1946
O
ARG
B
39
−33.995
−15.768
−38.558
1.00
41.00
8
O

ATOM
1947
N
GLY
B
40
−35.742
−15.175
−39.843
1.00
41.86
7
N

ATOM
1948
CA
GLY
B
40
−35.849
−16.479
−40.464
1.00
42.75
6
C

ATOM
1949
C
GLY
B
40
−34.620
−16.743
−41.309
1.00
43.43
6
C

ATOM
1950
O
GLY
B
40
−33.996
−17.798
−41.210
1.00
44.09
8
O

ATOM
1951
N
LEU
B
41
−34.265
−15.773
−42.142
1.00
43.47
7
N

ATOM
1952
CA
LEU
B
41
−33.093
−15.910
−42.992
1.00
43.77
6
C

ATOM
1953
CB
LEU
B
41
−33.485
−15.855
−44.473
1.00
43.92
6
C

ATOM
1954
CG
LEU
B
41
−32.312
−15.792
−45.454
1.00
45.32
6
C

ATOM
1955
CD1
LEU
B
41
−31.384
−16.988
−45.271
1.00
46.78
6
C

ATOM
1956
CD2
LEU
B
41
−32.814
−15.720
−46.887
1.00
46.32
6
C

ATOM
1957
C
LEU
B
41
−32.071
−14.827
−42.675
1.00
43.35
6
C

ATOM
1958
O
LEU
B
41
−32.352
−13.638
−42.805
1.00
43.80
8
O

ATOM
1959
N
VAL
B
42
−30.890
−15.249
−42.242
1.00
42.64
7
N

ATOM
1960
CA
VAL
B
42
−29.816
−14.325
−41.922
1.00
42.03
6
C

ATOM
1961
CB
VAL
B
42
−29.002
−14.809
−40.696
1.00
42.22
6
C

ATOM
1962
CG1
VAL
B
42
−27.760
−13.961
−40.511
1.00
41.23
6
C

ATOM
1963
CG2
VAL
B
42
−29.853
−14.784
−39.434
1.00
42.53
6
C

ATOM
1964
C
VAL
B
42
−28.882
−14.217
−43.119
1.00
41.54
6
C

ATOM
1965
O
VAL
B
42
−28.497
−15.233
−43.691
1.00
41.43
8
O

ATOM
1966
N
ARG
B
43
−28.540
−12.994
−43.513
1.00
40.84
7
N

ATOM
1967
CA
ARG
B
43
−27.606
−12.783
−44.620
1.00
40.38
6
C

ATOM
1968
CB
ARG
B
43
−28.335
−12.301
−45.876
1.00
40.78
6
C

ATOM
1969
CG
ARG
B
43
−29.070
−13.411
−46.622
1.00
43.34
6
C

ATOM
1970
CD
ARG
B
43
−29.934
−12.922
−47.784
1.00
46.71
6
C

ATOM
1971
NE
ARG
B
43
−31.124
−12.220
−47.306
1.00
50.29
7
N

ATOM
1972
CZ
ARG
B
43
−31.778
−11.288
−47.995
1.00
51.44
6
C

ATOM
1973
NH1
ARG
B
43
−31.363
−10.939
−49.204
1.00
51.72
7
N

ATOM
1974
NH2
ARG
B
43
−32.850
−10.704
−47.469
1.00
52.75
7
N

ATOM
1975
C
ARG
B
43
−26.512
−11.803
−44.205
1.00
39.52
6
C

ATOM
1976
O
ARG
B
43
−26.335
−10.743
−44.809
1.00
38.70
8
O

ATOM
1977
N
GLN
B
44
−25.778
−12.187
−43.166
1.00
38.29
7
N

ATOM
1978
CA
GLN
B
44
−24.723
−11.364
−42.592
1.00
37.43
6
C

ATOM
1979
CB
GLN
B
44
−24.009
−12.154
−41.498
1.00
37.56
6
C

ATOM
1980
CG
GLN
B
44
−23.236
−11.321
−40.507
1.00
39.18
6
C

ATOM
1981
CD
GLN
B
44
−22.846
−12.139
−39.295
1.00
40.77
6
C

ATOM
1982
OE1
GLN
B
44
−23.595
−13.024
−38.892
1.00
41.02
8
O

ATOM
1983
NE2
GLN
B
44
−21.674
−11.863
−38.726
1.00
41.75
7
N

ATOM
1984
C
GLN
B
44
−23.715
−10.889
−43.635
1.00
36.43
6
C

ATOM
1985
O
GLN
B
44
−23.247
−9.750
−43.585
1.00
35.59
8
O

ATOM
1986
N
ALA
B
45
−23.390
−11.762
−44.585
1.00
35.69
7
N

ATOM
1987
CA
ALA
B
45
−22.416
−11.419
−45.614
1.00
35.09
6
C

ATOM
1988
CB
ALA
B
45
−22.193
−12.599
−46.567
1.00
35.40
6
C

ATOM
1989
C
ALA
B
45
−22.774
−10.149
−46.395
1.00
34.46
6
C

ATOM
1990
O
ALA
B
45
−21.879
−9.416
−46.816
1.00
34.15
8
O

ATOM
1991
N
GLU
B
46
−24.068
−9.882
−46.575
1.00
33.79
7
N

ATOM
1992
CA
GLU
B
46
−24.499
−8.700
−47.329
1.00
33.73
6
C

ATOM
1993
CB
GLU
B
46
−25.999
−8.765
−47.662
1.00
33.80
6
C

ATOM
1994
CG
GLU
B
46
−26.421
−9.857
−48.641
1.00
35.36
6
C

ATOM
1995
CD
GLU
B
46
−25.928
−9.626
−50.060
1.00
37.74
6
C

ATOM
1996
OE1
GLU
B
46
−25.631
−8.468
−50.428
1.00
37.79
8
O

ATOM
1997
OE2
GLU
B
46
−25.832
−10.620
−50.814
1.00
39.71
8
O

ATOM
1998
C
GLU
B
46
−24.220
−7.389
−46.596
1.00
33.38
6
C

ATOM
1999
O
GLU
B
46
−24.357
−6.307
−47.177
1.00
32.62
8
O

ATOM
2000
N
ALA
B
47
−23.864
−7.481
−45.314
1.00
32.85
7
N

ATOM
2001
CA
ALA
B
47
−23.568
−6.290
−44.528
1.00
33.07
6
C

ATOM
2002
CB
ALA
B
47
−24.142
−6.413
−43.113
1.00
32.94
6
C

ATOM
2003
C
ALA
B
47
−22.073
−5.995
−44.471
1.00
33.26
6
C

ATOM
2004
O
ALA
B
47
−21.660
−4.966
−43.941
1.00
32.82
8
O

ATOM
2005
N
GLU
B
48
−21.265
−6.902
−45.007
1.00
33.94
7
N

ATOM
2006
CA
GLU
B
48
−19.821
−6.705
−45.013
1.00
35.23
6
C

ATOM
2007
CB
GLU
B
48
−19.107
−7.975
−45.482
1.00
35.50
6
C

ATOM
2008
CG
GLU
B
48
−19.178
−9.119
−44.489
1.00
37.08
6
C

ATOM
2009
CD
GLU
B
48
−18.470
−10.365
−44.981
1.00
39.75
6
C

ATOM
2010
OE1
GLU
B
48
−17.398
−10.228
−45.611
1.00
42.17
8
O

ATOM
2011
OE2
GLU
B
48
−18.981
−11.476
−44.734
1.00
39.89
8
O

ATOM
2012
C
GLU
B
48
−19.413
−5.532
−45.899
1.00
35.92
6
C

ATOM
2013
O
GLU
B
48
−19.998
−5.311
−46.953
1.00
35.83
8
O

ATOM
2014
N
ASP
B
49
−18.406
−4.783
−45.464
1.00
36.95
7
N

ATOM
2015
CA
ASP
B
49
−17.895
−3.667
−46.247
1.00
38.46
6
C

ATOM
2016
CB
ASP
B
49
−18.668
−2.378
−45.949
1.00
38.28
6
C

ATOM
2017
CG
ASP
B
49
−18.434
−1.306
−46.997
1.00
39.03
6
C

ATOM
2018
OD1
ASP
B
49
−17.482
−1.460
−47.788
1.00
39.15
8
O

ATOM
2019
OD2
ASP
B
49
−19.143
−0.282
−47.113
1.00
39.57
8
O

ATOM
2020
C
ASP
B
49
−16.403
−3.482
−45.977
1.00
39.53
6
C

ATOM
2021
O
ASP
B
49
−16.014
−2.811
−45.024
1.00
39.07
8
O

ATOM
2022
N
PRO
B
50
−15.581
−4.100
−46.818
1.00
41.19
7
N

ATOM
2023
CA
PRO
B
50
−14.114
−4.027
−46.710
1.00
42.47
6
C

ATOM
2024
CB
PRO
B
50
−13.637
−4.691
−48.005
1.00
42.40
6
C

ATOM
2025
CG
PRO
B
50
−14.750
−5.607
−48.395
1.00
42.26
6
C

ATOM
2026
CD
PRO
B
50
−16.017
−4.935
−47.951
1.00
41.29
6
C

ATOM
2027
C
PRO
B
50
−13.559
−2.605
−46.632
1.00
43.64
6
C

ATOM
2028
O
PRO
B
50
−12.559
−2.378
−45.949
1.00
44.29
8
O

ATOM
2029
N
ALA
B
51
−14.189
−1.663
−47.322
1.00
45.00
7
N

ATOM
2030
CA
ALA
B
51
−13.724
−0.278
−47.311
1.00
45.79
6
C

ATOM
2031
CB
ALA
B
51
−14.465
0.533
−48.344
1.00
46.07
6
C

ATOM
2032
C
ALA
B
51
−13.897
0.348
−45.939
1.00
46.52
6
C

ATOM
2033
O
ALA
B
51
−13.550
1.514
−45.726
1.00
46.62
8
O

ATOM
2034
N
CYS
B
52
−14.424
−0.442
−45.008
1.00
46.79
7
N

ATOM
2035
CA
CYS
B
52
−14.698
0.031
−43.662
1.00
47.44
6
C

ATOM
2036
CB
CYS
B
52
−16.079
−0.430
−43.223
1.00
47.69
6
C

ATOM
2037
SG
CYS
B
52
−17.315
0.800
−43.562
1.00
50.99
16
S

ATOM
2038
C
CYS
B
52
−13.702
−0.439
−42.633
1.00
46.96
6
C

ATOM
2039
O
CYS
B
52
−13.770
−0.026
−41.475
1.00
46.95
8
O

ATOM
2040
N
ILE
B
53
−12.809
−1.334
−43.034
1.00
46.55
7
N

ATOM
2041
CA
ILE
B
53
−11.809
−1.825
−42.107
1.00
46.54
6
C

ATOM
2042
CB
ILE
B
53
−10.772
−2.698
−42.833
1.00
46.69
6
C

ATOM
2043
CG1
ILE
B
53
−11.474
−3.857
−43.543
1.00
47.41
6
C

ATOM
2044
CD1
ILE
B
53
−10.540
−4.758
−44.349
1.00
47.75
6
C

ATOM
2045
CG2
ILE
B
53
−9.743
−3.236
−41.849
1.00
46.72
6
C

ATOM
2046
C
ILE
B
53
−11.170
−0.604
−41.462
1.00
46.15
6
C

ATOM
2047
O
ILE
B
53
−10.792
0.340
−42.151
1.00
46.05
8
O

ATOM
2048
N
PRO
B
54
−11.109
−0.600
−40.137
1.00
46.00
7
N

ATOM
2049
CA
PRO
B
54
−10.550
0.525
−39.384
1.00
45.97
6
C

ATOM
2050
CB
PRO
B
54
−10.600
0.025
−37.938
1.00
45.87
6
C

ATOM
2051
CG
PRO
B
54
−10.707
−1.466
−38.075
1.00
46.07
6
C

ATOM
2052
CD
PRO
B
54
−11.613
−1.657
−39.244
1.00
45.94
6
C

ATOM
2053
C
PRO
B
54
−9.114
0.854
−39.779
1.00
46.00
6
C

ATOM
2054
O
PRO
B
54
−8.362
−0.018
−40.220
1.00
46.11
8
O

ATOM
2055
N
ILE
B
55
−8.747
2.120
−39.637
1.00
45.93
7
N

ATOM
2056
CA
ILE
B
55
−7.383
2.536
−39.926
1.00
46.00
6
C

ATOM
2057
CB
ILE
B
55
−7.316
4.056
−40.160
1.00
46.03
6
C

ATOM
2058
CG1
ILE
B
55
−8.341
4.480
−41.213
1.00
46.99
6
C

ATOM
2059
CD1
ILE
B
55
−8.567
5.979
−41.276
1.00
47.02
6
C

ATOM
2060
CG2
ILE
B
55
−5.921
4.469
−40.603
1.00
46.63
6
C

ATOM
2061
C
ILE
B
55
−6.509
2.133
−38.742
1.00
45.49
6
C

ATOM
2062
O
ILE
B
55
−5.387
1.655
−38.921
1.00
45.69
8
O

ATOM
2063
N
PHE
B
56
−7.048
2.294
−37.536
1.00
44.71
7
N

ATOM
2064
CA
PHE
B
56
−6.321
1.969
−36.312
1.00
44.06
6
C

ATOM
2065
CB
PHE
B
56
−5.940
3.250
−35.561
1.00
44.46
6
C

ATOM
2066
CG
PHE
B
56
−5.109
4.209
−36.357
1.00
45.29
6
C

ATOM
2067
CD1
PHE
B
56
−5.662
5.375
−36.854
1.00
46.35
6
C

ATOM
2068
CE1
PHE
B
56
−4.893
6.274
−37.576
1.00
46.75
6
C

ATOM
2069
CZ
PHE
B
56
−3.557
6.008
−37.807
1.00
46.64
6
C

ATOM
2070
CE2
PHE
B
56
−2.993
4.850
−37.311
1.00
46.33
6
C

ATOM
2071
CD2
PHE
B
56
−3.766
3.958
−36.589
1.00
46.39
6
C

ATOM
2072
C
PHE
B
56
−7.125
1.112
−35.338
1.00
43.15
6
C

ATOM
2073
O
PHE
B
56
−8.349
1.247
−35.242
1.00
42.86
8
O

ATOM
2074
N
TRP
B
57
−6.422
0.240
−34.617
1.00
41.94
7
N

ATOM
2075
CA
TRP
B
57
−6.995
−0.506
−33.496
1.00
41.32
6
C

ATOM
2076
CB
TRP
B
57
−7.742
−1.778
−33.932
1.00
40.91
6
C

ATOM
2077
CG
TRP
B
57
−6.895
−2.795
−34.636
1.00
39.37
6
C

ATOM
2078
CD1
TRP
B
57
−6.168
−3.799
−34.069
1.00
39.14
6
C

ATOM
2079
NE1
TRP
B
57
−5.524
−4.528
−35.042
1.00
38.59
7
N

ATOM
2080
CE2
TRP
B
57
−5.840
−4.002
−36.268
1.00
39.25
6
C

ATOM
2081
CD2
TRP
B
57
−6.705
−2.912
−36.046
1.00
38.79
6
C

ATOM
2082
CE3
TRP
B
57
−7.175
−2.197
−37.153
1.00
38.60
6
C

ATOM
2083
CZ3
TRP
B
57
−6.779
−2.588
−38.420
1.00
39.95
6
C

ATOM
2084
CH2
TRP
B
57
−5.912
−3.673
−38.603
1.00
39.37
6
C

ATOM
2085
CZ2
TRP
B
57
−5.435
−4.390
−37.543
1.00
39.00
6
C

ATOM
2086
C
TRP
B
57
−5.890
−0.836
−32.493
1.00
41.24
6
C

ATOM
2087
O
TRP
B
57
−4.708
−0.772
−32.824
1.00
41.34
8
O

ATOM
2088
N
VAL
B
58
−6.277
−1.167
−31.267
1.00
40.90
7
N

ATOM
2089
CA
VAL
B
58
−5.322
−1.524
−30.229
1.00
40.95
6
C

ATOM
2090
CB
VAL
B
58
−5.938
−1.346
−28.827
1.00
40.92
6
C

ATOM
2091
CG1
VAL
B
58
−4.980
−1.810
−27.745
1.00
41.17
6
C

ATOM
2092
CG2
VAL
B
58
−6.335
0.112
−28.606
1.00
41.00
6
C

ATOM
2093
C
VAL
B
58
−4.852
−2.967
−30.424
1.00
40.88
6
C

ATOM
2094
O
VAL
B
58
−5.644
−3.906
−30.328
1.00
40.63
8
O

ATOM
2095
N
SER
B
59
−3.562
−3.137
−30.710
1.00
40.71
7
N

ATOM
2096
CA
SER
B
59
−3.004
−4.469
−30.963
1.00
40.82
6
C

ATOM
2097
CB
SER
B
59
−1.917
−4.400
−32.039
1.00
41.06
6
C

ATOM
2098
OG
SER
B
59
−1.090
−3.266
−31.856
1.00
41.44
8
O

ATOM
2099
C
SER
B
59
−2.473
−5.163
−29.708
1.00
40.52
6
C

ATOM
2100
O
SER
B
59
−2.444
−6.395
−29.631
1.00
40.41
8
O

ATOM
2101
N
LYS
B
60
−2.046
−4.362
−28.738
1.00
39.92
7
N

ATOM
2102
CA
LYS
B
60
−1.557
−4.854
−27.459
1.00
39.51
6
C

ATOM
2103
CB
LYS
B
60
−0.055
−5.160
−27.518
1.00
39.60
6
C

ATOM
2104
CG
LYS
B
60
0.448
−5.788
−28.809
1.00
40.15
6
C

ATOM
2105
CD
LYS
B
60
1.919
−6.195
−28.675
1.00
41.00
6
C

ATOM
2106
CE
LYS
B
60
2.458
−6.765
−29.982
1.00
41.88
6
C

ATOM
2107
NZ
LYS
B
60
3.869
−7.271
−29.822
1.00
42.94
7
N

ATOM
2108
C
LYS
B
60
−1.776
−3.763
−26.414
1.00
39.09
6
C

ATOM
2109
O
LYS
B
60
−1.808
−2.578
−26.745
1.00
38.83
8
O

ATOM

2110

N

TRP

B

61

−1.929

−4.167

−25.161

1.00

38.91

7

N

ATOM

2111

CA

TRP

B

61

−2.053

−3.211

−24.063

1.00

39.28

6

C

ATOM

2112

CB

TRP

B

61

−3.510

−2.759

−23.878

1.00

38.78

6

C

ATOM

2113

CG

TRP

B

61

−4.472

−3.888

−23.641

1.00

37.70

6

C

ATOM

2114

CD1

TRP

B

61

−5.204

−4.554

−24.586

1.00

35.95

6

C

ATOM

2115

NE1

TRP

B

61

−5.973

−5.524

−23.992

1.00

36.01

7

N

ATOM

2116

CE2

TRP

B

61

−5.754

−5.499

−22.641

1.00

36.60

6

C

ATOM

2117

CD2

TRP

B

61

−4.814

−4.479

−22.385

1.00

36.92

6

C

ATOM

2118

CE3

TRP

B

61

−4.426

−4.248

−21.060

1.00

38.31

6

C

ATOM

2119

CZ3

TRP

B

61

−4.966

−5.036

−20.061

1.00

37.79

6

C

ATOM

2120

CH2

TRP

B

61

−5.893

−6.038

−20.351

1.00

38.23

6

C

ATOM

2121

CZ2

TRP

B

61

−6.298

−6.286

−21.633

1.00

36.55

6

C

ATOM

2122

C

TRP

B

61

−1.501

−3.776

−22.758

1.00

39.88

6

C

ATOM

2123

O

TRP

B

61

−1.449

−4.993

−22.566

1.00

40.11

8

O

ATOM
2124
N
VAL
B
62
−1.084
−2.877
−21.871
1.00
40.59
7
N

ATOM
2125
CA
VAL
B
62
−0.560
−3.245
−20.563
1.00
41.45
6
C

ATOM
2126
CB
VAL
B
62
0.977
−3.140
−20.510
1.00
41.29
6
C

ATOM
2127
CG1
VAL
B
62
1.480
−3.465
−19.115
1.00
41.94
6
C

ATOM
2128
CG2
VAL
B
62
1.615
−4.067
−21.519
1.00
41.97
6
C

ATOM
2129
C
VAL
B
62
−1.156
−2.297
−19.524
1.00
41.74
6
C

ATOM
2130
O
VAL
B
62
−0.977
−1.081
−19.612
1.00
41.59
8
O

ATOM
2131
N
ASP
B
63
−1.863
−2.861
−18.551
1.00
42.53
7
N

ATOM
2132
CA
ASP
B
63
−2.522
−2.083
−17.512
1.00
43.68
6
C

ATOM
2133
CB
ASP
B
63
−3.831
−2.755
−17.093
1.00
43.55
6
C

ATOM
2134
CG
ASP
B
63
−4.525
−2.030
−15.956
1.00
43.95
6
C

ATOM
2135
OD1
ASP
B
63
−4.019
−0.969
−15.526
1.00
43.59
8
O

ATOM
2136
OD2
ASP
B
63
−5.576
−2.450
−15.421
1.00
43.71
8
O

ATOM
2137
C
ASP
B
63
−1.632
−1.893
−16.287
1.00
44.67
6
C

ATOM
2138
O
ASP
B
63
−1.605
−2.734
−15.387
1.00
44.34
8
O

ATOM
2139
N
TYR
B
64
−0.905
−0.784
−16.267
1.00
46.03
7
N

ATOM
2140
CA
TYR
B
64
−0.073
−0.445
−15.120
1.00
47.40
6
C

ATOM
2141
CB
TYR
B
64
1.380
−0.222
−15.540
1.00
47.82
6
C

ATOM
2142
CG
TYR
B
64
2.167
−1.506
−15.659
1.00
50.03
6
C

ATOM
2143
CD1
TYR
B
64
3.478
−1.504
−16.112
1.00
51.96
6
C

ATOM
2144
CE1
TYR
B
64
4.197
−2.688
−16.220
1.00
53.33
6
C

ATOM
2145
CZ
TYR
B
64
3.602
−3.884
−15.867
1.00
53.34
6
C

ATOM
2146
OH
TYR
B
64
4.307
−5.062
−15.968
1.00
54.77
8
O

ATOM
2147
CE2
TYR
B
64
2.305
−3.907
−15.412
1.00
52.75
6
C

ATOM
2148
CD2
TYR
B
64
1.596
−2.725
−15.311
1.00
51.72
6
C

ATOM
2149
C
TYR
B
64
−0.642
0.802
−14.462
1.00
47.57
6
C

ATOM
2150
O
TYR
B
64
0.101
1.633
−13.947
1.00
47.39
8
O

ATOM
2151
N
SER
B
65
−1.968
0.923
−14.501
1.00
47.80
7
N

ATOM
2152
CA
SER
B
65
−2.670
2.066
−13.918
1.00
48.23
6
C

ATOM
2153
CB
SER
B
65
−4.162
2.023
−14.277
1.00
48.08
6
C

ATOM
2154
OG
SER
B
65
−4.788
0.876
−13.726
1.00
47.43
8
O

ATOM
2155
C
SER
B
65
−2.492
2.106
−12.404
1.00
48.73
6
C

ATOM
2156
O
SER
B
65
−2.846
3.088
−11.749
1.00
48.86
8
O

ATOM
2157
N
ASP
B
66
−1.946
1.021
−11.864
1.00
49.45
7
N

ATOM
2158
CA
ASP
B
66
−1.649
0.888
−10.442
1.00
50.03
6
C

ATOM
2159
CB
ASP
B
66
−0.936
−0.444
−10.197
1.00
50.24
6
C

ATOM
2160
CG
ASP
B
66
−1.298
−1.071
−8.871
1.00
51.55
6
C

ATOM
2161
OD1
ASP
B
66
−1.291
−0.358
−7.843
1.00
53.29
8
O

ATOM
2162
OD2
ASP
B
66
−1.598
−2.281
−8.761
1.00
53.05
8
O

ATOM
2163
C
ASP
B
66
−0.740
2.020
−9.980
1.00
49.99
6
C

ATOM
2164
O
ASP
B
66
−0.898
2.552
−8.879
1.00
50.31
8
O

ATOM
2165
N
LYS
B
67
0.217
2.388
−10.824
1.00
49.83
7
N

ATOM
2166
CA
LYS
B
67
1.187
3.408
−10.445
1.00
49.88
6
C

ATOM
2167
CB
LYS
B
67
2.486
2.744
−9.968
1.00
50.16
6
C

ATOM
2168
CG
LYS
B
67
2.292
1.606
−8.970
1.00
50.67
6
C

ATOM
2169
CD
LYS
B
67
3.628
1.115
−8.427
1.00
52.01
6
C

ATOM
2170
CE
LYS
B
67
3.426
−0.029
−7.441
1.00
52.48
6
C

ATOM
2171
NZ
LYS
B
67
4.654
−0.315
−6.657
1.00
52.33
7
N

ATOM
2172
C
LYS
B
67
1.521
4.402
−11.555
1.00
49.63
6
C

ATOM
2173
O
LYS
B
67
1.918
5.534
−11.273
1.00
49.74
8
O

ATOM
2174
N
TYR
B
68
1.367
3.988
−12.811
1.00
49.08
7
N

ATOM
2175
CA
TYR
B
68
1.777
4.841
−13.924
1.00
48.69
6
C

ATOM
2176
CB
TYR
B
68
2.941
4.188
−14.664
1.00
48.97
6
C

ATOM
2177
CG
TYR
B
68
4.094
3.857
−13.750
1.00
50.19
6
C

ATOM
2178
CD1
TYR
B
68
4.546
2.553
−13.608
1.00
51.24
6
C

ATOM
2179
CE1
TYR
B
68
5.603
2.252
−12.761
1.00
52.27
6
C

ATOM
2180
CZ
TYR
B
68
6.207
3.265
−12.043
1.00
52.23
6
C

ATOM
2181
OH
TYR
B
68
7.254
2.982
−11.197
1.00
53.59
8
O

ATOM
2182
CE2
TYR
B
68
5.771
4.563
−12.167
1.00
51.84
6
C

ATOM
2183
CD2
TYR
B
68
4.719
4.852
−13.013
1.00
51.41
6
C

ATOM
2184
C
TYR
B
68
0.673
5.200
−14.911
1.00
48.02
6
C

ATOM
2185
O
TYR
B
68
0.413
6.376
−15.160
1.00
48.00
8
O

ATOM
2186
N
GLY
B
69
0.050
4.178
−15.490
1.00
47.28
7
N

ATOM
2187
CA
GLY
B
69
−0.998
4.379
−16.475
1.00
46.31
6
C

ATOM
2188
C
GLY
B
69
−1.173
3.145
−17.341
1.00
45.70
6
C

ATOM
2189
O
GLY
B
69
−0.731
2.056
−16.975
1.00
45.54
8
O

ATOM
2190
N
LEU
B
70
−1.823
3.308
−18.487
1.00
44.96
7
N

ATOM
2191
CA
LEU
B
70
−2.035
2.182
−19.389
1.00
44.33
6
C

ATOM
2192
CB
LEU
B
70
−3.526
1.973
−19.674
1.00
44.42
6
C

ATOM
2193
CG
LEU
B
70
−3.858
0.689
−20.448
1.00
44.65
6
C

ATOM
2194
CD1
LEU
B
70
−5.027
−0.062
−19.821
1.00
44.25
6
C

ATOM
2195
CD2
LEU
B
70
−4.105
0.990
−21.921
1.00
44.67
6
C

ATOM
2196
C
LEU
B
70
−1.257
2.377
−20.679
1.00
43.72
6
C

ATOM
2197
O
LEU
B
70
−1.379
3.405
−21.336
1.00
43.83
8
O

ATOM
2198
N
GLY
B
71
−0.443
1.387
−21.022
1.00
43.34
7
N

ATOM
2199
CA
GLY
B
71
0.350
1.426
−22.232
1.00
42.55
6
C

ATOM
2200
C
GLY
B
71
−0.299
0.531
−23.260
1.00
42.19
6
C

ATOM
2201
O
GLY
B
71
−0.919
−0.468
−22.919
1.00
41.85
8
O

ATOM
2202
N
TYR
B
72
−0.142
0.878
−24.526
1.00
42.15
7
N

ATOM
2203
CA
TYR
B
72
−0.806
0.131
−25.574
1.00
42.11
6
C

ATOM
2204
CB
TYR
B
72
−2.250
0.641
−25.732
1.00
41.72
6
C

ATOM
2205
CG
TYR
B
72
−2.334
2.099
−26.140
1.00
40.71
6
C

ATOM
2206
CD1
TYR
B
72
−2.322
2.467
−27.481
1.00
39.92
6
C

ATOM
2207
CE1
TYR
B
72
−2.383
3.792
−27.860
1.00
38.64
6
C

ATOM
2208
CZ
TYR
B
72
−2.457
4.776
−26.895
1.00
37.83
6
C

ATOM
2209
OH
TYR
B
72
−2.527
6.092
−27.283
1.00
37.64
8
O

ATOM
2210
CE2
TYR
B
72
−2.473
4.444
−25.562
1.00
37.39
6
C

ATOM
2211
CD2
TYR
B
72
−2.409
3.112
−25.188
1.00
40.00
6
C

ATOM
2212
C
TYR
B
72
−0.069
0.321
−26.877
1.00
42.59
6
C

ATOM
2213
O
TYR
B
72
0.690
1.276
−27.039
1.00
42.25
8
O

ATOM
2214
N
GLN
B
73
−0.296
−0.598
−27.804
1.00
43.00
7
N

ATOM
2215
CA
GLN
B
73
0.271
−0.479
−29.130
1.00
43.94
6
C

ATOM
2216
CB
GLN
B
73
1.159
−1.686
−29.457
1.00
43.99
6
C

ATOM
2217
CG
GLN
B
73
1.751
−1.635
−30.868
1.00
44.38
6
C

ATOM
2218
CD
GLN
B
73
2.136
−3.000
−31.409
1.00
45.38
6
C

ATOM
2219
OE1
GLN
B
73
1.291
−3.892
−31.519
1.00
45.59
8
O

ATOM
2220
NE2
GLN
B
73
3.407
−3.162
−31.764
1.00
44.82
7
N

ATOM
2221
C
GLN
B
73
−0.870
−0.395
−30.130
1.00
44.38
6
C

ATOM
2222
O
GLN
B
73
−1.900
−1.046
−29.961
1.00
44.15
8
O

ATOM
2223
N
LEU
B
74
−0.700
0.435
−31.151
1.00
45.26
7
N

ATOM
2224
CA
LEU
B
74
−1.666
0.500
−32.233
1.00
46.12
6
C

ATOM
2225
CB
LEU
B
74
−1.819
1.928
−32.749
1.00
46.00
6
C

ATOM
2226
CG
LEU
B
74
−2.463
2.946
−31.806
1.00
45.43
6
C

ATOM
2227
CD1
LEU
B
74
−2.767
4.221
−32.563
1.00
45.01
6
C

ATOM
2228
CD2
LEU
B
74
−3.730
2.382
−31.184
1.00
45.18
6
C

ATOM
2229
C
LEU
B
74
−1.137
−0.407
−33.335
1.00
47.07
6
C

ATOM
2230
O
LEU
B
74
0.076
−0.536
−33.502
1.00
47.34
8
O

ATOM
2231
N
CYS
B
75
−2.036
−1.048
−34.074
1.00
47.91
7
N

ATOM
2232
CA
CYS
B
75
−1.637
−1.956
−35.149
1.00
48.87
6
C

ATOM
2233
CB
CYS
B
75
−2.858
−2.361
−35.967
1.00
48.61
6
C

ATOM
2234
SG
CYS
B
75
−3.706
−0.962
−36.722
1.00
49.27
16
S

ATOM
2235
C
CYS
B
75
−0.617
−1.289
−36.066
1.00
49.39
6
C

ATOM
2236
O
CYS
B
75
−0.059
−1.916
−36.966
1.00
49.37
8
O

ATOM
2237
N
ASP
B
76
−0.396
−0.003
−35.820
1.00
50.09
7
N

ATOM
2238
CA
ASP
B
76
0.507
0.828
−36.602
1.00
50.55
6
C

ATOM
2239
CB
ASP
B
76
0.195
2.297
−36.309
1.00
50.73
6
C

ATOM
2240
CG
ASP
B
76
0.719
3.229
−37.378
1.00
51.81
6
C

ATOM
2241
OD1
ASP
B
76
0.891
2.775
−38.531
1.00
53.10
8
O

ATOM
2242
OD2
ASP
B
76
0.976
4.430
−37.163
1.00
51.77
8
O

ATOM
2243
C
ASP
B
76
1.954
0.560
−36.233
1.00
50.56
6
C

ATOM
2244
O
ASP
B
76
2.876
0.983
−36.936
1.00
50.71
8
O

ATOM
2245
N
ASN
B
77
2.147
−0.147
−35.124
1.00
50.40
7
N

ATOM
2246
CA
ASN
B
77
3.473
−0.393
−34.582
1.00
50.27
6
C

ATOM
2247
CB
ASN
B
77
4.505
−0.595
−35.690
1.00
50.38
6
C

ATOM
2248
CG
ASN
B
77
4.327
−1.907
−36.410
1.00
51.17
6
C

ATOM
2249
OD1
ASN
B
77
4.226
−2.962
−35.783
1.00
52.10
8
O

ATOM
2250
ND2
ASN
B
77
4.283
−1.854
−37.736
1.00
51.93
7
N

ATOM
2251
C
ASN
B
77
3.849
0.784
−33.701
1.00
49.87
6
C

ATOM
2252
O
ASN
B
77
4.838
0.746
−32.966
1.00
49.97
8
O

ATOM
2253
N
SER
B
78
3.051
1.843
−33.795
1.00
49.30
7
N

ATOM
2254
CA
SER
B
78
3.225
3.000
−32.939
1.00
48.55
6
C

ATOM
2255
CB
SER
B
78
2.419
4.187
−33.464
1.00
48.82
6
C

ATOM
2256
OG
SER
B
78
1.025
3.928
−33.415
1.00
49.14
8
O

ATOM
2257
C
SER
B
78
2.729
2.588
−31.564
1.00
47.91
6
C

ATOM
2258
O
SER
B
78
1.936
1.657
−31.445
1.00
47.73
8
O

ATOM
2259
N
VAL
B
79
3.205
3.264
−30.526
1.00
47.11
7
N

ATOM
2260
CA
VAL
B
79
2.797
2.940
−29.165
1.00
46.32
6
C

ATOM
2261
CB
VAL
B
79
3.923
2.246
−28.365
1.00
46.43
6
C

ATOM
2262
CG1
VAL
B
79
4.309
0.926
−29.008
1.00
46.40
6
C

ATOM
2263
CG2
VAL
B
79
5.137
3.155
−28.243
1.00
46.59
6
C

ATOM
2264
C
VAL
B
79
2.361
4.197
−28.440
1.00
45.82
6
C

ATOM
2265
O
VAL
B
79
2.638
5.315
−28.885
1.00
45.91
8
O

ATOM
2266
N
GLY
B
80
1.674
4.017
−27.323
1.00
45.07
7
N

ATOM
2267
CA
GLY
B
80
1.177
5.149
−26.574
1.00
44.81
6
C

ATOM
2268
C
GLY
B
80
0.838
4.787
−25.148
1.00
44.62
6
C

ATOM
2269
O
GLY
B
80
0.876
3.620
−24.755
1.00
44.13
8
O

ATOM
2270
N
VAL
B
81
0.491
5.802
−24.370
1.00
44.84
7
N

ATOM
2271
CA
VAL
B
81
0.161
5.594
−22.976
1.00
45.14
6
C

ATOM
2272
CB
VAL
B
81
1.441
5.569
−22.116
1.00
45.19
6
C

ATOM
2273
CG1
VAL
B
81
2.311
6.762
−22.452
1.00
45.41
6
C

ATOM
2274
CG2
VAL
B
81
1.112
5.535
−20.626
1.00
45.15
6
C

ATOM
2275
C
VAL
B
81
−0.759
6.695
−22.480
1.00
45.31
6
C

ATOM
2276
O
VAL
B
81
−0.682
7.842
−22.926
1.00
45.36
8
O

ATOM
2277
N
LEU
B
82
−1.653
6.327
−21.573
1.00
45.75
7
N

ATOM
2278
CA
LEU
B
82
−2.521
7.290
−20.919
1.00
46.32
6
C

ATOM
2279
CB
LEU
B
82
−3.994
6.893
−21.052
1.00
46.22
6
C

ATOM
2280
CG
LEU
B
82
−4.986
7.694
−20.203
1.00
46.72
6
C

ATOM
2281
CD1
LEU
B
82
−4.728
9.186
−20.324
1.00
46.40
6
C

ATOM
2282
CD2
LEU
B
82
−6.428
7.359
−20.587
1.00
47.47
6
C

ATOM
2283
C
LEU
B
82
−2.087
7.267
−19.469
1.00
46.70
6
C

ATOM
2284
O
LEU
B
82
−2.391
6.323
−18.737
1.00
46.54
8
O

ATOM
2285
N
PHE
B
83
−1.338
8.290
−19.071
1.00
47.21
7
N

ATOM
2286
CA
PHE
B
83
−0.816
8.379
−17.713
1.00
48.06
6
C

ATOM
2287
CB
PHE
B
83
0.311
9.411
−17.644
1.00
47.70
6
C

ATOM
2288
CG
PHE
B
83
1.547
9.006
−18.400
1.00
47.11
6
C

ATOM
2289
CD1
PHE
B
83
1.882
9.627
−19.590
1.00
46.02
6
C

ATOM
2290
CE1
PHE
B
83
3.021
9.251
−20.284
1.00
45.84
6
C

ATOM
2291
CZ
PHE
B
83
3.835
8.251
−19.788
1.00
45.40
6
C

ATOM
2292
CE2
PHE
B
83
3.511
7.626
−18.603
1.00
45.30
6
C

ATOM
2293
CD2
PHE
B
83
2.375
8.002
−17.915
1.00
46.06
6
C

ATOM
2294
C
PHE
B
83
−1.907
8.711
−16.704
1.00
48.88
6
C

ATOM
2295
O
PHE
B
83
−2.950
9.256
−17.065
1.00
48.97
8
O

ATOM
2296
N
ASN
B
84
−1.650
8.386
−15.438
1.00
49.94
7
N

ATOM
2297
CA
ASN
B
84
−2.614
8.594
−14.361
1.00
50.92
6
C

ATOM
2298
CB
ASN
B
84
−2.119
7.949
−13.068
1.00
50.84
6
C

ATOM
2299
CG
ASN
B
84
−2.358
6.457
−13.036
1.00
51.37
6
C

ATOM
2300
OD1
ASN
B
84
−2.907
5.882
−13.979
1.00
51.80
8
O

ATOM
2301
ND2
ASN
B
84
−1.947
5.816
−11.946
1.00
51.25
7
N

ATOM
2302
C
ASN
B
84
−3.010
10.044
−14.095
1.00
51.66
6
C

ATOM
2303
O
ASN
B
84
−3.951
10.302
−13.348
1.00
51.79
8
O

ATOM
2304
N
ASN
B
85
−2.290
10.990
−14.688
1.00
52.45
7
N

ATOM
2305
CA
ASN
B
85
−2.637
12.397
−14.524
1.00
53.40
6
C

ATOM
2306
CB
ASN
B
85
−1.383
13.253
−14.343
1.00
53.43
6
C

ATOM
2307
CG
ASN
B
85
−0.268
12.849
−15.281
1.00
54.35
6
C

ATOM
2308
OD1
ASN
B
85
−0.458
12.781
−16.495
1.00
54.69
8
O

ATOM
2309
ND2
ASN
B
85
0.905
12.563
−14.721
1.00
55.14
7
N

ATOM
2310
C
ASN
B
85
−3.463
12.901
−15.701
1.00
53.75
6
C

ATOM
2311
O
ASN
B
85
−3.652
14.105
−15.872
1.00
53.84
8
O

ATOM
2312
N
SER
B
86
−3.938
11.964
−16.517
1.00
54.18
7
N

ATOM
2313
CA
SER
B
86
−4.770
12.285
−17.672
1.00
54.59
6
C

ATOM
2314
CB
SER
B
86
−5.810
13.348
−17.308
1.00
54.75
6
C

ATOM
2315
OG
SER
B
86
−6.539
12.975
−16.148
1.00
55.35
8
O

ATOM
2316
C
SER
B
86
−3.972
12.713
−18.911
1.00
54.75
6
C

ATOM
2317
O
SER
B
86
−4.550
12.927
−19.977
1.00
54.87
8
O

ATOM
2318
N
THR
B
87
−2.654
12.846
−18.782
1.00
54.78
7
N

ATOM
2319
CA
THR
B
87
−1.838
13.224
−19.935
1.00
54.86
6
C

ATOM
2320
CB
THR
B
87
−0.513
13.884
−19.510
1.00
54.79
6
C

ATOM
2321
OG1
THR
B
87
0.303
12.929
−18.821
1.00
54.67
8
O

ATOM
2322
CG2
THR
B
87
−0.761
14.972
−18.477
1.00
55.12
6
C

ATOM
2323
C
THR
B
87
−1.548
12.008
−20.803
1.00
54.96
6
C

ATOM
2324
O
THR
B
87
−1.619
10.873
−20.338
1.00
54.54
8
O

ATOM
2325
N
ARG
B
88
−1.210
12.257
−22.063
1.00
55.37
7
N

ATOM
2326
CA
ARG
B
88
−0.937
11.181
−23.003
1.00
55.98
6
C

ATOM
2327
CB
ARG
B
88
−2.129
10.996
−23.944
1.00
56.11
6
C

ATOM
2328
CG
ARG
B
88
−3.465
11.001
−23.216
1.00
56.90
6
C

ATOM
2329
CD
ARG
B
88
−4.642
11.404
−24.076
1.00
58.32
6
C

ATOM
2330
NE
ARG
B
88
−5.564
12.295
−23.375
1.00
59.85
7
N

ATOM
2331
CZ
ARG
B
88
−6.502
11.895
−22.528
1.00
60.67
6
C

ATOM
2332
NH1
ARG
B
88
−6.657
10.607
−22.259
1.00
61.31
7
N

ATOM
2333
NH2
ARG
B
88
−7.288
12.785
−21.944
1.00
61.21
7
N

ATOM
2334
C
ARG
B
88
0.338
11.438
−23.798
1.00
56.15
6
C

ATOM
2335
O
ARG
B
88
0.687
12.585
−24.083
1.00
56.09
8
O

ATOM
2336
N
LEU
B
89
1.030
10.359
−24.146
1.00
56.45
7
N

ATOM
2337
CA
LEU
B
89
2.266
10.442
−24.910
1.00
56.73
6
C

ATOM
2338
CB
LEU
B
89
3.468
10.266
−23.986
1.00
56.69
6
C

ATOM
2339
CG
LEU
B
89
4.845
10.423
−24.630
1.00
56.69
6
C

ATOM
2340
CD1
LEU
B
89
4.954
11.755
−25.358
1.00
56.62
6
C

ATOM
2341
CD2
LEU
B
89
5.934
10.287
−23.578
1.00
56.68
6
C

ATOM
2342
C
LEU
B
89
2.275
9.372
−25.996
1.00
56.98
6
C

ATOM
2343
O
LEU
B
89
2.106
8.188
−25.711
1.00
56.74
8
O

ATOM
2344
N
ILE
B
90
2.468
9.798
−27.240
1.00
57.42
7
N

ATOM
2345
CA
ILE
B
90
2.462
8.887
−28.380
1.00
58.03
6
C

ATOM
2346
CB
ILE
B
90
1.402
9.332
−29.412
1.00
57.99
6
C

ATOM
2347
CG1
ILE
B
90
0.034
8.739
−29.072
1.00
58.00
6
C

ATOM
2348
CD1
ILE
B
90
−0.523
9.185
−27.745
1.00
58.27
6
C

ATOM
2349
CG2
ILE
B
90
1.803
8.899
−30.809
1.00
57.79
6
C

ATOM
2350
C
ILE
B
90
3.825
8.778
−29.064
1.00
58.55
6
C

ATOM
2351
O
ILE
B
90
4.454
9.788
−29.377
1.00
58.58
8
O

ATOM
2352
N
LEU
B
91
4.268
7.546
−29.298
1.00
59.14
7
N

ATOM
2353
CA
LEU
B
91
5.530
7.296
−29.985
1.00
59.96
6
C

ATOM
2354
CB
LEU
B
91
6.424
6.379
−29.146
1.00
59.95
6
C

ATOM
2355
CG
LEU
B
91
7.784
5.982
−29.726
1.00
60.22
6
C

ATOM
2356
CD1
LEU
B
91
8.661
7.203
−29.972
1.00
60.08
6
C

ATOM
2357
CD2
LEU
B
91
8.488
4.989
−28.812
1.00
60.59
6
C

ATOM
2358
C
LEU
B
91
5.280
6.681
−31.363
1.00
60.42
6
C

ATOM
2359
O
LEU
B
91
4.936
5.505
−31.468
1.00
60.57
8
O

ATOM
2360
N
TYR
B
92
5.461
7.483
−32.411
1.00
61.14
7
N

ATOM
2361
CA
TYR
B
92
5.237
7.050
−33.797
1.00
61.87
6
C

ATOM
2362
CB
TYR
B
92
5.565
8.187
−34.769
1.00
61.89
6
C

ATOM
2363
CG
TYR
B
92
4.556
9.313
−34.755
1.00
62.35
6
C

ATOM
2364
CD1
TYR
B
92
4.621
10.320
−33.799
1.00
62.74
6
C

ATOM
2365
CE1
TYR
B
92
3.698
11.351
−33.781
1.00
63.08
6
C

ATOM
2366
CZ
TYR
B
92
2.694
11.382
−34.728
1.00
63.28
6
C

ATOM
2367
OH
TYR
B
92
1.772
12.407
−34.716
1.00
63.24
8
O

ATOM
2368
CE2
TYR
B
92
2.611
10.393
−35.689
1.00
62.82
6
C

ATOM
2369
CD2
TYR
B
92
3.535
9.368
−35.696
1.00
62.47
6
C

ATOM
2370
C
TYR
B
92
5.998
5.780
−34.198
1.00
62.32
6
C

ATOM
2371
O
TYR
B
92
6.924
5.354
−33.507
1.00
62.29
8
O

ATOM
2372
N
ASN
B
93
5.609
5.185
−35.325
1.00
62.98
7
N

ATOM
2373
CA
ASN
B
93
6.243
3.949
−35.790
1.00
63.67
6
C

ATOM
2374
CB
ASN
B
93
5.471
3.297
−36.952
1.00
63.71
6
C

ATOM
2375
CG
ASN
B
93
5.126
4.275
−38.067
1.00
63.98
6
C

ATOM
2376
OD1
ASN
B
93
5.878
5.205
−38.360
1.00
64.09
8
O

ATOM
2377
ND2
ASN
B
93
3.982
4.054
−38.705
1.00
64.30
7
N

ATOM
2378
C
ASN
B
93
7.736
4.084
−36.106
1.00
64.13
6
C

ATOM
2379
O
ASN
B
93
8.316
3.243
−36.790
1.00
64.16
8
O

ATOM
2380
N
ASP
B
94
8.342
5.156
−35.604
1.00
64.70
7
N

ATOM
2381
CA
ASP
B
94
9.783
5.361
−35.701
1.00
65.19
6
C

ATOM
2382
CB
ASP
B
94
10.155
6.394
−36.779
1.00
65.18
6
C

ATOM
2383
CG
ASP
B
94
9.643
7.798
−36.473
1.00
65.18
6
C

ATOM
2384
OD1
ASP
B
94
9.338
8.103
−35.304
1.00
65.04
8
O

ATOM
2385
OD2
ASP
B
94
9.524
8.677
−37.352
1.00
65.48
8
O

ATOM
2386
C
ASP
B
94
10.287
5.771
−34.321
1.00
65.50
6
C

ATOM
2387
O
ASP
B
94
10.165
6.927
−33.925
1.00
65.67
8
O

ATOM
2388
N
GLY
B
95
10.829
4.809
−33.581
1.00
65.83
7
N

ATOM
2389
CA
GLY
B
95
11.301
5.041
−32.226
1.00
66.28
6
C

ATOM
2390
C
GLY
B
95
11.863
6.420
−31.919
1.00
66.59
6
C

ATOM
2391
O
GLY
B
95
12.755
6.552
−31.080
1.00
66.62
8
O

ATOM
2392
N
ASP
B
96
11.338
7.450
−32.578
1.00
66.88
7
N

ATOM
2393
CA
ASP
B
96
11.809
8.813
−32.356
1.00
67.18
6
C

ATOM
2394
CB
ASP
B
96
12.768
9.236
−33.474
1.00
67.27
6
C

ATOM
2395
CG
ASP
B
96
13.823
10.219
−32.997
1.00
67.45
6
C

ATOM
2396
OD1
ASP
B
96
13.594
10.896
−31.971
1.00
67.40
8
O

ATOM
2397
OD2
ASP
B
96
14.916
10.377
−33.581
1.00
68.03
8
O

ATOM
2398
C
ASP
B
96
10.672
9.835
−32.208
1.00
67.32
6
C

ATOM
2399
O
ASP
B
96
10.487
10.406
−31.134
1.00
67.40
8
O

ATOM
2400
N
SER
B
97
9.915
10.061
−33.281
1.00
67.42
7
N

ATOM
2401
CA
SER
B
97
8.832
11.053
−33.275
1.00
67.54
6
C

ATOM
2402
CB
SER
B
97
8.043
10.995
−34.584
1.00
67.55
6
C

ATOM
2403
OG
SER
B
97
8.855
11.364
−35.686
1.00
67.59
8
O

ATOM
2404
C
SER
B
97
7.880
10.931
−32.080
1.00
67.68
6
C

ATOM
2405
O
SER
B
97
7.582
9.827
−31.622
1.00
67.61
8
O

ATOM
2406
N
LEU
B
98
7.400
12.072
−31.586
1.00
67.81
7
N

ATOM
2407
CA
LEU
B
98
6.516
12.089
−30.422
1.00
68.00
6
C

ATOM
2408
CB
LEU
B
98
7.319
12.318
−29.140
1.00
67.93
6
C

ATOM
2409
CG
LEU
B
98
8.190
11.213
−28.554
1.00
67.95
6
C

ATOM
2410
CD1
LEU
B
98
9.010
11.789
−27.417
1.00
67.95
6
C

ATOM
2411
CD2
LEU
B
98
7.352
10.048
−28.070
1.00
67.97
6
C

ATOM
2412
C
LEU
B
98
5.420
13.146
−30.470
1.00
68.19
6
C

ATOM
2413
O
LEU
B
98
5.580
14.210
−31.069
1.00
68.13
8
O

ATOM
2414
N
GLN
B
99
4.309
12.832
−29.812
1.00
68.43
7
N

ATOM
2415
CA
GLN
B
99
3.202
13.759
−29.639
1.00
68.66
6
C

ATOM
2416
CB
GLN
B
99
2.012
13.382
−30.521
1.00
68.63
6
C

ATOM
2417
CG
GLN
B
99
0.804
14.293
−30.335
1.00
68.59
6
C

ATOM
2418
CD
GLN
B
99
−0.424
13.810
−31.085
1.00
68.71
6
C

ATOM
2419
OE1
GLN
B
99
−1.170
12.968
−30.587
1.00
68.60
8
O

ATOM
2420
NE2
GLN
B
99
−0.641
14.347
−32.278
1.00
68.61
7
N

ATOM
2421
C
GLN
B
99
2.802
13.701
−28.170
1.00
68.91
6
C

ATOM
2422
O
GLN
B
99
2.559
12.619
−27.634
1.00
68.84
8
O

ATOM
2423
N
TYR
B
100
2.757
14.858
−27.517
1.00
69.20
7
N

ATOM
2424
CA
TYR
B
100
2.380
14.927
−26.109
1.00
69.56
6
C

ATOM
2425
CB
TYR
B
100
3.503
15.557
−25.282
1.00
69.32
6
C

ATOM
2426
CG
TYR
B
100
3.236
15.589
−23.794
1.00
68.56
6
C

ATOM
2427
CD1
TYR
B
100
3.109
14.415
−23.065
1.00
67.74
6
C

ATOM
2428
CE1
TYR
B
100
2.868
14.441
−21.703
1.00
67.47
6
C

ATOM
2429
CZ
TYR
B
100
2.752
15.655
−21.054
1.00
67.56
6
C

ATOM
2430
OH
TYR
B
100
2.513
15.691
−19.699
1.00
67.02
8
O

ATOM
2431
CE2
TYR
B
100
2.879
16.834
−21.758
1.00
67.71
6
C

ATOM
2432
CD2
TYR
B
100
3.119
16.796
−23.119
1.00
68.03
6
C

ATOM
2433
C
TYR
B
100
1.083
15.713
−25.941
1.00
70.07
6
C

ATOM
2434
O
TYR
B
100
0.976
16.854
−26.392
1.00
70.07
8
O

ATOM
2435
N
ILE
B
101
0.099
15.093
−25.298
1.00
70.74
7
N

ATOM
2436
CA
ILE
B
101
−1.202
15.720
−25.093
1.00
71.51
6
C

ATOM
2437
CB
ILE
B
101
−2.311
14.944
−25.840
1.00
71.47
6
C

ATOM
2438
CG1
ILE
B
101
−1.852
14.522
−27.240
1.00
71.40
6
C

ATOM
2439
CD1
ILE
B
101
−1.252
13.132
−27.298
1.00
71.02
6
C

ATOM
2440
CG2
ILE
B
101
−3.583
15.769
−25.915
1.00
71.48
6
C

ATOM
2441
C
ILE
B
101
−1.551
15.793
−23.613
1.00
72.16
6
C

ATOM
2442
O
ILE
B
101
−1.736
14.764
−22.966
1.00
72.17
8
O

ATOM
2443
N
GLU
B
102
−1.650
17.009
−23.083
1.00
73.07
7
N

ATOM
2444
CA
GLU
B
102
−1.990
17.206
−21.674
1.00
73.99
6
C

ATOM
2445
CB
GLU
B
102
−1.534
18.587
−21.189
1.00
73.96
6
C

ATOM
2446
CG
GLU
B
102
−0.031
18.699
−20.984
1.00
74.39
6
C

ATOM
2447
CD
GLU
B
102
0.396
20.050
−20.444
1.00
74.91
6
C

ATOM
2448
OE1
GLU
B
102
0.630
20.157
−19.222
1.00
75.31
8
O

ATOM
2449
OE2
GLU
B
102
0.506
21.005
−21.242
1.00
74.86
8
O

ATOM
2450
C
GLU
B
102
−3.483
17.004
−21.403
1.00
74.52
6
C

ATOM
2451
O
GLU
B
102
−4.286
16.922
−22.334
1.00
74.54
8
O

ATOM
2452
N
ARG
B
103
−3.842
16.923
−20.123
1.00
75.28
7
N

ATOM
2453
CA
ARG
B
103
−5.227
16.706
−19.704
1.00
76.03
6
C

ATOM
2454
CB
ARG
B
103
−5.381
16.978
−18.206
1.00
76.14
6
C

ATOM
2455
CG
ARG
B
103
−4.074
17.057
−17.434
1.00
76.75
6
C

ATOM
2456
CD
ARG
B
103
−4.244
17.442
−15.969
1.00
77.80
6
C

ATOM
2457
NE
ARG
B
103
−4.767
16.336
−15.170
1.00
78.42
7
N

ATOM
2458
CZ
ARG
B
103
−5.251
16.462
−13.941
1.00
78.66
6
C

ATOM
2459
NH1
ARG
B
103
−5.289
17.653
−13.357
1.00
78.73
7
N

ATOM
2460
NH2
ARG
B
103
−5.699
15.395
−13.294
1.00
78.70
7
N

ATOM
2461
C
ARG
B
103
−6.174
17.624
−20.460
1.00
76.38
6
C

ATOM
2462
O
ARG
B
103
−7.267
17.222
−20.862
1.00
76.42
8
O

ATOM
2463
N
ASP
B
104
−5.734
18.864
−20.646
1.00
76.80
7
N

ATOM
2464
CA
ASP
B
104
−6.528
19.884
−21.315
1.00
77.26
6
C

ATOM
2465
CB
ASP
B
104
−5.940
21.265
−21.024
1.00
77.33
6
C

ATOM
2466
CG
ASP
B
104
−5.645
21.468
−19.548
1.00
77.74
6
C

ATOM
2467
OD1
ASP
B
104
−6.428
20.963
−18.713
1.00
78.08
8
O

ATOM
2468
OD2
ASP
B
104
−4.656
22.107
−19.126
1.00
78.02
8
O

ATOM
2469
C
ASP
B
104
−6.639
19.654
−22.821
1.00
77.45
6
C

ATOM
2470
O
ASP
B
104
−7.158
20.502
−23.548
1.00
77.53
8
O

ATOM
2471
N
GLY
B
105
−6.149
18.506
−23.284
1.00
77.62
7
N

ATOM
2472
CA
GLY
B
105
−6.222
18.146
−24.689
1.00
77.76
6
C

ATOM
2473
C
GLY
B
105
−5.246
18.881
−25.589
1.00
77.96
6
C

ATOM
2474
O
GLY
B
105
−5.221
18.656
−26.801
1.00
77.92
8
O

ATOM
2475
N
THR
B
106
−4.438
19.758
−25.001
1.00
78.10
7
N

ATOM
2476
CA
THR
B
106
−3.467
20.533
−25.766
1.00
78.26
6
C

ATOM
2477
CB
THR
B
106
−2.848
21.640
−24.890
1.00
78.26
6
C

ATOM
2478
OG1
THR
B
106
−3.860
22.589
−24.530
1.00
78.31
8
O

ATOM
2479
CG2
THR
B
106
−1.861
22.471
−25.697
1.00
78.25
6
C

ATOM
2480
C
THR
B
106
−2.371
19.645
−26.351
1.00
78.36
6
C

ATOM
2481
O
THR
B
106
−1.687
18.921
−25.625
1.00
78.39
8
O

ATOM
2482
N
GLU
B
107
−2.211
19.710
−27.669
1.00
78.42
7
N

ATOM
2483
CA
GLU
B
107
−1.207
18.915
−28.365
1.00
78.51
6
C

ATOM
2484
CB
GLU
B
107
−1.568
18.797
−29.847
1.00
78.54
6
C

ATOM
2485
CG
GLU
B
107
−3.050
18.591
−30.124
1.00
78.80
6
C

ATOM
2486
CD
GLU
B
107
−3.453
17.130
−30.142
1.00
79.26
6
C

ATOM
2487
OE1
GLU
B
107
−2.556
16.267
−30.240
1.00
79.50
8
O

ATOM
2488
OE2
GLU
B
107
−4.667
16.844
−30.065
1.00
79.53
8
O

ATOM
2489
C
GLU
B
107
0.180
19.541
−28.226
1.00
78.51
6
C

ATOM
2490
O
GLU
B
107
0.318
20.665
−27.748
1.00
78.62
8
O

ATOM
2491
N
SER
B
108
1.199
18.800
−28.652
1.00
78.50
7
N

ATOM
2492
CA
SER
B
108
2.585
19.260
−28.626
1.00
78.45
6
C

ATOM
2493
CB
SER
B
108
3.051
19.543
−27.199
1.00
78.46
6
C

ATOM
2494
OG
SER
B
108
3.040
18.365
−26.415
1.00
78.62
8
O

ATOM
2495
C
SER
B
108
3.461
18.193
−29.270
1.00
78.43
6
C

ATOM
2496
O
SER
B
108
3.515
17.057
−28.803
1.00
78.45
8
O

ATOM
2497
N
TYR
B
109
4.152
18.564
−30.341
1.00
78.38
7
N

ATOM
2498
CA
TYR
B
109
4.948
17.608
−31.101
1.00
78.34
6
C

ATOM
2499
CB
TYR
B
109
4.589
17.716
−32.585
1.00
78.39
6
C

ATOM
2500
CG
TYR
B
109
3.099
17.874
−32.805
1.00
78.59
6
C

ATOM
2501
CD1
TYR
B
109
2.483
19.113
−32.660
1.00
78.75
6
C

ATOM
2502
CE1
TYR
B
109
1.121
19.262
−32.845
1.00
78.95
6
C

ATOM
2503
CZ
TYR
B
109
0.353
18.164
−33.175
1.00
79.00
6
C

ATOM
2504
OH
TYR
B
109
−1.003
18.310
−33.361
1.00
79.25
8
O

ATOM
2505
CE2
TYR
B
109
0.938
16.923
−33.319
1.00
78.96
6
C

ATOM
2506
CD2
TYR
B
109
2.303
16.782
−33.129
1.00
78.86
6
C

ATOM
2507
C
TYR
B
109
6.449
17.775
−30.881
1.00
78.23
6
C

ATOM
2508
O
TYR
B
109
7.040
18.780
−31.276
1.00
78.29
8
O

ATOM
2509
N
LEU
B
110
7.057
16.780
−30.243
1.00
78.03
7
N

ATOM
2510
CA
LEU
B
110
8.485
16.813
−29.947
1.00
77.83
6
C

ATOM
2511
CB
LEU
B
110
8.724
16.946
−28.439
1.00
77.87
6
C

ATOM
2512
CG
LEU
B
110
8.115
15.884
−27.517
1.00
77.92
6
C

ATOM
2513
CD1
LEU
B
110
8.880
15.816
−26.203
1.00
77.91
6
C

ATOM
2514
CD2
LEU
B
110
6.636
16.148
−27.269
1.00
77.94
6
C

ATOM
2515
C
LEU
B
110
9.209
15.583
−30.490
1.00
77.67
6
C

ATOM
2516
O
LEU
B
110
8.751
14.952
−31.443
1.00
77.63
8
O

ATOM
2517
N
THR
B
111
10.342
15.251
−29.877
1.00
77.46
7
N

ATOM
2518
CA
THR
B
111
11.153
14.119
−30.311
1.00
77.27
6
C

ATOM
2519
CB
THR
B
111
12.266
14.600
−31.267
1.00
77.30
6
C

ATOM
2520
OG1
THR
B
111
11.832
15.769
−31.973
1.00
77.54
8
O

ATOM
2521
CG2
THR
B
111
12.498
13.589
−32.373
1.00
77.32
6
C

ATOM
2522
C
THR
B
111
11.787
13.416
−29.115
1.00
77.06
6
C

ATOM
2523
O
THR
B
111
11.875
13.985
−28.027
1.00
77.07
8
O

ATOM
2524
N
VAL
B
112
12.218
12.174
−29.313
1.00
76.82
7
N

ATOM
2525
CA
VAL
B
112
12.906
11.438
−28.260
1.00
76.62
6
C

ATOM
2526
CB
VAL
B
112
12.893
9.915
−28.505
1.00
76.66
6
C

ATOM
2527
CG1
VAL
B
112
13.780
9.202
−27.498
1.00
76.61
6
C

ATOM
2528
CG2
VAL
B
112
11.473
9.370
−28.433
1.00
76.72
6
C

ATOM
2529
C
VAL
B
112
14.341
11.943
−28.224
1.00
76.45
6
C

ATOM
2530
O
VAL
B
112
15.005
11.911
−27.186
1.00
76.43
8
O

ATOM
2531
N
SER
B
113
14.806
12.419
−29.375
1.00
76.23
7
N

ATOM
2532
CA
SER
B
113
16.147
12.972
−29.501
1.00
76.03
6
C

ATOM
2533
CB
SER
B
113
16.628
12.901
−30.953
1.00
76.07
6
C

ATOM
2534
OG
SER
B
113
15.670
13.448
−31.844
1.00
76.02
8
O

ATOM
2535
C
SER
B
113
16.169
14.411
−28.996
1.00
75.80
6
C

ATOM
2536
O
SER
B
113
17.219
15.051
−28.953
1.00
75.73
8
O

ATOM
2537
N
SER
B
114
14.996
14.911
−28.616
1.00
75.52
7
N

ATOM
2538
CA
SER
B
114
14.871
16.254
−28.068
1.00
75.28
6
C

ATOM
2539
CB
SER
B
114
13.576
16.910
−28.540
1.00
75.35
6
C

ATOM
2540
OG
SER
B
114
12.455
16.348
−27.882
1.00
75.61
8
O

ATOM
2541
C
SER
B
114
14.879
16.160
−26.551
1.00
75.02
6
C

ATOM
2542
O
SER
B
114
14.202
16.931
−25.869
1.00
74.97
8
O

ATOM
2543
N
HIS
B
115
15.647
15.197
−26.046
1.00
74.69
7
N

ATOM
2544
CA
HIS
B
115
15.804
14.914
−24.617
1.00
74.31
6
C

ATOM
2545
CB
HIS
B
115
17.292
14.925
−24.250
1.00
74.43
6
C

ATOM
2546
CG
HIS
B
115
17.641
14.033
−23.099
1.00
74.97
6
C

ATOM
2547
ND1
HIS
B
115
18.750
13.214
−23.104
1.00
75.40
7
N

ATOM
2548
CE1
HIS
B
115
18.808
12.547
−21.965
1.00
75.64
6
C

ATOM
2549
NE2
HIS
B
115
17.776
12.904
−21.221
1.00
75.94
7
N

ATOM
2550
CD2
HIS
B
115
17.030
13.833
−21.907
1.00
75.53
6
C

ATOM
2551
C
HIS
B
115
15.029
15.834
−23.669
1.00
73.80
6
C

ATOM
2552
O
HIS
B
115
15.630
16.638
−22.954
1.00
73.83
8
O

ATOM
2553
N
PRO
B
116
13.703
15.710
−23.656
1.00
73.26
7
N

ATOM
2554
CA
PRO
B
116
12.855
16.537
−22.789
1.00
72.72
6
C

ATOM
2555
CB
PRO
B
116
11.443
16.154
−23.236
1.00
72.81
6
C

ATOM
2556
CG
PRO
B
116
11.605
14.775
−23.740
1.00
73.08
6
C

ATOM
2557
CD
PRO
B
116
12.900
14.797
−24.492
1.00
73.17
6
C

ATOM
2558
C
PRO
B
116
13.037
16.192
−21.317
1.00
72.09
6
C

ATOM
2559
O
PRO
B
116
12.667
15.096
−20.904
1.00
72.06
8
O

ATOM
2560
N
ASN
B
117
13.598
17.114
−20.540
1.00
71.24
7
N

ATOM
2561
CA
ASN
B
117
13.809
16.872
−19.119
1.00
70.39
6
C

ATOM
2562
CB
ASN
B
117
14.694
17.958
−18.509
1.00
70.52
6
C

ATOM
2563
CG
ASN
B
117
16.127
17.877
−18.994
1.00
70.84
6
C

ATOM
2564
OD1
ASN
B
117
16.923
17.091
−18.480
1.00
71.28
8
O

ATOM
2565
ND2
ASN
B
117
16.462
18.685
−19.994
1.00
70.97
7
N

ATOM
2566
C
ASN
B
117
12.489
16.767
−18.367
1.00
69.59
6
C

ATOM
2567
O
ASN
B
117
12.315
15.894
−17.516
1.00
69.52
8
O

ATOM
2568
N
ALA
B
118
11.560
17.660
−18.691
1.00
68.62
7
N

ATOM
2569
CA
ALA
B
118
10.242
17.645
−18.073
1.00
67.70
6
C

ATOM
2570
CB
ALA
B
118
9.438
18.858
−18.511
1.00
67.77
6
C

ATOM
2571
C
ALA
B
118
9.509
16.354
−18.434
1.00
66.98
6
C

ATOM
2572
O
ALA
B
118
8.786
15.786
−17.614
1.00
66.84
8
O

ATOM
2573
N
LEU
B
119
9.715
15.890
−19.664
1.00
66.05
7
N

ATOM
2574
CA
LEU
B
119
9.073
14.670
−20.148
1.00
65.14
6
C

ATOM
2575
CB
LEU
B
119
8.456
14.902
−21.531
1.00
65.24
6
C

ATOM
2576
CG
LEU
B
119
7.357
15.965
−21.601
1.00
65.57
6
C

ATOM
2577
CD1
LEU
B
119
6.827
16.114
−23.020
1.00
65.85
6
C

ATOM
2578
CD2
LEU
B
119
6.231
15.626
−20.636
1.00
65.80
6
C

ATOM
2579
C
LEU
B
119
10.033
13.486
−20.204
1.00
64.34
6
C

ATOM
2580
O
LEU
B
119
9.884
12.604
−21.046
1.00
64.28
8
O

ATOM
2581
N
MET
B
120
11.013
13.466
−19.305
1.00
63.32
7
N

ATOM
2582
CA
MET
B
120
11.990
12.378
−19.269
1.00
62.27
6
C

ATOM
2583
CB
MET
B
120
13.271
12.809
−18.551
1.00
62.47
6
C

ATOM
2584
CG
MET
B
120
14.453
13.018
−19.473
1.00
63.24
6
C

ATOM
2585
SD
MET
B
120
14.822
11.543
−20.454
1.00
65.13
16
S

ATOM
2586
CE
MET
B
120
15.182
10.353
−19.163
1.00
65.06
6
C

ATOM
2587
C
MET
B
120
11.445
11.118
−18.616
1.00
61.30
6
C

ATOM
2588
O
MET
B
120
11.685
10.011
−19.096
1.00
61.03
8
O

ATOM
2589
N
LYS
B
121
10.728
11.288
−17.512
1.00
60.15
7
N

ATOM
2590
CA
LYS
B
121
10.168
10.152
−16.795
1.00
59.20
6
C

ATOM
2591
CB
LYS
B
121
9.682
10.571
−15.403
1.00
59.38
6
C

ATOM
2592
CG
LYS
B
121
10.772
11.136
−14.489
1.00
59.63
6
C

ATOM
2593
CD
LYS
B
121
10.256
11.300
−13.062
1.00
60.08
6
C

ATOM
2594
CE
LYS
B
121
11.338
11.818
−12.119
1.00
60.74
6
C

ATOM
2595
NZ
LYS
B
121
11.682
13.251
−12.368
1.00
60.83
7
N

ATOM
2596
C
LYS
B
121
9.026
9.523
−17.588
1.00
58.39
6
C

ATOM
2597
O
LYS
B
121
8.823
8.310
−17.536
1.00
58.28
8
O

ATOM
2598
N
LYS
B
122
8.285
10.348
−18.324
1.00
57.44
7
N

ATOM
2599
CA
LYS
B
122
7.166
9.852
−19.125
1.00
56.58
6
C

ATOM
2600
CB
LYS
B
122
6.204
10.987
−19.499
1.00
56.59
6
C

ATOM
2601
CG
LYS
B
122
5.258
11.375
−18.362
1.00
56.35
6
C

ATOM
2602
CD
LYS
B
122
4.421
12.604
−18.684
1.00
56.34
6
C

ATOM
2603
CE
LYS
B
122
3.443
12.907
−17.547
1.00
56.68
6
C

ATOM
2604
NZ
LYS
B
122
2.607
14.115
−17.796
1.00
55.88
7
N

ATOM
2605
C
LYS
B
122
7.655
9.097
−20.363
1.00
56.03
6
C

ATOM
2606
O
LYS
B
122
7.179
8.000
−20.660
1.00
55.84
8
O

ATOM
2607
N
ILE
B
123
8.613
9.686
−21.074
1.00
55.27
7
N

ATOM
2608
CA
ILE
B
123
9.200
9.045
−22.247
1.00
54.71
6
C

ATOM
2609
CB
ILE
B
123
10.249
9.962
−22.902
1.00
54.74
6
C

ATOM
2610
CG1
ILE
B
123
9.563
11.042
−23.736
1.00
55.03
6
C

ATOM
2611
CD1
ILE
B
123
10.521
11.855
−24.568
1.00
55.34
6
C

ATOM
2612
CG2
ILE
B
123
11.199
9.156
−23.773
1.00
54.92
6
C

ATOM
2613
C
ILE
B
123
9.837
7.718
−21.863
1.00
54.03
6
C

ATOM
2614
O
ILE
B
123
9.676
6.712
−22.555
1.00
54.00
8
O

ATOM
2615
N
THR
B
124
10.560
7.721
−20.750
1.00
53.37
7
N

ATOM
2616
CA
THR
B
124
11.200
6.510
−20.255
1.00
52.73
6
C

ATOM
2617
CB
THR
B
124
12.011
6.810
−18.976
1.00
52.82
6
C

ATOM
2618
OG1
THR
B
124
13.171
7.577
−19.317
1.00
51.93
8
O

ATOM
2619
CG2
THR
B
124
12.587
5.531
−18.391
1.00
52.26
6
C

ATOM
2620
C
THR
B
124
10.154
5.439
−19.991
1.00
52.58
6
C

ATOM
2621
O
THR
B
124
10.320
4.285
−20.388
1.00
52.40
8
O

ATOM
2622
N
LEU
B
125
9.072
5.827
−19.324
1.00
52.52
7
N

ATOM
2623
CA
LEU
B
125
7.981
4.900
−19.051
1.00
52.53
6
C

ATOM
2624
CB
LEU
B
125
6.868
5.589
−18.264
1.00
52.34
6
C

ATOM
2625
CG
LEU
B
125
6.919
5.415
−16.746
1.00
52.83
6
C

ATOM
2626
CD1
LEU
B
125
5.986
6.401
−16.059
1.00
52.57
6
C

ATOM
2627
CD2
LEU
B
125
6.568
3.981
−16.361
1.00
52.79
6
C

ATOM
2628
C
LEU
B
125
7.433
4.328
−20.351
1.00
52.47
6
C

ATOM
2629
O
LEU
B
125
7.090
3.149
−20.422
1.00
52.35
8
O

ATOM
2630
N
LEU
B
126
7.369
5.164
−21.382
1.00
52.61
7
N

ATOM
2631
CA
LEU
B
126
6.852
4.735
−22.678
1.00
52.77
6
C

ATOM
2632
CB
LEU
B
126
6.673
5.927
−23.617
1.00
52.93
6
C

ATOM
2633
CG
LEU
B
126
5.952
5.609
−24.928
1.00
53.38
6
C

ATOM
2634
CD1
LEU
B
126
4.676
4.832
−24.644
1.00
54.11
6
C

ATOM
2635
CD2
LEU
B
126
5.642
6.875
−25.714
1.00
53.94
6
C

ATOM
2636
C
LEU
B
126
7.759
3.693
−23.326
1.00
52.83
6
C

ATOM
2637
O
LEU
B
126
7.284
2.795
−24.021
1.00
52.65
8
O

ATOM
2638
N
LYS
B
127
9.064
3.819
−23.095
1.00
52.84
7
N

ATOM
2639
CA
LYS
B
127
10.030
2.872
−23.640
1.00
52.93
6
C

ATOM
2640
CB
LYS
B
127
11.457
3.327
−23.346
1.00
53.06
6
C

ATOM
2641
CG
LYS
B
127
11.788
4.730
−23.807
1.00
53.98
6
C

ATOM
2642
CD
LYS
B
127
12.228
4.752
−25.255
1.00
55.23
6
C

ATOM
2643
CE
LYS
B
127
13.246
5.862
−25.484
1.00
55.58
6
C

ATOM
2644
NZ
LYS
B
127
13.877
5.784
−26.833
1.00
56.15
7
N

ATOM
2645
C
LYS
B
127
9.815
1.497
−23.024
1.00
52.76
6
C

ATOM
2646
O
LYS
B
127
9.772
0.489
−23.732
1.00
52.74
8
O

ATOM
2647
N
TYR
B
128
9.693
1.467
−21.701
1.00
52.52
7
N

ATOM
2648
CA
TYR
B
128
9.475
0.221
−20.977
1.00
52.44
6
C

ATOM
2649
CB
TYR
B
128
9.321
0.493
−19.478
1.00
52.59
6
C

ATOM
2650
CG
TYR
B
128
8.920
−0.707
−18.643
1.00
53.23
6
C

ATOM
2651
CD1
TYR
B
128
7.627
−0.829
−18.151
1.00
54.30
6
C

ATOM
2652
CE1
TYR
B
128
7.252
−1.915
−17.385
1.00
54.61
6
C

ATOM
2653
CZ
TYR
B
128
8.171
−2.894
−17.095
1.00
54.75
6
C

ATOM
2654
OH
TYR
B
128
7.788
−3.972
−16.329
1.00
55.15
8
O

ATOM
2655
CE2
TYR
B
128
9.466
−2.798
−17.563
1.00
54.54
6
C

ATOM
2656
CD2
TYR
B
128
9.834
−1.706
−18.332
1.00
54.10
6
C

ATOM
2657
C
TYR
B
128
8.258
−0.515
−21.530
1.00
52.27
6
C

ATOM
2658
O
TYR
B
128
8.344
−1.694
−21.867
1.00
52.20
8
O

ATOM
2659
N
PHE
B
129
7.132
0.188
−21.635
1.00
51.96
7
N

ATOM
2660
CA
PHE
B
129
5.907
−0.407
−22.162
1.00
51.77
6
C

ATOM
2661
CB
PHE
B
129
4.800
0.646
−22.261
1.00
51.70
6
C

ATOM
2662
CG
PHE
B
129
4.180
0.997
−20.942
1.00
51.57
6
C

ATOM
2663
CD1
PHE
B
129
3.995
2.320
−20.577
1.00
51.51
6
C

ATOM
2664
CE1
PHE
B
129
3.420
2.647
−19.358
1.00
51.58
6
C

ATOM
2665
CZ
PHE
B
129
3.024
1.648
−18.495
1.00
51.40
6
C

ATOM
2666
CE2
PHE
B
129
3.203
0.325
−18.850
1.00
51.91
6
C

ATOM
2667
CD2
PHE
B
129
3.778
0.003
−20.067
1.00
51.64
6
C

ATOM
2668
C
PHE
B
129
6.154
−1.040
−23.527
1.00
51.63
6
C

ATOM
2669
O
PHE
B
129
5.849
−2.210
−23.742
1.00
51.43
8
O

ATOM
2670
N
ARG
B
130
6.716
−0.251
−24.438
1.00
51.76
7
N

ATOM
2671
CA
ARG
B
130
7.029
−0.712
−25.782
1.00
52.09
6
C

ATOM
2672
CB
ARG
B
130
7.826
0.350
−26.540
1.00
52.12
6
C

ATOM
2673
CG
ARG
B
130
8.353
−0.128
−27.886
1.00
52.68
6
C

ATOM
2674
CD
ARG
B
130
9.224
0.881
−28.613
1.00
53.70
6
C

ATOM
2675
NE
ARG
B
130
10.443
1.189
−27.868
1.00
54.71
7
N

ATOM
2676
CZ
ARG
B
130
11.375
2.032
−28.287
1.00
55.07
6
C

ATOM
2677
NH1
ARG
B
130
11.230
2.654
−29.451
1.00
55.48
7
N

ATOM
2678
NH2
ARG
B
130
12.454
2.256
−27.547
1.00
55.58
7
N

ATOM
2679
C
ARG
B
130
7.811
−2.015
−25.750
1.00
52.16
6
C

ATOM
2680
O
ARG
B
130
7.408
−3.007
−26.359
1.00
52.02
8
O

ATOM
2681
N
ASN
B
131
8.933
−2.005
−25.035
1.00
52.22
7
N

ATOM
2682
CA
ASN
B
131
9.780
−3.184
−24.921
1.00
52.32
6
C

ATOM
2683
CB
ASN
B
131
11.061
−2.854
−24.144
1.00
52.50
6
C

ATOM
2684
CG
ASN
B
131
11.847
−1.714
−24.768
1.00
53.32
6
C

ATOM
2685
OD1
ASN
B
131
11.504
−1.220
−25.845
1.00
54.22
8
O

ATOM
2686
ND2
ASN
B
131
12.910
−1.291
−24.092
1.00
54.67
7
N

ATOM
2687
C
ASN
B
131
9.054
−4.352
−24.260
1.00
52.15
6
C

ATOM
2688
O
ASN
B
131
9.245
−5.507
−24.644
1.00
52.01
8
O

ATOM
2689
N
TYR
B
132
8.222
−4.052
−23.266
1.00
51.97
7
N

ATOM
2690
CA
TYR
B
132
7.481
−5.100
−22.571
1.00
52.06
6
C

ATOM
2691
CB
TYR
B
132
6.742
−4.542
−21.354
1.00
52.17
6
C

ATOM
2692
CG
TYR
B
132
5.904
−5.575
−20.632
1.00
52.63
6
C

ATOM
2693
CD1
TYR
B
132
6.388
−6.228
−19.510
1.00
52.75
6
C

ATOM
2694
CE1
TYR
B
132
5.626
−7.173
−18.849
1.00
53.37
6
C

ATOM
2695
CZ
TYR
B
132
4.363
−7.479
−19.313
1.00
53.58
6
C

ATOM
2696
OH
TYR
B
132
3.603
−8.421
−18.658
1.00
54.12
8
O

ATOM
2697
CE2
TYR
B
132
3.859
−6.847
−20.427
1.00
53.55
6
C

ATOM
2698
CD2
TYR
B
132
4.628
−5.901
−21.080
1.00
53.51
6
C

ATOM
2699
C
TYR
B
132
6.490
−5.799
−23.499
1.00
51.98
6
C

ATOM
2700
O
TYR
B
132
6.414
−7.023
−23.527
1.00
51.86
8
O

ATOM
2701
N
MET
B
133
5.727
−5.015
−24.252
1.00
52.02
7
N

ATOM
2702
CA
MET
B
133
4.738
−5.578
−25.167
1.00
52.00
6
C

ATOM
2703
CB
MET
B
133
3.871
−4.468
−25.768
1.00
51.99
6
C

ATOM
2704
CG
MET
B
133
3.024
−3.728
−24.737
1.00
51.75
6
C

ATOM
2705
SD
MET
B
133
2.056
−2.335
−25.406
1.00
50.86
16
S

ATOM
2706
CE
MET
B
133
3.353
−1.216
−25.922
1.00
51.88
6
C

ATOM
2707
C
MET
B
133
5.424
−6.395
−26.261
1.00
52.18
6
C

ATOM
2708
O
MET
B
133
5.016
−7.517
−26.563
1.00
52.10
8
O

ATOM
2709
N
SER
B
134
6.486
−5.829
−26.829
1.00
52.40
7
N

ATOM
2710
CA
SER
B
134
7.260
−6.479
−27.880
1.00
52.62
6
C

ATOM
2711
CB
SER
B
134
8.332
−5.523
−28.410
1.00
52.61
6
C

ATOM
2712
OG
SER
B
134
9.154
−6.146
−29.382
1.00
53.06
8
O

ATOM
2713
C
SER
B
134
7.904
−7.782
−27.408
1.00
52.68
6
C

ATOM
2714
O
SER
B
134
8.330
−8.601
−28.223
1.00
52.83
8
O

ATOM
2715
N
GLU
B
135
7.953
−7.977
−26.095
1.00
52.77
7
N

ATOM
2716
CA
GLU
B
135
8.563
−9.171
−25.517
1.00
52.92
6
C

ATOM
2717
CB
GLU
B
135
9.353
−8.814
−24.256
1.00
53.26
6
C

ATOM
2718
CG
GLU
B
135
10.839
−8.604
−24.488
1.00
54.61
6
C

ATOM
2719
CD
GLU
B
135
11.646
−8.804
−23.224
1.00
56.43
6
C

ATOM
2720
OE1
GLU
B
135
11.356
−8.121
−22.219
1.00
57.82
8
O

ATOM
2721
OE2
GLU
B
135
12.563
−9.650
−23.234
1.00
57.68
8
O

ATOM
2722
C
GLU
B
135
7.597
−10.309
−25.187
1.00
52.64
6
C

ATOM
2723
O
GLU
B
135
7.868
−11.463
−25.518
1.00
52.69
8
O

ATOM
2724
N
HIS
B
136
6.480
−9.988
−24.539
1.00
52.18
7
N

ATOM
2725
CA
HIS
B
136
5.550
−11.015
−24.066
1.00
51.81
6
C

ATOM
2726
CB
HIS
B
136
5.302
−10.838
−22.564
1.00
52.15
6
C

ATOM
2727
CG
HIS
B
136
6.531
−10.962
−21.717
1.00
52.92
6
C

ATOM
2728
ND1
HIS
B
136
6.978
−12.170
−21.230
1.00
53.81
7
N

ATOM
2729
CE1
HIS
B
136
8.069
−11.975
−20.509
1.00
54.28
6
C

ATOM
2730
NE2
HIS
B
136
8.340
−10.681
−20.505
1.00
54.06
7
N

ATOM
2731
CD2
HIS
B
136
7.391
−10.025
−21.252
1.00
53.73
6
C

ATOM
2732
C
HIS
B
136
4.178
−11.053
−24.744
1.00
51.31
6
C

ATOM
2733
O
HIS
B
136
3.456
−12.044
−24.620
1.00
51.17
8
O

ATOM
2734
N
LEU
B
137
3.812
−9.988
−25.447
1.00
50.72
7
N

ATOM
2735
CA
LEU
B
137
2.442
−9.878
−25.960
1.00
50.21
6
C

ATOM
2736
CB
LEU
B
137
1.858
−8.517
−25.569
1.00
49.98
6
C

ATOM
2737
CG
LEU
B
137
1.925
−8.177
−24.078
1.00
49.21
6
C

ATOM
2738
CD1
LEU
B
137
1.125
−6.918
−23.785
1.00
48.39
6
C

ATOM
2739
CD2
LEU
B
137
1.421
−9.335
−23.224
1.00
48.56
6
C

ATOM
2740
C
LEU
B
137
2.217
−10.138
−27.451
1.00
49.97
6
C

ATOM
2741
O
LEU
B
137
3.000
−9.713
−28.298
1.00
49.87
8
O

ATOM

2742

N

LEU

B

138

1.111

−10.818

−27.749

1.00

49.94

7

N

ATOM

2743

CA

LEU

B

138

0.698

−11.120

−29.120

1.00

49.71

6

C

ATOM

2744

CB

LEU

B

138

−0.210

−12.351

−29.129

1.00

49.67

6

C

ATOM

2745

CG

LEU

B

138

−0.887

−12.698

−30.458

1.00

49.70

6

C

ATOM

2746

CD1

LEU

B

138

0.106

−13.338

−31.416

1.00

49.27

6

C

ATOM

2747

CD2

LEU

B

138

−2.073

−13.618

−30.222

1.00

49.98

6

C

ATOM

2748

C

LEU

B

138

−0.035

−9.942

−29.776

1.00

49.63

6

C

ATOM

2749

O

LEU

B

138

−0.911

−9.329

−29.172

1.00

49.34

8

O

ATOM
2750
N
LYS
B
139
0.329
−9.645
−31.018
1.00
49.67
7
N

ATOM
2751
CA
LYS
B
139
−0.260
−8.546
−31.775
1.00
50.01
6
C

ATOM
2752
CB
LYS
B
139
0.691
−8.140
−32.904
1.00
49.94
6
C

ATOM
2753
CG
LYS
B
139
0.324
−6.867
−33.656
1.00
50.31
6
C

ATOM
2754
CD
LYS
B
139
1.516
−6.389
−34.478
1.00
50.49
6
C

ATOM
2755
CE
LYS
B
139
1.096
−5.589
−35.696
1.00
50.83
6
C

ATOM
2756
NZ
LYS
B
139
0.523
−4.273
−35.345
1.00
51.00
7
N

ATOM
2757
C
LYS
B
139
−1.634
−8.921
−32.342
1.00
50.29
6
C

ATOM
2758
O
LYS
B
139
−1.737
−9.750
−33.252
1.00
50.15
8
O

ATOM
2759
N
ALA
B
140
−2.682
−8.306
−31.796
1.00
50.50
7
N

ATOM
2760
CA
ALA
B
140
−4.054
−8.562
−32.234
1.00
50.83
6
C

ATOM
2761
CB
ALA
B
140
−5.050
−7.982
−31.239
1.00
50.79
6
C

ATOM
2762
C
ALA
B
140
−4.322
−8.010
−33.626
1.00
51.19
6
C

ATOM
2763
O
ALA
B
140
−3.855
−6.930
−33.979
1.00
51.15
8
O

ATOM
2764
N
GLY
B
141
−5.086
−8.759
−34.414
1.00
51.87
7
N

ATOM
2765
CA
GLY
B
141
−5.411
−8.348
−35.765
1.00
52.43
6
C

ATOM
2766
C
GLY
B
141
−4.195
−8.378
−36.665
1.00
53.11
6
C

ATOM
2767
O
GLY
B
141
−4.118
−7.639
−37.644
1.00
52.86
8
O

ATOM
2768
N
ALA
B
142
−3.239
−9.236
−36.329
1.00
53.86
7
N

ATOM
2769
CA
ALA
B
142
−2.027
−9.374
−37.125
1.00
54.88
6
C

ATOM
2770
CB
ALA
B
142
−1.108
−10.422
−36.516
1.00
54.80
6
C

ATOM
2771
C
ALA
B
142
−2.354
−9.729
−38.575
1.00
55.58
6
C

ATOM
2772
O
ALA
B
142
−1.614
−9.369
−39.490
1.00
55.55
8
O

ATOM
2773
N
ASN
B
143
−3.472
−10.424
−38.774
1.00
56.60
7
N

ATOM
2774
CA
ASN
B
143
−3.899
−10.838
−40.109
1.00
57.64
6
C

ATOM
2775
CB
ASN
B
143
−4.584
−12.208
−40.057
1.00
57.56
6
C

ATOM
2776
CG
ASN
B
143
−5.751
−12.246
−39.084
1.00
57.75
6
C

ATOM
2777
OD1
ASN
B
143
−5.854
−11.411
−38.182
1.00
57.67
8
O

ATOM
2778
ND2
ASN
B
143
−6.634
−13.224
−39.259
1.00
57.49
7
N

ATOM
2779
C
ASN
B
143
−4.808
−9.829
−40.806
1.00
58.43
6
C

ATOM
2780
O
ASN
B
143
−5.391
−10.130
−41.850
1.00
58.60
8
O

ATOM
2781
N
ILE
B
144
−4.927
−8.636
−40.231
1.00
59.33
7
N

ATOM
2782
CA
ILE
B
144
−5.757
−7.587
−40.814
1.00
60.27
6
C

ATOM
2783
CB
ILE
B
144
−6.665
−6.953
−39.735
1.00
60.15
6
C

ATOM
2784
CG1
ILE
B
144
−7.501
−8.025
−39.033
1.00
60.27
6
C

ATOM
2785
CD1
ILE
B
144
−8.419
−7.489
−37.949
1.00
59.65
6
C

ATOM
2786
CG2
ILE
B
144
−7.560
−5.887
−40.344
1.00
60.18
6
C

ATOM
2787
C
ILE
B
144
−4.896
−6.510
−41.466
1.00
61.06
6
C

ATOM
2788
O
ILE
B
144
−3.826
−6.175
−40.960
1.00
61.18
8
O

ATOM
2789
N
THR
B
145
−5.362
−5.971
−42.589
1.00
62.15
7
N

ATOM
2790
CA
THR
B
145
−4.656
−4.894
−43.280
1.00
63.24
6
C

ATOM
2791
CB
THR
B
145
−4.454
−5.233
−44.775
1.00
63.29
6
C

ATOM
2792
OG1
THR
B
145
−3.538
−6.327
−44.907
1.00
63.28
8
O

ATOM
2793
CG2
THR
B
145
−3.741
−4.092
−45.489
1.00
63.12
6
C

ATOM
2794
C
THR
B
145
−5.422
−3.578
−43.138
1.00
64.10
6
C

ATOM
2795
O
THR
B
145
−6.539
−3.447
−43.641
1.00
63.93
8
O

ATOM
2796
N
PRO
B
146
−4.812
−2.614
−42.449
1.00
65.06
7
N

ATOM
2797
CA
PRO
B
146
−5.414
−1.293
−42.203
1.00
65.86
6
C

ATOM
2798
CB
PRO
B
146
−4.308
−0.542
−41.457
1.00
65.86
6
C

ATOM
2799
CG
PRO
B
146
−3.478
−1.613
−40.847
1.00
65.50
6
C

ATOM
2800
CD
PRO
B
146
−3.484
−2.743
−41.826
1.00
64.99
6
C

ATOM
2801
C
PRO
B
146
−5.832
−0.513
−43.457
1.00
66.81
6
C

ATOM
2802
O
PRO
B
146
−5.676
−0.994
−44.581
1.00
66.84
8
O

ATOM
2803
N
ARG
B
147
−6.324
0.707
−43.244
1.00
67.90
7
N

ATOM
2804
CA
ARG
B
147
−6.918
1.522
−44.303
1.00
68.90
6
C

ATOM
2805
CB
ARG
B
147
−8.351
1.872
−43.891
1.00
68.76
6
C

ATOM
2806
CG
ARG
B
147
−9.312
2.113
−45.034
1.00
68.48
6
C

ATOM
2807
CD
ARG
B
147
−10.729
2.421
−44.577
1.00
68.19
6
C

ATOM
2808
NE
ARG
B
147
−10.853
3.743
−43.970
1.00
67.59
7
N

ATOM
2809
CZ
ARG
B
147
−11.582
4.006
−42.891
1.00
67.44
6
C

ATOM
2810
NH1
ARG
B
147
−12.251
3.036
−42.283
1.00
67.26
7
N

ATOM
2811
NH2
ARG
B
147
−11.642
5.240
−42.413
1.00
67.45
7
N

ATOM
2812
C
ARG
B
147
−6.155
2.809
−44.642
1.00
69.68
6
C

ATOM
2813
O
ARG
B
147
−4.973
2.944
−44.333
1.00
69.95
8
O

ATOM
2814
N
GLU
B
148
−6.854
3.750
−45.280
1.00
70.69
7
N

ATOM
2815
CA
GLU
B
148
−6.277
5.031
−45.699
1.00
71.50
6
C

ATOM
2816
CB
GLU
B
148
−6.978
5.552
−46.957
1.00
71.67
6
C

ATOM
2817
CG
GLU
B
148
−7.163
4.544
−48.078
1.00
72.40
6
C

ATOM
2818
CD
GLU
B
148
−8.089
5.069
−49.160
1.00
73.44
6
C

ATOM
2819
OE1
GLU
B
148
−8.789
6.073
−48.902
1.00
73.72
8
O

ATOM
2820
OE2
GLU
B
148
−8.116
4.482
−50.265
1.00
73.75
8
O

ATOM
2821
C
GLU
B
148
−6.388
6.110
−44.624
1.00
71.87
6
C

ATOM
2822
O
GLU
B
148
−7.396
6.199
−43.923
1.00
72.05
8
O

ATOM
2823
N
GLY
B
149
−5.359
6.947
−44.523
1.00
72.24
7
N

ATOM
2824
CA
GLY
B
149
−5.339
8.038
−43.564
1.00
72.61
6
C

ATOM
2825
C
GLY
B
149
−4.753
9.306
−44.162
1.00
72.89
6
C

ATOM
2826
O
GLY
B
149
−4.102
9.263
−45.209
1.00
72.91
8
O

ATOM
2827
N
ASP
B
150
−4.985
10.438
−43.500
1.00
73.09
7
N

ATOM
2828
CA
ASP
B
150
−4.475
11.726
−43.968
1.00
73.25
6
C

ATOM
2829
CB
ASP
B
150
−5.458
12.850
−43.637
1.00
73.46
6
C

ATOM
2830
CG
ASP
B
150
−6.855
12.569
−44.151
1.00
74.14
6
C

ATOM
2831
OD1
ASP
B
150
−6.979
11.880
−45.187
1.00
74.66
8
O

ATOM
2832
OD2
ASP
B
150
−7.886
12.992
−43.584
1.00
75.11
8
O

ATOM
2833
C
ASP
B
150
−3.102
12.022
−43.370
1.00
73.12
6
C

ATOM
2834
O
ASP
B
150
−2.960
12.868
−42.484
1.00
73.15
8
O

ATOM
2835
N
GLU
B
151
−2.099
11.315
−43.882
1.00
72.84
7
N

ATOM
2836
CA
GLU
B
151
−0.708
11.406
−43.435
1.00
72.56
6
C

ATOM
2837
CB
GLU
B
151
0.229
11.118
−44.613
1.00
72.70
6
C

ATOM
2838
CG
GLU
B
151
−0.106
9.841
−45.369
1.00
73.28
6
C

ATOM
2839
CD
GLU
B
151
0.772
9.637
−46.589
1.00
74.10
6
C

ATOM
2840
OE1
GLU
B
151
1.606
10.521
−46.876
1.00
74.33
8
O

ATOM
2841
OE2
GLU
B
151
0.627
8.593
−47.262
1.00
74.53
8
O

ATOM
2842
C
GLU
B
151
−0.274
12.704
−42.744
1.00
72.11
6
C

ATOM
2843
O
GLU
B
151
0.538
12.670
−41.817
1.00
72.16
8
O

ATOM
2844
N
LEU
B
152
−0.805
13.840
−43.189
1.00
71.49
7
N

ATOM
2845
CA
LEU
B
152
−0.397
15.141
−42.647
1.00
70.86
6
C

ATOM
2846
CB
LEU
B
152
−0.871
16.279
−43.557
1.00
71.02
6
C

ATOM
2847
CG
LEU
B
152
−0.300
16.285
−44.977
1.00
71.31
6
C

ATOM
2848
CD1
LEU
B
152
−0.840
17.469
−45.771
1.00
71.66
6
C

ATOM
2849
CD2
LEU
B
152
1.222
16.305
−44.946
1.00
71.67
6
C

ATOM
2850
C
LEU
B
152
−0.829
15.410
−41.201
1.00
70.20
6
C

ATOM
2851
O
LEU
B
152
−0.299
16.313
−40.551
1.00
70.25
8
O

ATOM
2852
N
ALA
B
153
−1.784
14.630
−40.703
1.00
69.21
7
N

ATOM
2853
CA
ALA
B
153
−2.261
14.796
−39.334
1.00
68.16
6
C

ATOM
2854
CB
ALA
B
153
−2.827
16.197
−39.132
1.00
68.31
6
C

ATOM
2855
C
ALA
B
153
−3.307
13.746
−38.978
1.00
67.34
6
C

ATOM
2856
O
ALA
B
153
−4.502
14.042
−38.936
1.00
67.43
8
O

ATOM
2857
N
ARG
B
154
−2.851
12.524
−38.720
1.00
66.04
7
N

ATOM
2858
CA
ARG
B
154
−3.748
11.428
−38.365
1.00
64.75
6
C

ATOM
2859
CB
ARG
B
154
−4.341
10.776
−39.617
1.00
64.95
6
C

ATOM
2860
CG
ARG
B
154
−5.369
11.623
−40.344
1.00
65.66
6
C

ATOM
2861
CD
ARG
B
154
−6.610
11.944
−39.533
1.00
66.89
6
C

ATOM
2862
NE
ARG
B
154
−7.517
12.814
−40.275
1.00
67.71
7
N

ATOM
2863
CZ
ARG
B
154
−8.735
13.142
−39.866
1.00
68.02
6
C

ATOM
2864
NH1
ARG
B
154
−9.197
12.675
−38.715
1.00
68.34
7
N

ATOM
2865
NH2
ARG
B
154
−9.493
13.940
−40.607
1.00
68.13
7
N

ATOM
2866
C
ARG
B
154
−3.048
10.365
−37.528
1.00
63.44
6
C

ATOM
2867
O
ARG
B
154
−2.103
9.717
−37.983
1.00
63.52
8
O

ATOM
2868
N
LEU
B
155
−3.532
10.194
−36.305
1.00
61.63
7
N

ATOM
2869
CA
LEU
B
155
−3.025
9.193
−35.375
1.00
59.76
6
C

ATOM
2870
CB
LEU
B
155
−1.506
9.246
−35.247
1.00
59.94
6
C

ATOM
2871
CG
LEU
B
155
−0.903
7.990
−34.617
1.00
60.13
6
C

ATOM
2872
CD1
LEU
B
155
−0.654
6.935
−35.679
1.00
60.60
6
C

ATOM
2873
CD2
LEU
B
155
0.385
8.318
−33.897
1.00
60.79
6
C

ATOM
2874
C
LEU
B
155
−3.670
9.501
−34.041
1.00
58.15
6
C

ATOM
2875
O
LEU
B
155
−3.347
10.505
−33.406
1.00
58.10
8
O

ATOM
2876
N
PRO
B
156
−4.587
8.635
−33.625
1.00
56.42
7
N

ATOM
2877
CA
PRO
B
156
−5.373
8.850
−32.413
1.00
54.95
6
C

ATOM
2878
CB
PRO
B
156
−6.534
7.884
−32.621
1.00
55.01
6
C

ATOM
2879
CG
PRO
B
156
−5.843
6.718
−33.220
1.00
55.56
6
C

ATOM
2880
CD
PRO
B
156
−4.956
7.366
−34.279
1.00
56.24
6
C

ATOM
2881
C
PRO
B
156
−4.633
8.472
−31.145
1.00
53.41
6
C

ATOM
2882
O
PRO
B
156
−3.687
7.689
−31.174
1.00
53.43
8
O

ATOM
2883
N
TYR
B
157
−5.075
9.041
−30.034
1.00
51.51
7
N

ATOM
2884
CA
TYR
B
157
−4.530
8.697
−28.735
1.00
49.68
6
C

ATOM
2885
CB
TYR
B
157
−3.965
9.931
−28.031
1.00
49.53
6
C

ATOM
2886
CG
TYR
B
157
−4.937
11.084
−27.898
1.00
48.93
6
C

ATOM
2887
CD1
TYR
B
157
−5.843
11.139
−26.849
1.00
48.66
6
C

ATOM
2888
CE1
TYR
B
157
−6.727
12.196
−26.721
1.00
47.81
6
C

ATOM
2889
CZ
TYR
B
157
−6.708
13.217
−27.646
1.00
47.98
6
C

ATOM
2890
OH
TYR
B
157
−7.583
14.276
−27.522
1.00
47.77
8
O

ATOM
2891
CE2
TYR
B
157
−5.814
13.188
−28.693
1.00
48.01
6
C

ATOM
2892
CD2
TYR
B
157
−4.934
12.128
−28.813
1.00
48.72
6
C

ATOM
2893
C
TYR
B
157
−5.664
8.102
−27.926
1.00
48.53
6
C

ATOM
2894
O
TYR
B
157
−6.829
8.241
−28.294
1.00
48.31
8
O

ATOM
2895
N
LEU
B
158
−5.332
7.439
−26.827
1.00
47.21
7
N

ATOM
2896
CA
LEU
B
158
−6.353
6.853
−25.975
1.00
46.08
6
C

ATOM
2897
CB
LEU
B
158
−5.738
5.803
−25.068
1.00
46.07
6
C

ATOM
2898
CG
LEU
B
158
−6.731
5.066
−24.180
1.00
45.30
6
C

ATOM
2899
CD1
LEU
B
158
−7.716
4.278
−25.042
1.00
45.13
6
C

ATOM
2900
CD2
LEU
B
158
−5.978
4.150
−23.247
1.00
44.78
6
C

ATOM
2901
C
LEU
B
158
−7.028
7.918
−25.122
1.00
45.64
6
C

ATOM
2902
O
LEU
B
158
−6.416
8.460
−24.201
1.00
45.58
8
O

ATOM
2903
N
ARG
B
159
−8.288
8.217
−25.422
1.00
44.66
7
N

ATOM
2904
CA
ARG
B
159
−9.013
9.220
−24.650
1.00
44.17
6
C

ATOM
2905
CB
ARG
B
159
−10.315
9.610
−25.341
1.00
44.51
6
C

ATOM
2906
CG
ARG
B
159
−10.975
10.832
−24.730
1.00
47.41
6
C

ATOM
2907
CD
ARG
B
159
−12.466
10.915
−24.992
1.00
51.06
6
C

ATOM
2908
NE
ARG
B
159
−12.853
12.232
−25.485
1.00
53.52
7
N

ATOM
2909
CZ
ARG
B
159
−12.653
12.641
−26.733
1.00
55.08
6
C

ATOM
2910
NH1
ARG
B
159
−12.065
11.835
−27.613
1.00
55.53
7
N

ATOM
2911
NH2
ARG
B
159
−13.039
13.855
−27.105
1.00
55.21
7
N

ATOM
2912
C
ARG
B
159
−9.315
8.688
−23.259
1.00
43.00
6
C

ATOM
2913
O
ARG
B
159
−9.125
9.375
−22.257
1.00
42.82
8
O

ATOM
2914
N
THR
B
160
−9.799
7.457
−23.203
1.00
41.62
7
N

ATOM
2915
CA
THR
B
160
−10.102
6.827
−21.929
1.00
40.46
6
C

ATOM
2916
CB
THR
B
160
−11.342
7.472
−21.271
1.00
40.57
6
C

ATOM
2917
OG1
THR
B
160
−11.439
7.048
−19.902
1.00
40.80
8
O

ATOM
2918
CG2
THR
B
160
−12.633
6.962
−21.911
1.00
41.26
6
C

ATOM
2919
C
THR
B
160
−10.288
5.327
−22.108
1.00
39.68
6
C

ATOM
2920
O
THR
B
160
−10.299
4.818
−23.231
1.00
38.81
8
O

ATOM
2921
N
TRP
B
161
−10.424
4.625
−20.993
1.00
38.77
7
N

ATOM
2922
CA
TRP
B
161
−10.577
3.185
−21.019
1.00
38.55
6
C

ATOM
2923
CB
TRP
B
161
−9.227
2.508
−21.276
1.00
38.41
6
C

ATOM
2924
CG
TRP
B
161
−8.281
2.658
−20.114
1.00
39.64
6
C

ATOM
2925
CD1
TRP
B
161
−7.445
3.708
−19.870
1.00
40.03
6
C

ATOM
2926
NE1
TRP
B
161
−6.749
3.499
−18.702
1.00
41.04
7
N

ATOM
2927
CE2
TRP
B
161
−7.135
2.300
−18.163
1.00
40.87
6
C

ATOM
2928
CD2
TRP
B
161
−8.105
1.745
−19.024
1.00
39.93
6
C

ATOM
2929
CE3
TRP
B
161
−8.657
0.503
−18.694
1.00
40.64
6
C

ATOM
2930
CZ3
TRP
B
161
−8.240
−0.130
−17.537
1.00
41.98
6
C

ATOM
2931
CH2
TRP
B
161
−7.276
0.450
−16.702
1.00
41.80
6
C

ATOM
2932
CZ2
TRP
B
161
−6.712
1.661
−16.999
1.00
41.26
6
C

ATOM
2933
C
TRP
B
161
−11.096
2.741
−19.676
1.00
37.76
6
C

ATOM
2934
O
TRP
B
161
−11.030
3.491
−18.706
1.00
37.63
8
O

ATOM
2935
N
PHE
B
162
−11.637
1.531
−19.625
1.00
37.15
7
N

ATOM
2936
CA
PHE
B
162
−12.058
0.936
−18.359
1.00
36.85
6
C

ATOM
2937
CB
PHE
B
162
−13.321
1.594
−17.770
1.00
37.15
6
C

ATOM
2938
CG
PHE
B
162
−14.592
1.319
−18.540
1.00
36.74
6
C

ATOM
2939
CD1
PHE
B
162
−15.378
0.213
−18.246
1.00
36.22
6
C

ATOM
2940
CE1
PHE
B
162
−16.549
−0.033
−18.944
1.00
37.13
6
C

ATOM
2941
CZ
PHE
B
162
−16.956
0.835
−19.935
1.00
35.85
6
C

ATOM
2942
CE2
PHE
B
162
−16.191
1.945
−20.235
1.00
36.28
6
C

ATOM
2943
CD2
PHE
B
162
−15.011
2.185
−19.533
1.00
36.01
6
C

ATOM
2944
C
PHE
B
162
−12.215
−0.566
−18.510
1.00
36.88
6
C

ATOM
2945
O
PHE
B
162
−12.373
−1.070
−19.617
1.00
36.24
8
O

ATOM
2946
N
ARG
B
163
−12.133
−1.285
−17.398
1.00
36.54
7
N

ATOM
2947
CA
ARG
B
163
−12.281
−2.732
−17.446
1.00
36.80
6
C

ATOM
2948
CB
ARG
B
163
−11.065
−3.427
−16.822
1.00
36.93
6
C

ATOM
2949
CG
ARG
B
163
−10.863
−3.121
−15.340
1.00
37.83
6
C

ATOM
2950
CD
ARG
B
163
−9.774
−3.974
−14.676
1.00
40.09
6
C

ATOM
2951
NE
ARG
B
163
−8.523
−3.931
−15.431
1.00
40.80
7
N

ATOM
2952
CZ
ARG
B
163
−7.968
−4.978
−16.032
1.00
41.56
6
C

ATOM
2953
NH1
ARG
B
163
−8.543
−6.173
−15.976
1.00
41.79
7
N

ATOM
2954
NH2
ARG
B
163
−6.826
−4.831
−16.689
1.00
41.68
7
N

ATOM
2955
C
ARG
B
163
−13.541
−3.151
−16.714
1.00
36.35
6
C

ATOM
2956
O
ARG
B
163
−14.005
−2.451
−15.820
1.00
36.32
8
O

ATOM
2957
N
THR
B
164
−14.121
−4.268
−17.141
1.00
36.11
7
N

ATOM
2958
CA
THR
B
164
−15.242
−4.881
−16.440
1.00
36.21
6
C

ATOM
2959
CB
THR
B
164
−16.510
−4.900
−17.302
1.00
36.10
6
C

ATOM
2960
OG1
THR
B
164
−16.329
−5.800
−18.409
1.00
36.37
8
O

ATOM
2961
CG2
THR
B
164
−16.727
−3.544
−17.965
1.00
36.23
6
C

ATOM
2962
C
THR
B
164
−14.785
−6.311
−16.163
1.00
36.56
6
C

ATOM
2963
O
THR
B
164
−13.656
−6.665
−16.492
1.00
36.28
8
O

ATOM
2964
N
ARG
B
165
−15.651
−7.130
−15.582
1.00
36.60
7
N

ATOM
2965
CA
ARG
B
165
−15.292
−8.518
−15.306
1.00
37.60
6
C

ATOM
2966
CB
ARG
B
165
−16.314
−9.166
−14.370
1.00
37.85
6
C

ATOM
2967
CG
ARG
B
165
−16.517
−8.457
−13.039
1.00
38.71
6
C

ATOM
2968
CD
ARG
B
165
−17.309
−9.283
−12.047
1.00
40.03
6
C

ATOM
2969
NE
ARG
B
165
−16.648
−10.561
−11.799
1.00
42.08
7
N

ATOM
2970
CZ
ARG
B
165
−17.224
−11.605
−11.210
1.00
43.13
6
C

ATOM
2971
NH1
ARG
B
165
−18.485
−11.529
−10.799
1.00
43.55
7
N

ATOM
2972
NH2
ARG
B
165
−16.538
−12.725
−11.028
1.00
42.32
7
N

ATOM
2973
C
ARG
B
165
−15.202
−9.372
−16.564
1.00
37.61
6
C

ATOM
2974
O
ARG
B
165
−14.737
−10.510
−16.502
1.00
38.14
8
O

ATOM
2975
N
SER
B
166
−15.666
−8.843
−17.696
1.00
37.20
7
N

ATOM
2976
CA
SER
B
166
−15.697
−9.618
−18.936
1.00
36.59
6
C

ATOM
2977
CB
SER
B
166
−17.138
−9.809
−19.422
1.00
36.76
6
C

ATOM
2978
OG
SER
B
166
−17.986
−10.287
−18.396
1.00
37.78
8
O

ATOM
2979
C
SER
B
166
−14.885
−9.027
−20.076
1.00
35.94
6
C

ATOM
2980
O
SER
B
166
−14.591
−9.721
−21.036
1.00
35.60
8
O

ATOM
2981
N
ALA
B
167
−14.525
−7.751
−19.986
1.00
35.33
7
N

ATOM
2982
CA
ALA
B
167
−13.796
−7.124
−21.082
1.00
35.07
6
C

ATOM
2983
CB
ALA
B
167
−14.776
−6.801
−22.223
1.00
35.05
6
C

ATOM
2984
C
ALA
B
167
−13.039
−5.862
−20.692
1.00
34.47
6
C

ATOM
2985
O
ALA
B
167
−13.225
−5.328
−19.607
1.00
34.75
8
O

ATOM
2986
N
ILE
B
168
−12.165
−5.411
−21.585
1.00
34.39
7
N

ATOM
2987
CA
ILE
B
168
−11.539
−4.101
−21.450
1.00
34.20
6
C

ATOM
2988
CB
ILE
B
168
−9.998
−4.158
−21.534
1.00
34.23
6
C

ATOM
2989
CG1
ILE
B
168
−9.404
−2.744
−21.420
1.00
34.54
6
C

ATOM
2990
CD1
ILE
B
168
−7.881
−2.715
−21.338
1.00
36.87
6
C

ATOM
2991
CG2
ILE
B
168
−9.533
−4.816
−22.826
1.00
34.54
6
C

ATOM
2992
C
ILE
B
168
−12.127
−3.246
−22.574
1.00
34.13
6
C

ATOM
2993
O
ILE
B
168
−12.311
−3.739
−23.698
1.00
33.84
8
O

ATOM
2994
N
ILE
B
169
−12.468
−1.997
−22.249
1.00
33.64
7
N

ATOM
2995
CA
ILE
B
169
−13.064
−1.057
−23.202
1.00
33.32
6
C

ATOM
2996
CB
ILE
B
169
−14.420
−0.521
−22.676
1.00
33.61
6
C

ATOM
2997
CG1
ILE
B
169
−15.469
−1.635
−22.639
1.00
33.72
6
C

ATOM
2998
CD1
ILE
B
169
−15.300
−2.621
−21.494
1.00
36.49
6
C

ATOM
2999
CG2
ILE
B
169
−14.922
0.618
−23.555
1.00
33.32
6
C

ATOM
3000
C
ILE
B
169
−12.099
0.095
−23.474
1.00
33.28
6
C

ATOM
3001
O
ILE
B
169
−11.649
0.774
−22.552
1.00
33.30
8
O

ATOM
3002
N
LEU
B
170
−11.765
0.292
−24.740
1.00
33.25
7
N

ATOM
3003
CA
LEU
B
170
−10.801
1.310
−25.137
1.00
33.87
6
C

ATOM
3004
CB
LEU
B
170
−9.625
0.642
−25.845
1.00
33.84
6
C

ATOM
3005
CG
LEU
B
170
−8.876
−0.317
−24.920
1.00
34.72
6
C

ATOM
3006
CD1
LEU
B
170
−8.437
−1.581
−25.646
1.00
36.16
6
C

ATOM
3007
CD2
LEU
B
170
−7.686
0.400
−24.301
1.00
35.50
6
C

ATOM
3008
C
LEU
B
170
−11.434
2.353
−26.043
1.00
34.26
6
C

ATOM
3009
O
LEU
B
170
−11.923
2.033
−27.118
1.00
33.92
8
O

ATOM
3010
N
HIS
B
171
−11.406
3.606
−25.602
1.00
35.06
7
N

ATOM
3011
CA
HIS
B
171
−11.995
4.704
−26.364
1.00
35.98
6
C

ATOM
3012
CB
HIS
B
171
−12.955
5.487
−25.460
1.00
35.84
6
C

ATOM
3013
CG
HIS
B
171
−13.669
6.609
−26.147
1.00
36.49
6
C

ATOM
3014
ND1
HIS
B
171
−14.038
6.562
−27.473
1.00
37.25
7
N

ATOM
3015
CE1
HIS
B
171
−14.645
7.690
−27.800
1.00
37.12
6
C

ATOM
3016
NE2
HIS
B
171
−14.690
8.463
−26.731
1.00
36.55
7
N

ATOM
3017
CD2
HIS
B
171
−14.087
7.811
−25.683
1.00
36.20
6
C

ATOM
3018
C
HIS
B
171
−10.918
5.622
−26.955
1.00
36.33
6
C

ATOM
3019
O
HIS
B
171
−10.269
6.368
−26.227
1.00
36.67
8
O

ATOM
3020
N
LEU
B
172
−10.742
5.561
−28.274
1.00
36.92
7
N

ATOM
3021
CA
LEU
B
172
−9.744
6.368
−28.976
1.00
37.64
6
C

ATOM
3022
CB
LEU
B
172
−9.212
5.612
−30.201
1.00
37.85
6
C

ATOM
3023
CG
LEU
B
172
−8.422
4.324
−29.951
1.00
37.61
6
C

ATOM
3024
CD1
LEU
B
172
−8.052
3.648
−31.266
1.00
38.15
6
C

ATOM
3025
CD2
LEU
B
172
−7.169
4.620
−29.132
1.00
38.00
6
C

ATOM
3026
C
LEU
B
172
−10.285
7.735
−29.400
1.00
38.28
6
C

ATOM
3027
O
LEU
B
172
−11.499
7.924
−29.529
1.00
38.09
8
O

ATOM
3028
N
SER
B
173
−9.372
8.675
−29.642
1.00
38.69
7
N

ATOM
3029
CA
SER
B
173
−9.727
10.056
−29.982
1.00
39.00
6
C

ATOM
3030
CB
SER
B
173
−8.496
10.959
−29.858
1.00
39.22
6
C

ATOM
3031
OG
SER
B
173
−7.450
10.474
−30.683
1.00
38.70
8
O

ATOM
3032
C
SER
B
173
−10.358
10.237
−31.360
1.00
39.22
6
C

ATOM
3033
O
SER
B
173
−10.952
11.283
−31.639
1.00
39.75
8
O

ATOM
3034
N
ASN
B
174
−10.222
9.241
−32.231
1.00
38.69
7
N

ATOM
3035
CA
ASN
B
174
−10.866
9.325
−33.533
1.00
38.56
6
C

ATOM
3036
CB
ASN
B
174
−10.104
8.543
−34.607
1.00
38.62
6
C

ATOM
3037
CG
ASN
B
174
−10.080
7.046
−34.351
1.00
39.22
6
C

ATOM
3038
OD1
ASN
B
174
−10.567
6.561
−33.322
1.00
39.24
8
O

ATOM
3039
ND2
ASN
B
174
−9.499
6.302
−35.293
1.00
37.86
7
N

ATOM
3040
C
ASN
B
174
−12.323
8.874
−33.439
1.00
37.88
6
C

ATOM
3041
O
ASN
B
174
−13.026
8.800
−34.443
1.00
37.60
8
O

ATOM
3042
N
GLY
B
175
−12.754
8.565
−32.220
1.00
37.14
7
N

ATOM
3043
CA
GLY
B
175
−14.126
8.163
−31.968
1.00
36.74
6
C

ATOM
3044
C
GLY
B
175
−14.361
6.664
−31.880
1.00
36.07
6
C

ATOM
3045
O
GLY
B
175
−15.415
6.231
−31.431
1.00
35.97
8
O

ATOM
3046
N
SER
B
176
−13.386
5.871
−32.308
1.00
35.44
7
N

ATOM
3047
CA
SER
B
176
−13.526
4.419
−32.266
1.00
34.98
6
C

ATOM
3048
CB
SER
B
176
−12.402
3.737
−33.039
1.00
34.77
6
C

ATOM
3049
OG
SER
B
176
−12.541
3.974
−34.423
1.00
35.05
8
O

ATOM
3050
C
SER
B
176
−13.557
3.885
−30.847
1.00
34.41
6
C

ATOM
3051
O
SER
B
176
−12.898
4.415
−29.954
1.00
34.86
8
O

ATOM
3052
N
VAL
B
177
−14.339
2.832
−30.646
1.00
33.80
7
N

ATOM
3053
CA
VAL
B
177
−14.424
2.178
−29.355
1.00
32.73
6
C

ATOM
3054
CB
VAL
B
177
−15.810
2.340
−28.734
1.00
33.19
6
C

ATOM
3055
CG1
VAL
B
177
−15.914
1.543
−27.446
1.00
32.36
6
C

ATOM
3056
CG2
VAL
B
177
−16.107
3.823
−28.466
1.00
32.87
6
C

ATOM
3057
C
VAL
B
177
−14.103
0.698
−29.568
1.00
32.66
6
C

ATOM
3058
O
VAL
B
177
−14.716
0.032
−30.407
1.00
31.91
8
O

ATOM
3059
N
GLN
B
178
−13.120
0.200
−28.829
1.00
31.88
7
N

ATOM
3060
CA
GLN
B
178
−12.699
−1.185
−28.973
1.00
32.15
6
C

ATOM
3061
CB
GLN
B
178
−11.198
−1.274
−29.260
1.00
32.48
6
C

ATOM
3062
CG
GLN
B
178
−10.692
−2.708
−29.355
1.00
32.29
6
C

ATOM
3063
CD
GLN
B
178
−9.298
−2.798
−29.915
1.00
32.59
6
C

ATOM
3064
OE1
GLN
B
178
−8.862
−1.923
−30.671
1.00
32.25
8
O

ATOM
3065
NE2
GLN
B
178
−8.589
−3.859
−29.552
1.00
33.06
7
N

ATOM
3066
C
GLN
B
178
−13.029
−1.947
−27.715
1.00
31.55
6
C

ATOM
3067
O
GLN
B
178
−12.812
−1.449
−26.609
1.00
31.51
8
O

ATOM
3068
N
ILE
B
179
−13.587
−3.144
−27.885
1.00
31.35
7
N

ATOM
3069
CA
ILE
B
179
−13.932
−3.995
−26.755
1.00
30.76
6
C

ATOM
3070
CB
ILE
B
179
−15.466
−4.117
−26.602
1.00
30.79
6
C

ATOM
3071
CG1
ILE
B
179
−16.121
−2.739
−26.491
1.00
30.81
6
C

ATOM
3072
CD1
ILE
B
179
−17.647
−2.801
−26.483
1.00
31.41
6
C

ATOM
3073
CG2
ILE
B
179
−15.828
−4.966
−25.389
1.00
30.53
6
C

ATOM
3074
C
ILE
B
179
−13.299
−5.378
−26.934
1.00
31.25
6
C

ATOM
3075
O
ILE
B
179
−13.595
−6.082
−27.909
1.00
30.19
8
O

ATOM
3076
N
ASN
B
180
−12.419
−5.750
−26.004
1.00
31.44
7
N

ATOM
3077
CA
ASN
B
180
−11.754
−7.059
−26.029
1.00
32.18
6
C

ATOM
3078
CB
ASN
B
180
−10.245
−6.933
−25.787
1.00
32.08
6
C

ATOM
3079
CG
ASN
B
180
−9.514
−6.306
−26.943
1.00
33.32
6
C

ATOM
3080
OD1
ASN
B
180
−10.120
−5.723
−27.832
1.00
33.17
8
O

ATOM
3081
ND2
ASN
B
180
−8.187
−6.419
−26.936
1.00
33.68
7
N

ATOM
3082
C
ASN
B
180
−12.322
−7.922
−24.930
1.00
32.14
6
C

ATOM
3083
O
ASN
B
180
−12.172
−7.597
−23.750
1.00
32.05
8
O

ATOM
3084
N
PHE
B
181
−12.977
−9.016
−25.302
1.00
32.45
7
N

ATOM
3085
CA
PHE
B
181
−13.565
−9.919
−24.318
1.00
32.92
6
C

ATOM
3086
CB
PHE
B
181
−14.752
−10.685
−24.921
1.00
32.53
6
C

ATOM
3087
CG
PHE
B
181
−15.944
−9.809
−25.216
1.00
33.45
6
C

ATOM
3088
CD1
PHE
B
181
−16.174
−9.336
−26.498
1.00
31.84
6
C

ATOM
3089
CE1
PHE
B
181
−17.263
−8.509
−26.770
1.00
32.71
6
C

ATOM
3090
CZ
PHE
B
181
−18.130
−8.158
−25.757
1.00
31.84
6
C

ATOM
3091
CE2
PHE
B
181
−17.904
−8.617
−24.474
1.00
32.79
6
C

ATOM
3092
CD2
PHE
B
181
−16.815
−9.435
−24.204
1.00
32.67
6
C

ATOM
3093
C
PHE
B
181
−12.505
−10.875
−23.766
1.00
33.64
6
C

ATOM
3094
O
PHE
B
181
−11.829
−11.555
−24.525
1.00
33.85
8
O

ATOM
3095
N
PHE
B
182
−12.376
−10.912
−22.442
1.00
34.71
7
N

ATOM
3096
CA
PHE
B
182
−11.360
−11.722
−21.755
1.00
35.89
6
C

ATOM
3097
CB
PHE
B
182
−11.391
−11.442
−20.245
1.00
35.83
6
C

ATOM
3098
CG
PHE
B
182
−11.052
−10.023
−19.877
1.00
35.30
6
C

ATOM
3099
CD1
PHE
B
182
−11.754
−9.373
−18.879
1.00
34.96
6
C

ATOM
3100
CE1
PHE
B
182
−11.449
−8.081
−18.533
1.00
35.25
6
C

ATOM
3101
CZ
PHE
B
182
−10.421
−7.413
−19.185
1.00
35.90
6
C

ATOM
3102
CE2
PHE
B
182
−9.717
−8.045
−20.180
1.00
35.75
6
C

ATOM
3103
CD2
PHE
B
182
−10.033
−9.348
−20.521
1.00
35.84
6
C

ATOM
3104
C
PHE
B
182
−11.426
−13.235
−21.960
1.00
36.43
6
C

ATOM
3105
O
PHE
B
182
−10.459
−13.841
−22.408
1.00
37.17
8
O

ATOM
3106
N
GLN
B
183
−12.554
−13.847
−21.622
1.00
37.30
7
N

ATOM
3107
CA
GLN
B
183
−12.657
−15.315
−21.637
1.00
37.89
6
C

ATOM
3108
CB
GLN
B
183
−13.905
−15.795
−20.885
1.00
38.49
6
C

ATOM
3109
CG
GLN
B
183
−13.600
−16.691
−19.676
1.00
42.03
6
C

ATOM
3110
CD
GLN
B
183
−14.591
−17.834
−19.544
1.00
45.34
6
C

ATOM
3111
OE1
GLN
B
183
−15.503
−17.964
−20.365
1.00
47.51
8
O

ATOM
3112
NE2
GLN
B
183
−14.417
−18.665
−18.516
1.00
46.40
7
N

ATOM
3113
C
GLN
B
183
−12.576
−16.025
−22.988
1.00
37.30
6
C

ATOM
3114
O
GLN
B
183
−11.961
−17.090
−23.093
1.00
36.80
8
O

ATOM
3115
N
ASP
B
184
−13.191
−15.455
−24.019
1.00
36.51
7
N

ATOM
3116
CA
ASP
B
184
−13.233
−16.129
−25.314
1.00
35.92
6
C

ATOM
3117
CB
ASP
B
184
−14.667
−16.189
−25.830
1.00
36.35
6
C

ATOM
3118
CG
ASP
B
184
−15.262
−14.814
−26.017
1.00
36.30
6
C

ATOM
3119
OD1
ASP
B
184
−14.506
−13.900
−26.397
1.00
35.71
8
O

ATOM
3120
OD2
ASP
B
184
−16.456
−14.545
−25.775
1.00
39.16
8
O

ATOM
3121
C
ASP
B
184
−12.333
−15.493
−26.357
1.00
35.04
6
C

ATOM
3122
O
ASP
B
184
−12.241
−15.984
−27.475
1.00
35.28
8
O

ATOM

3123

N

HIS

B

185

−11.689

−14.388

−26.001

1.00

34.06

7

N

ATOM

3124

CA

HIS

B

185

−10.746

−13.735

−26.902

1.00

33.53

6

C

ATOM

3125

CB

HIS

B

185

−9.706

−14.749

−27.362

1.00

34.21

6

C

ATOM

3126

CG

HIS

B

185

−8.925

−15.342

−26.232

1.00

36.01

6

C

ATOM

3127

ND1

HIS

B

185

−8.240

−14.567

−25.321

1.00

37.51

7

N

ATOM

3128

CE1

HIS

B

185

−7.657

−15.349

−24.430

1.00

38.12

6

C

ATOM

3129

NE2

HIS

B

185

−7.946

−16.603

−24.726

1.00

38.61

7

N

ATOM

3130

CD2

HIS

B

185

−8.742

−16.627

−25.848

1.00

37.72

6

C

ATOM

3131

C

HIS

B

185

−11.354

−13.017

−28.121

1.00

32.46

6

C

ATOM

3132

O

HIS

B

185

−10.627

−12.603

−29.026

1.00

31.72

8

O

ATOM
3133
N
THR
B
186
−12.674
−12.876
−28.149
1.00
31.83
7
N

ATOM
3134
CA
THR
B
186
−13.302
−12.146
−29.250
1.00
31.41
6
C

ATOM
3135
CB
THR
B
186
−14.777
−12.539
−29.430
1.00
31.18
6
C

ATOM
3136
OG1
THR
B
186
−15.477
−12.376
−28.196
1.00
30.58
8
O

ATOM
3137
CG2
THR
B
186
−14.921
−14.034
−29.737
1.00
31.70
6
C

ATOM
3138
C
THR
B
186
−13.178
−10.638
−29.001
1.00
31.34
6
C

ATOM
3139
O
THR
B
186
−13.050
−10.200
−27.855
1.00
31.21
8
O

ATOM

3140

N

LYS

B

187

−13.212

−9.852

−30.075

1.00

30.85

7

N

ATOM

3141

CA

LYS

B

187

−13.076

−8.406

−29.958

1.00

30.83

6

C

ATOM

3142

CB

LYS

B

187

−11.626

−7.977

−30.210

1.00

31.19

6

C

ATOM

3143

CG

LYS

B

187

−10.576

−8.793

−29.461

1.00

31.58

6

C

ATOM

3144

CD

LYS

B

187

−9.185

−8.345

−29.866

1.00

34.32

6

C

ATOM

3145

CE

LYS

B

187

−8.109

−8.980

−28.981

1.00

34.39

6

C

ATOM

3146

NZ

LYS

B

187

−8.082

−10.456

−29.126

1.00

34.97

7

N

ATOM

3147

C

LYS

B

187

−13.961

−7.685

−30.966

1.00

30.47

6

C

ATOM

3148

O

LYS

B

187

−14.236

−8.211

−32.048

1.00

29.89

8

O

ATOM
3149
N
LEU
B
188
−14.387
−6.480
−30.598
1.00
29.76
7
N

ATOM
3150
CA
LEU
B
188
−15.140
−5.618
−31.493
1.00
30.16
6
C

ATOM
3151
CB
LEU
B
188
−16.533
−5.320
−30.954
1.00
29.59
6
C

ATOM
3152
CG
LEU
B
188
−17.545
−6.424
−30.679
1.00
30.75
6
C

ATOM
3153
CD1
LEU
B
188
−18.701
−5.817
−29.923
1.00
31.35
6
C

ATOM
3154
CD2
LEU
B
188
−18.039
−7.056
−31.959
1.00
32.85
6
C

ATOM
3155
C
LEU
B
188
−14.406
−4.292
−31.639
1.00
30.15
6
C

ATOM
3156
O
LEU
B
188
−13.838
−3.775
−30.678
1.00
30.39
8
O

ATOM
3157
N
ILE
B
189
−14.404
−3.753
−32.845
1.00
30.49
7
N

ATOM
3158
CA
ILE
B
189
−13.876
−2.418
−33.076
1.00
30.85
6
C

ATOM
3159
CB
ILE
B
189
−12.663
−2.446
−34.002
1.00
30.93
6
C

ATOM
3160
CG1
ILE
B
189
−11.558
−3.346
−33.422
1.00
31.25
6
C

ATOM
3161
CD1
ILE
B
189
−10.629
−3.901
−34.473
1.00
33.02
6
C

ATOM
3162
CG2
ILE
B
189
−12.149
−1.025
−34.211
1.00
31.24
6
C

ATOM
3163
C
ILE
B
189
−15.010
−1.620
−33.712
1.00
31.21
6
C

ATOM
3164
O
ILE
B
189
−15.417
−1.912
−34.832
1.00
31.08
8
O

ATOM
3165
N
LEU
B
190
−15.527
−0.632
−32.988
1.00
31.34
7
N

ATOM
3166
CA
LEU
B
190
−16.656
0.159
−33.478
1.00
31.95
6
C

ATOM
3167
CB
LEU
B
190
−17.745
0.256
−32.404
1.00
31.74
6
C

ATOM
3168
CG
LEU
B
190
−18.423
−1.062
−31.992
1.00
31.40
6
C

ATOM
3169
CD1
LEU
B
190
−18.772
−1.078
−30.505
1.00
32.33
6
C

ATOM
3170
CD2
LEU
B
190
−19.668
−1.321
−32.823
1.00
30.49
6
C

ATOM
3171
C
LEU
B
190
−16.216
1.553
−33.908
1.00
32.26
6
C

ATOM
3172
O
LEU
B
190
−15.495
2.233
−33.187
1.00
32.36
8
O

ATOM
3173
N
CYS
B
191
−16.648
1.970
−35.093
1.00
32.91
7
N

ATOM
3174
CA
CYS
B
191
−16.347
3.310
−35.593
1.00
33.26
6
C

ATOM
3175
CB
CYS
B
191
−15.517
3.243
−36.873
1.00
33.24
6
C

ATOM
3176
SG
CYS
B
191
−15.232
4.869
−37.646
1.00
34.76
16
S

ATOM
3177
C
CYS
B
191
−17.657
4.032
−35.879
1.00
33.25
6
C

ATOM
3178
O
CYS
B
191
−18.436
3.575
−36.703
1.00
33.43
8
O

ATOM
3179
N
PRO
B
192
−17.904
5.141
−35.187
1.00
33.55
7
N

ATOM
3180
CA
PRO
B
192
−19.135
5.919
−35.361
1.00
34.06
6
C

ATOM
3181
CB
PRO
B
192
−19.162
6.758
−34.090
1.00
33.94
6
C

ATOM
3182
CG
PRO
B
192
−17.714
7.062
−33.888
1.00
33.71
6
C

ATOM
3183
CD
PRO
B
192
−17.042
5.729
−34.146
1.00
33.40
6
C

ATOM
3184
C
PRO
B
192
−19.128
6.817
−36.600
1.00
34.90
6
C

ATOM
3185
O
PRO
B
192
−20.185
7.339
−36.978
1.00
34.87
8
O

ATOM
3186
N
LEU
B
193
−17.959
7.003
−37.212
1.00
35.55
7
N

ATOM
3187
CA
LEU
B
193
−17.849
7.806
−38.429
1.00
36.44
6
C

ATOM
3188
CB
LEU
B
193
−16.401
8.228
−38.670
1.00
36.83
6
C

ATOM
3189
CG
LEU
B
193
−15.830
9.480
−38.002
1.00
38.55
6
C

ATOM
3190
CD1
LEU
B
193
−16.082
9.509
−36.509
1.00
38.97
6
C

ATOM
3191
CD2
LEU
B
193
−14.338
9.579
−38.290
1.00
39.57
6
C

ATOM
3192
C
LEU
B
193
−18.339
6.986
−39.609
1.00
36.47
6
C

ATOM
3193
O
LEU
B
193
−19.032
7.490
−40.488
1.00
37.34
8
O

ATOM
3194
N
MET
B
194
−17.987
5.706
−39.617
1.00
36.22
7
N

ATOM
3195
CA
MET
B
194
−18.396
4.804
−40.684
1.00
35.99
6
C

ATOM
3196
CB
MET
B
194
−17.258
3.833
−41.006
1.00
36.75
6
C

ATOM
3197
CG
MET
B
194
−15.974
4.507
−41.482
1.00
40.37
6
C

ATOM
3198
SD
MET
B
194
−16.263
5.440
−42.975
1.00
48.21
16
S

ATOM
3199
CE
MET
B
194
−16.542
4.127
−44.115
1.00
45.23
6
C

ATOM
3200
C
MET
B
194
−19.637
4.001
−40.297
1.00
34.72
6
C

ATOM
3201
O
MET
B
194
−20.161
3.232
−41.110
1.00
34.69
8
O

ATOM
3202
N
ALA
B
195
−20.102
4.187
−39.061
1.00
32.98
7
N

ATOM
3203
CA
ALA
B
195
−21.232
3.421
−38.532
1.00
31.57
6
C

ATOM
3204
CB
ALA
B
195
−22.562
3.901
−39.143
1.00
31.57
6
C

ATOM
3205
C
ALA
B
195
−21.001
1.938
−38.810
1.00
30.28
6
C

ATOM
3206
O
ALA
B
195
−21.856
1.254
−39.373
1.00
29.74
8
O

ATOM
3207
N
ALA
B
196
−19.836
1.447
−38.394
1.00
29.57
7
N

ATOM
3208
CA
ALA
B
196
−19.434
0.076
−38.667
1.00
29.18
6
C

ATOM
3209
CB
ALA
B
196
−18.423
0.049
−39.814
1.00
29.60
6
C

ATOM
3210
C
ALA
B
196
−18.852
−0.646
−37.458
1.00
29.28
6
C

ATOM
3211
O
ALA
B
196
−18.476
−0.023
−36.457
1.00
29.26
8
O

ATOM
3212
N
VAL
B
197
−18.774
−1.965
−37.574
1.00
28.98
7
N

ATOM
3213
CA
VAL
B
197
−18.202
−2.794
−36.526
1.00
28.77
6
C

ATOM
3214
CB
VAL
B
197
−19.281
−3.482
−35.649
1.00
28.71
6
C

ATOM
3215
CG1
VAL
B
197
−20.183
−4.404
−36.484
1.00
28.89
6
C

ATOM
3216
CG2
VAL
B
197
−18.624
−4.270
−34.509
1.00
29.06
6
C

ATOM
3217
C
VAL
B
197
−17.329
−3.851
−37.170
1.00
29.08
6
C

ATOM
3218
O
VAL
B
197
−17.703
−4.458
−38.172
1.00
28.66
8
O

ATOM
3219
N
THR
B
198
−16.147
−4.045
−36.599
1.00
29.05
7
N

ATOM
3220
CA
THR
B
198
−15.260
−5.115
−37.017
1.00
29.47
6
C

ATOM
3221
CB
THR
B
198
−13.830
−4.589
−37.150
1.00
29.50
6
C

ATOM
3222
OG1
THR
B
198
−13.763
−3.713
−38.279
1.00
30.50
8
O

ATOM
3223
CG2
THR
B
198
−12.858
−5.715
−37.527
1.00
29.77
6
C

ATOM
3224
C
THR
B
198
−15.334
−6.159
−35.923
1.00
29.99
6
C

ATOM
3225
O
THR
B
198
−15.176
−5.832
−34.745
1.00
29.34
8
O

ATOM
3226
N
TYR
B
199
−15.615
−7.400
−36.308
1.00
29.93
7
N

ATOM
3227
CA
TYR
B
199
−15.720
−8.472
−35.341
1.00
30.91
6
C

ATOM
3228
CB
TYR
B
199
−17.046
−9.224
−35.521
1.00
30.44
6
C

ATOM
3229
CG
TYR
B
199
−17.254
−10.364
−34.558
1.00
30.96
6
C

ATOM
3230
CD1
TYR
B
199
−16.804
−10.285
−33.247
1.00
31.88
6
C

ATOM
3231
CE1
TYR
B
199
−16.991
−11.325
−32.365
1.00
32.55
6
C

ATOM
3232
CZ
TYR
B
199
−17.647
−12.459
−32.774
1.00
33.17
6
C

ATOM
3233
OH
TYR
B
199
−17.840
−13.489
−31.878
1.00
36.31
8
O

ATOM
3234
CE2
TYR
B
199
−18.110
−12.564
−34.067
1.00
33.56
6
C

ATOM
3235
CD2
TYR
B
199
−17.915
−11.519
−34.952
1.00
32.17
6
C

ATOM
3236
C
TYR
B
199
−14.554
−9.419
−35.560
1.00
31.41
6
C

ATOM
3237
O
TYR
B
199
−14.332
−9.887
−36.673
1.00
31.16
8
O

ATOM
3238
N
ILE
B
200
−13.794
−9.662
−34.502
1.00
32.55
7
N

ATOM
3239
CA
ILE
B
200
−12.714
−10.635
−34.534
1.00
33.59
6
C

ATOM
3240
CB
ILE
B
200
−11.414
−10.031
−33.962
1.00
33.63
6
C

ATOM
3241
CG1
ILE
B
200
−10.948
−8.870
−34.843
1.00
33.74
6
C

ATOM
3242
CD1
ILE
B
200
−9.712
−8.133
−34.330
1.00
35.27
6
C

ATOM
3243
CG2
ILE
B
200
−10.325
−11.108
−33.866
1.00
33.81
6
C

ATOM
3244
C
ILE
B
200
−13.198
−11.801
−33.693
1.00
34.45
6
C

ATOM
3245
O
ILE
B
200
−13.412
−11.656
−32.496
1.00
34.78
8
O

ATOM
3246
N
ASP
B
201
−13.410
−12.953
−34.318
1.00
35.60
7
N

ATOM
3247
CA
ASP
B
201
−13.973
−14.086
−33.597
1.00
36.61
6
C

ATOM
3248
CB
ASP
B
201
−14.868
−14.925
−34.508
1.00
36.72
6
C

ATOM
3249
CG
ASP
B
201
−14.106
−15.575
−35.654
1.00
37.71
6
C

ATOM
3250
OD1
ASP
B
201
−12.851
−15.613
−35.626
1.00
37.60
8
O

ATOM
3251
OD2
ASP
B
201
−14.696
−16.088
−36.627
1.00
38.71
8
O

ATOM
3252
C
ASP
B
201
−12.908
−14.948
−32.924
1.00
37.15
6
C

ATOM
3253
O
ASP
B
201
−11.729
−14.620
−32.962
1.00
37.08
8
O

ATOM
3254
N
GLU
B
202
−13.335
−16.045
−32.311
1.00
38.32
7
N

ATOM
3255
CA
GLU
B
202
−12.408
−16.924
−31.599
1.00
39.84
6
C

ATOM
3256
CB
GLU
B
202
−13.148
−17.883
−30.658
1.00
40.25
6
C

ATOM
3257
CG
GLU
B
202
−14.367
−18.567
−31.256
1.00
42.60
6
C

ATOM
3258
CD
GLU
B
202
−15.614
−17.703
−31.162
1.00
45.06
6
C

ATOM
3259
OE1
GLU
B
202
−16.208
−17.626
−30.059
1.00
46.48
8
O

ATOM
3260
OE2
GLU
B
202
−15.989
−17.097
−32.186
1.00
44.47
8
O

ATOM
3261
C
GLU
B
202
−11.434
−17.690
−32.503
1.00
40.07
6
C

ATOM
3262
O
GLU
B
202
−10.446
−18.235
−32.013
1.00
40.52
8
O

ATOM
3263
N
LYS
B
203
−11.700
−17.730
−33.808
1.00
40.52
7
N

ATOM
3264
CA
LYS
B
203
−10.784
−18.372
−34.754
1.00
41.15
6
C

ATOM
3265
CB
LYS
B
203
−11.518
−18.902
−35.986
1.00
41.26
6
C

ATOM
3266
CG
LYS
B
203
−12.568
−19.953
−35.768
1.00
42.20
6
C

ATOM
3267
CD
LYS
B
203
−13.049
−20.405
−37.141
1.00
43.91
6
C

ATOM
3268
CE
LYS
B
203
−14.421
−21.032
−37.111
1.00
45.01
6
C

ATOM
3269
NZ
LYS
B
203
−14.983
−21.106
−38.487
1.00
45.80
7
N

ATOM
3270
C
LYS
B
203
−9.806
−17.329
−35.254
1.00
41.28
6
C

ATOM
3271
O
LYS
B
203
−8.943
−17.619
−36.080
1.00
41.11
8
O

ATOM
3272
N
ARG
B
204
−9.971
−16.106
−34.761
1.00
41.53
7
N

ATOM
3273
CA
ARG
B
204
−9.168
−14.958
−35.179
1.00
42.04
6
C

ATOM
3274
CB
ARG
B
204
−7.666
−15.235
−35.094
1.00
42.26
6
C

ATOM
3275
CG
ARG
B
204
−7.154
−15.387
−33.682
1.00
44.06
6
C

ATOM
3276
CD
ARG
B
204
−5.672
−15.068
−33.530
1.00
47.27
6
C

ATOM
3277
NE
ARG
B
204
−5.037
−15.848
−32.470
1.00
49.18
7
N

ATOM
3278
CZ
ARG
B
204
−5.371
−15.794
−31.189
1.00
50.09
6
C

ATOM
3279
NH1
ARG
B
204
−6.347
−14.989
−30.787
1.00
51.10
7
N

ATOM
3280
NH2
ARG
B
204
−4.729
−16.549
−30.302
1.00
50.21
7
N

ATOM
3281
C
ARG
B
204
−9.552
−14.443
−36.564
1.00
41.94
6
C

ATOM
3282
O
ARG
B
204
−8.838
−13.641
−37.163
1.00
41.39
8
O

ATOM
3283
N
ASP
B
205
−10.680
−14.912
−37.082
1.00
42.30
7
N

ATOM
3284
CA
ASP
B
205
−11.159
−14.383
−38.343
1.00
42.63
6
C

ATOM
3285
CB
ASP
B
205
−12.084
−15.355
−39.059
1.00
43.15
6
C

ATOM
3286
CG
ASP
B
205
−11.363
−16.147
−40.116
1.00
45.20
6
C

ATOM
3287
OD1
ASP
B
205
−11.487
−15.791
−41.308
1.00
48.54
8
O

ATOM
3288
OD2
ASP
B
205
−10.636
−17.126
−39.847
1.00
46.34
8
O

ATOM
3289
C
ASP
B
205
−11.847
−13.064
−38.078
1.00
42.42
6
C

ATOM
3290
O
ASP
B
205
−12.376
−12.828
−36.988
1.00
42.54
8
O

ATOM
3291
N
PHE
B
206
−11.827
−12.202
−39.080
1.00
41.94
7
N

ATOM
3292
CA
PHE
B
206
−12.350
−10.863
−38.928
1.00
41.63
6
C

ATOM
3293
CB
PHE
B
206
−11.191
−9.880
−38.826
1.00
41.67
6
C

ATOM
3294
CG
PHE
B
206
−10.306
−9.887
−40.032
1.00
43.86
6
C

ATOM
3295
CD1
PHE
B
206
−10.387
−8.874
−40.974
1.00
45.15
6
C

ATOM
3296
CE1
PHE
B
206
−9.581
−8.890
−42.094
1.00
45.57
6
C

ATOM
3297
CZ
PHE
B
206
−8.691
−9.931
−42.294
1.00
46.16
6
C

ATOM
3298
CE2
PHE
B
206
−8.608
−10.952
−41.367
1.00
46.38
6
C

ATOM
3299
CD2
PHE
B
206
−9.416
−10.929
−40.245
1.00
45.29
6
C

ATOM
3300
C
PHE
B
206
−13.214
−10.475
−40.111
1.00
40.74
6
C

ATOM
3301
O
PHE
B
206
−12.981
−10.896
−41.253
1.00
40.86
8
O

ATOM
3302
N
ARG
B
207
−14.203
−9.644
−39.828
1.00
39.32
7
N

ATOM
3303
CA
ARG
B
207
−15.099
−9.141
−40.843
1.00
38.06
6
C

ATOM
3304
CB
ARG
B
207
−16.283
−10.086
−41.014
1.00
38.93
6
C

ATOM
3305
CG
ARG
B
207
−15.921
−11.425
−41.649
1.00
41.23
6
C

ATOM
3306
CD
ARG
B
207
−15.806
−11.365
−43.161
1.00
45.10
6
C

ATOM
3307
NE
ARG
B
207
−15.000
−12.445
−43.720
1.00
47.95
7
N

ATOM
3308
CZ
ARG
B
207
−15.354
−13.725
−43.735
1.00
49.42
6
C

ATOM
3309
NH1
ARG
B
207
−16.504
−14.113
−43.204
1.00
50.39
7
N

ATOM
3310
NH2
ARG
B
207
−14.548
−14.624
−44.281
1.00
49.46
7
N

ATOM
3311
C
ARG
B
207
−15.575
−7.786
−40.365
1.00
36.54
6
C

ATOM
3312
O
ARG
B
207
−15.753
−7.576
−39.167
1.00
35.56
8
O

ATOM
3313
N
THR
B
208
−15.750
−6.864
−41.299
1.00
34.48
7
N

ATOM
3314
CA
THR
B
208
−16.203
−5.526
−40.975
1.00
33.17
6
C

ATOM
3315
CB
THR
B
208
−15.233
−4.497
−41.561
1.00
33.38
6
C

ATOM
3316
OG1
THR
B
208
−13.963
−4.594
−40.887
1.00
32.93
8
O

ATOM
3317
CG2
THR
B
208
−15.715
−3.091
−41.253
1.00
33.01
6
C

ATOM
3318
C
THR
B
208
−17.590
−5.349
−41.577
1.00
32.35
6
C

ATOM
3319
O
THR
B
208
−17.776
−5.583
−42.777
1.00
32.13
8
O

ATOM
3320
N
TYR
B
209
−18.547
−4.929
−40.751
1.00
30.97
7
N

ATOM
3321
CA
TYR
B
209
−19.938
−4.776
−41.175
1.00
30.20
6
C

ATOM
3322
CB
TYR
B
209
−20.846
−5.716
−40.362
1.00
29.85
6
C

ATOM
3323
CG
TYR
B
209
−20.446
−7.168
−40.380
1.00
30.61
6
C

ATOM
3324
CD1
TYR
B
209
−19.672
−7.719
−39.351
1.00
30.41
6
C

ATOM
3325
CE1
TYR
B
209
−19.302
−9.053
−39.377
1.00
31.94
6
C

ATOM
3326
CZ
TYR
B
209
−19.718
−9.844
−40.433
1.00
31.53
6
C

ATOM
3327
OH
TYR
B
209
−19.368
−11.171
−40.498
1.00
30.94
8
O

ATOM
3328
CE2
TYR
B
209
−20.476
−9.310
−41.455
1.00
31.95
6
C

ATOM
3329
CD2
TYR
B
209
−20.830
−7.989
−41.424
1.00
31.27
6
C

ATOM
3330
C
TYR
B
209
−20.480
−3.377
−40.969
1.00
29.27
6
C

ATOM
3331
O
TYR
B
209
−20.145
−2.726
−39.989
1.00
29.32
8
O

ATOM
3332
N
ARG
B
210
−21.345
−2.925
−41.878
1.00
28.16
7
N

ATOM
3333
CA
ARG
B
210
−22.070
−1.676
−41.658
1.00
27.88
6
C

ATOM
3334
CB
ARG
B
210
−22.643
−1.138
−42.969
1.00
27.74
6
C

ATOM
3335
CG
ARG
B
210
−21.594
−0.722
−43.996
1.00
29.12
6
C

ATOM
3336
CD
ARG
B
210
−20.835
0.545
−43.644
1.00
30.87
6
C

ATOM
3337
NE
ARG
B
210
−20.084
1.055
−44.796
1.00
30.84
7
N

ATOM
3338
CZ
ARG
B
210
−19.609
2.288
−44.889
1.00
31.64
6
C

ATOM
3339
NH1
ARG
B
210
−19.792
3.152
−43.899
1.00
31.10
7
N

ATOM
3340
NH2
ARG
B
210
−18.953
2.671
−45.983
1.00
31.71
7
N

ATOM
3341
C
ARG
B
210
−23.216
−1.973
−40.701
1.00
27.62
6
C

ATOM
3342
O
ARG
B
210
−24.034
−2.859
−40.967
1.00
27.36
8
O

ATOM
3343
N
LEU
B
211
−23.311
−1.219
−39.609
1.00
27.26
7
N

ATOM
3344
CA
LEU
B
211
−24.358
−1.463
−38.613
1.00
27.06
6
C

ATOM
3345
CB
LEU
B
211
−24.214
−0.482
−37.445
1.00
26.85
6
C

ATOM
3346
CG
LEU
B
211
−22.968
−0.712
−36.575
1.00
26.35
6
C

ATOM
3347
CD1
LEU
B
211
−22.809
0.428
−35.570
1.00
27.27
6
C

ATOM
3348
CD2
LEU
B
211
−23.061
−2.065
−35.860
1.00
27.54
6
C

ATOM
3349
C
LEU
B
211
−25.774
−1.379
−39.201
1.00
27.36
6
C

ATOM
3350
O
LEU
B
211
−26.637
−2.200
−38.891
1.00
26.70
8
O

ATOM
3351
N
SER
B
212
−26.013
−0.377
−40.039
1.00
27.73
7
N

ATOM
3352
CA
SER
B
212
−27.332
−0.234
−40.668
1.00
28.63
6
C

ATOM
3353
CB
SER
B
212
−27.441
1.079
−41.449
1.00
28.91
6
C

ATOM
3354
OG
SER
B
212
−27.316
2.220
−40.608
1.00
32.24
8
O

ATOM
3355
C
SER
B
212
−27.653
−1.420
−41.582
1.00
28.29
6
C

ATOM
3356
O
SER
B
212
−28.819
−1.789
−41.763
1.00
28.79
8
O

ATOM
3357
N
LEU
B
213
−26.624
−2.010
−42.183
1.00
28.05
7
N

ATOM
3358
CA
LEU
B
213
−26.835
−3.180
−43.029
1.00
27.68
6
C

ATOM
3359
CB
LEU
B
213
−25.668
−3.372
−43.994
1.00
27.38
6
C

ATOM
3360
CG
LEU
B
213
−25.551
−2.331
−45.113
1.00
26.68
6
C

ATOM
3361
CD1
LEU
B
213
−24.458
−2.754
−46.068
1.00
26.26
6
C

ATOM
3362
CD2
LEU
B
213
−26.893
−2.195
−45.857
1.00
26.57
6
C

ATOM
3363
C
LEU
B
213
−27.095
−4.462
−42.216
1.00
28.03
6
C

ATOM
3364
O
LEU
B
213
−27.747
−5.397
−42.695
1.00
27.81
8
O

ATOM
3365
N
LEU
B
214
−26.560
−4.532
−41.001
1.00
27.99
7
N

ATOM
3366
CA
LEU
B
214
−26.835
−5.696
−40.163
1.00
28.19
6
C

ATOM
3367
CB
LEU
B
214
−25.940
−5.723
−38.914
1.00
27.94
6
C

ATOM
3368
CG
LEU
B
214
−24.447
−6.003
−39.141
1.00
28.61
6
C

ATOM
3369
CD1
LEU
B
214
−23.665
−5.834
−37.824
1.00
29.52
6
C

ATOM
3370
CD2
LEU
B
214
−24.214
−7.392
−39.722
1.00
28.82
6
C

ATOM
3371
C
LEU
B
214
−28.312
−5.679
−39.788
1.00
28.08
6
C

ATOM
3372
O
LEU
B
214
−28.947
−6.720
−39.636
1.00
28.21
8
O

ATOM
3373
N
GLU
B
215
−28.858
−4.476
−39.673
1.00
29.11
7
N

ATOM
3374
CA
GLU
B
215
−30.260
−4.263
−39.352
1.00
30.18
6
C

ATOM
3375
CB
GLU
B
215
−30.453
−2.758
−39.187
1.00
30.65
6
C

ATOM
3376
CG
GLU
B
215
−31.844
−2.267
−38.869
1.00
33.74
6
C

ATOM
3377
CD
GLU
B
215
−31.793
−0.878
−38.253
1.00
37.26
6
C

ATOM
3378
OE1
GLU
B
215
−30.680
−0.444
−37.846
1.00
38.83
8
O

ATOM
3379
OE2
GLU
B
215
−32.852
−0.223
−38.186
1.00
39.07
8
O

ATOM
3380
C
GLU
B
215
−31.190
−4.810
−40.442
1.00
30.23
6
C

ATOM
3381
O
GLU
B
215
−32.238
−5.425
−40.171
1.00
30.85
8
O

ATOM
3382
N
GLU
B
216
−30.792
−4.601
−41.685
1.00
29.38
7
N

ATOM
3383
CA
GLU
B
216
−31.588
−5.031
−42.815
1.00
29.69
6
C

ATOM
3384
CB
GLU
B
216
−31.238
−4.165
−44.036
1.00
29.56
6
C

ATOM
3385
CG
GLU
B
216
−31.732
−2.726
−43.962
1.00
31.01
6
C

ATOM
3386
CD
GLU
B
216
−33.244
−2.621
−43.913
1.00
32.22
6
C

ATOM
3387
OE1
GLU
B
216
−33.815
−2.644
−42.796
1.00
35.17
8
O

ATOM
3388
OE2
GLU
B
216
−33.867
−2.525
−44.989
1.00
32.31
8
O

ATOM
3389
C
GLU
B
216
−31.405
−6.514
−43.155
1.00
29.31
6
C

ATOM
3390
O
GLU
B
216
−32.372
−7.203
−43.479
1.00
30.41
8
O

ATOM
3391
N
TYR
B
217
−30.180
−7.013
−43.056
1.00
29.30
7
N

ATOM
3392
CA
TYR
B
217
−29.882
−8.370
−43.499
1.00
29.29
6
C

ATOM
3393
CB
TYR
B
217
−28.592
−8.380
−44.321
1.00
29.39
6
C

ATOM
3394
CG
TYR
B
217
−28.745
−7.699
−45.664
1.00
29.10
6
C

ATOM
3395
CD1
TYR
B
217
−28.194
−6.449
−45.891
1.00
29.63
6
C

ATOM
3396
CE1
TYR
B
217
−28.339
−5.814
−47.126
1.00
29.43
6
C

ATOM
3397
CZ
TYR
B
217
−29.042
−6.431
−48.146
1.00
31.15
6
C

ATOM
3398
OH
TYR
B
217
−29.169
−5.788
−49.369
1.00
31.03
8
O

ATOM
3399
CE2
TYR
B
217
−29.604
−7.672
−47.947
1.00
31.19
6
C

ATOM
3400
CD2
TYR
B
217
−29.461
−8.301
−46.699
1.00
30.94
6
C

ATOM
3401
C
TYR
B
217
−29.807
−9.420
−42.399
1.00
29.70
6
C

ATOM
3402
O
TYR
B
217
−29.875
−10.616
−42.678
1.00
29.44
8
O

ATOM
3403
N
GLY
B
218
−29.648
−8.961
−41.163
1.00
30.30
7
N

ATOM
3404
CA
GLY
B
218
−29.556
−9.833
−40.008
1.00
30.67
6
C

ATOM
3405
C
GLY
B
218
−28.126
−10.233
−39.703
1.00
31.35
6
C

ATOM
3406
O
GLY
B
218
−27.209
−9.917
−40.466
1.00
30.66
8
O

ATOM
3407
N
CYS
B
219
−27.930
−10.913
−38.572
1.00
31.71
7
N

ATOM
3408
CA
CYS
B
219
−26.612
−11.439
−38.224
1.00
32.39
6
C

ATOM
3409
CB
CYS
B
219
−25.646
−10.339
−37.797
1.00
32.73
6
C

ATOM
3410
SG
CYS
B
219
−25.858
−9.654
−36.140
1.00
34.72
16
S

ATOM
3411
C
CYS
B
219
−26.697
−12.566
−37.193
1.00
32.51
6
C

ATOM
3412
O
CYS
B
219
−27.754
−12.803
−36.628
1.00
32.57
8
O

ATOM
3413
N
CYS
B
220
−25.587
−13.262
−36.970
1.00
32.50
7
N

ATOM
3414
CA
CYS
B
220
−25.554
−14.380
−36.027
1.00
32.97
6
C

ATOM
3415
CB
CYS
B
220
−24.231
−15.146
−36.150
1.00
33.12
6
C

ATOM
3416
SG
CYS
B
220
−22.768
−14.149
−35.739
1.00
37.64
16
S

ATOM
3417
C
CYS
B
220
−25.690
−13.925
−34.584
1.00
32.49
6
C

ATOM
3418
O
CYS
B
220
−25.447
−12.759
−34.262
1.00
31.34
8
O

ATOM
3419
N
LYS
B
221
−26.046
−14.871
−33.717
1.00
32.02
7
N

ATOM
3420
CA
LYS
B
221
−26.174
−14.607
−32.286
1.00
32.77
6
C

ATOM
3421
CB
LYS
B
221
−26.625
−15.876
−31.552
1.00
32.93
6
C

ATOM
3422
CG
LYS
B
221
−28.120
−16.043
−31.463
1.00
36.27
6
C

ATOM
3423
CD
LYS
B
221
−28.473
−17.215
−30.528
1.00
39.66
6
C

ATOM
3424
CE
LYS
B
221
−29.913
−17.111
−30.032
1.00
41.43
6
C

ATOM
3425
NZ
LYS
B
221
−30.246
−18.141
−29.000
1.00
43.73
7
N

ATOM
3426
C
LYS
B
221
−24.868
−14.109
−31.674
1.00
32.01
6
C

ATOM
3427
O
LYS
B
221
−24.887
−13.267
−30.777
1.00
32.02
8
O

ATOM
3428
N
GLU
B
222
−23.749
−14.666
−32.136
1.00
31.92
7
N

ATOM
3429
CA
GLU
B
222
−22.404
−14.273
−31.700
1.00
31.80
6
C

ATOM
3430
CB
GLU
B
222
−21.364
−14.845
−32.672
1.00
32.80
6
C

ATOM
3431
CG
GLU
B
222
−20.442
−15.939
−32.158
1.00
37.22
6
C

ATOM
3432
CD
GLU
B
222
−19.162
−16.024
−32.990
1.00
41.59
6
C

ATOM
3433
OE1
GLU
B
222
−18.071
−15.787
−32.426
1.00
42.25
8
O

ATOM
3434
OE2
GLU
B
222
−19.245
−16.296
−34.220
1.00
44.47
8
O

ATOM
3435
C
GLU
B
222
−22.222
−12.758
−31.725
1.00
30.78
6
C

ATOM
3436
O
GLU
B
222
−21.904
−12.116
−30.715
1.00
30.00
8
O

ATOM
3437
N
LEU
B
223
−22.388
−12.189
−32.913
1.00
29.53
7
N

ATOM
3438
CA
LEU
B
223
−22.209
−10.757
−33.092
1.00
28.77
6
C

ATOM
3439
CB
LEU
B
223
−22.097
−10.417
−34.586
1.00
29.52
6
C

ATOM
3440
CG
LEU
B
223
−21.901
−8.933
−34.861
1.00
30.50
6
C

ATOM
3441
CD1
LEU
B
223
−20.755
−8.407
−34.013
1.00
31.04
6
C

ATOM
3442
CD2
LEU
B
223
−21.651
−8.685
−36.358
1.00
32.50
6
C

ATOM
3443
C
LEU
B
223
−23.330
−9.950
−32.453
1.00
28.10
6
C

ATOM
3444
O
LEU
B
223
−23.075
−8.936
−31.809
1.00
27.14
8
O

ATOM
3445
N
ALA
B
224
−24.574
−10.394
−32.631
1.00
27.70
7
N

ATOM
3446
CA
ALA
B
224
−25.721
−9.676
−32.070
1.00
26.94
6
C

ATOM
3447
CB
ALA
B
224
−27.044
−10.393
−32.420
1.00
27.22
6
C

ATOM
3448
C
ALA
B
224
−25.609
−9.496
−30.556
1.00
27.06
6
C

ATOM
3449
O
ALA
B
224
−25.849
−8.408
−30.034
1.00
26.24
8
O

ATOM
3450
N
SER
B
225
−25.269
−10.575
−29.854
1.00
26.87
7
N

ATOM
3451
CA
SER
B
225
−25.155
−10.529
−28.399
1.00
27.50
6
C

ATOM
3452
CB
SER
B
225
−24.989
−11.945
−27.823
1.00
27.52
6
C

ATOM
3453
OG
SER
B
225
−23.738
−12.509
−28.196
1.00
29.93
8
O

ATOM
3454
C
SER
B
225
−24.023
−9.587
−27.964
1.00
27.29
6
C

ATOM
3455
O
SER
B
225
−24.152
−8.853
−26.979
1.00
27.90
8
O

ATOM
3456
N
ARG
B
226
−22.929
−9.563
−28.712
1.00
26.68
7
N

ATOM
3457
CA
ARG
B
226
−21.837
−8.660
−28.355
1.00
26.54
6
C

ATOM
3458
CB
ARG
B
226
−20.537
−9.070
−29.038
1.00
26.73
6
C

ATOM
3459
CG
ARG
B
226
−19.945
−10.333
−28.437
1.00
26.92
6
C

ATOM
3460
CD
ARG
B
226
−18.949
−11.028
−29.331
1.00
28.63
6
C

ATOM
3461
NE
ARG
B
226
−18.380
−12.205
−28.675
1.00
28.84
7
N

ATOM
3462
CZ
ARG
B
226
−19.015
−13.364
−28.558
1.00
30.18
6
C

ATOM
3463
NH1
ARG
B
226
−20.231
−13.511
−29.066
1.00
29.81
7
N

ATOM
3464
NH2
ARG
B
226
−18.428
−14.386
−27.938
1.00
30.49
7
N

ATOM
3465
C
ARG
B
226
−22.171
−7.188
−28.621
1.00
26.70
6
C

ATOM
3466
O
ARG
B
226
−21.670
−6.307
−27.917
1.00
26.13
8
O

ATOM
3467
N
LEU
B
227
−23.018
−6.932
−29.625
1.00
26.74
7
N

ATOM
3468
CA
LEU
B
227
−23.500
−5.573
−29.911
1.00
27.07
6
C

ATOM
3469
CB
LEU
B
227
−24.206
−5.510
−31.277
1.00
27.06
6
C

ATOM
3470
CG
LEU
B
227
−23.289
−5.649
−32.505
1.00
27.87
6
C

ATOM
3471
CD1
LEU
B
227
−24.049
−5.763
−33.847
1.00
29.73
6
C

ATOM
3472
CD2
LEU
B
227
−22.303
−4.492
−32.555
1.00
28.95
6
C

ATOM
3473
C
LEU
B
227
−24.424
−5.054
−28.799
1.00
27.49
6
C

ATOM
3474
O
LEU
B
227
−24.465
−3.852
−28.523
1.00
27.09
8
O

ATOM
3475
N
ARG
B
228
−25.183
−5.960
−28.178
1.00
27.37
7
N

ATOM
3476
CA
ARG
B
228
−26.016
−5.593
−27.045
1.00
27.38
6
C

ATOM
3477
CB
ARG
B
228
−26.915
−6.751
−26.610
1.00
28.17
6
C

ATOM
3478
CG
ARG
B
228
−28.199
−6.972
−27.404
1.00
28.52
6
C

ATOM
3479
CD
ARG
B
228
−29.157
−7.933
−26.667
1.00
33.60
6
C

ATOM
3480
NE
ARG
B
228
−28.917
−9.320
−27.044
1.00
36.16
7
N

ATOM
3481
CZ
ARG
B
228
−28.486
−10.282
−26.252
1.00
38.65
6
C

ATOM
3482
NH1
ARG
B
228
−28.239
−10.052
−24.961
1.00
42.93
7
N

ATOM
3483
NH2
ARG
B
228
−28.316
−11.500
−26.753
1.00
36.19
7
N

ATOM
3484
C
ARG
B
228
−25.111
−5.184
−25.875
1.00
27.29
6
C

ATOM
3485
O
ARG
B
228
−25.394
−4.213
−25.187
1.00
26.61
8
O

ATOM
3486
N
TYR
B
229
−24.031
−5.932
−25.643
1.00
26.96
7
N

ATOM
3487
CA
TYR
B
229
−23.086
−5.588
−24.573
1.00
27.38
6
C

ATOM
3488
CB
TYR
B
229
−22.025
−6.686
−24.396
1.00
27.60
6
C

ATOM
3489
CG
TYR
B
229
−21.122
−6.500
−23.184
1.00
28.68
6
C

ATOM
3490
CD1
TYR
B
229
−21.572
−6.796
−21.909
1.00
30.32
6
C

ATOM
3491
CE1
TYR
B
229
−20.760
−6.625
−20.798
1.00
30.49
6
C

ATOM
3492
CZ
TYR
B
229
−19.479
−6.162
−20.961
1.00
32.12
6
C

ATOM
3493
OH
TYR
B
229
−18.669
−5.989
−19.858
1.00
33.30
8
O

ATOM
3494
CE2
TYR
B
229
−19.007
−5.850
−22.218
1.00
31.11
6
C

ATOM
3495
CD2
TYR
B
229
−19.832
−6.017
−23.321
1.00
30.01
6
C

ATOM
3496
C
TYR
B
229
−22.410
−4.261
−24.906
1.00
26.96
6
C

ATOM
3497
O
TYR
B
229
−22.198
−3.409
−24.031
1.00
27.00
8
O

ATOM
3498
N
ALA
B
230
−22.070
−4.093
−26.178
1.00
26.19
7
N

ATOM
3499
CA
ALA
B
230
−21.397
−2.879
−26.612
1.00
26.77
6
C

ATOM
3500
CB
ALA
B
230
−21.096
−2.930
−28.094
1.00
25.91
6
C

ATOM
3501
C
ALA
B
230
−22.232
−1.651
−26.285
1.00
26.84
6
C

ATOM
3502
O
ALA
B
230
−21.705
−0.652
−25.830
1.00
27.61
8
O

ATOM
3503
N
ARG
B
231
−23.533
−1.723
−26.523
1.00
27.30
7
N

ATOM
3504
CA
ARG
B
231
−24.383
−0.574
−26.241
1.00
27.93
6
C

ATOM
3505
CB
ARG
B
231
−25.831
−0.838
−26.671
1.00
27.44
6
C

ATOM
3506
CG
ARG
B
231
−26.767
0.364
−26.489
1.00
28.31
6
C

ATOM
3507
CD
ARG
B
231
−27.516
0.376
−25.153
1.00
29.17
6
C

ATOM
3508
NE
ARG
B
231
−28.261
1.625
−24.958
1.00
31.21
7
N

ATOM
3509
CZ
ARG
B
231
−29.409
1.917
−25.571
1.00
32.23
6
C

ATOM
3510
NH1
ARG
B
231
−29.949
1.054
−26.419
1.00
32.15
7
N

ATOM
3511
NH2
ARG
B
231
−30.017
3.079
−25.343
1.00
32.54
7
N

ATOM
3512
C
ARG
B
231
−24.293
−0.226
−24.756
1.00
28.42
6
C

ATOM
3513
O
ARG
B
231
−24.230
0.953
−24.382
1.00
28.53
8
O

ATOM
3514
N
THR
B
232
−24.291
−1.249
−23.903
1.00
28.73
7
N

ATOM
3515
CA
THR
B
232
−24.150
−1.006
−22.466
1.00
29.42
6
C

ATOM
3516
CB
THR
B
232
−24.227
−2.319
−21.670
1.00
29.47
6
C

ATOM
3517
OG1
THR
B
232
−25.451
−2.985
−21.987
1.00
29.28
8
O

ATOM
3518
CG2
THR
B
232
−24.353
−2.026
−20.173
1.00
30.08
6
C

ATOM
3519
C
THR
B
232
−22.855
−0.264
−22.141
1.00
29.68
6
C

ATOM
3520
O
THR
B
232
−22.860
0.682
−21.341
1.00
29.95
8
O

ATOM
3521
N
MET
B
233
−21.754
−0.687
−22.762
1.00
30.00
7
N

ATOM
3522
CA
MET
B
233
−20.441
−0.069
−22.544
1.00
30.54
6
C

ATOM
3523
CB
MET
B
233
−19.337
−0.872
−23.245
1.00
30.12
6
C

ATOM
3524
CG
MET
B
233
−19.145
−2.306
−22.732
1.00
30.73
6
C

ATOM
3525
SD
MET
B
233
−18.887
−2.407
−20.932
1.00
31.99
16
S

ATOM
3526
CE
MET
B
233
−20.446
−2.980
−20.352
1.00
26.77
6
C

ATOM
3527
C
MET
B
233
−20.402
1.387
−23.018
1.00
31.10
6
C

ATOM
3528
O
MET
B
233
−19.817
2.257
−22.364
1.00
30.77
8
O

ATOM
3529
N
VAL
B
234
−21.016
1.647
−24.166
1.00
31.50
7
N

ATOM
3530
CA
VAL
B
234
−21.057
3.010
−24.691
1.00
32.56
6
C

ATOM
3531
CB
VAL
B
234
−21.588
3.031
−26.133
1.00
32.17
6
C

ATOM
3532
CG1
VAL
B
234
−21.888
4.459
−26.576
1.00
32.60
6
C

ATOM
3533
CG2
VAL
B
234
−20.567
2.359
−27.047
1.00
31.40
6
C

ATOM
3534
C
VAL
B
234
−21.859
3.927
−23.760
1.00
33.51
6
C

ATOM
3535
O
VAL
B
234
−21.476
5.082
−23.522
1.00
33.37
8
O

ATOM
3536
N
ASP
B
235
−22.952
3.403
−23.211
1.00
34.93
7
N

ATOM
3537
CA
ASP
B
235
−23.728
4.142
−22.225
1.00
36.74
6
C

ATOM
3538
CB
ASP
B
235
−24.939
3.335
−21.758
1.00
37.07
6
C

ATOM
3539
CG
ASP
B
235
−26.181
3.614
−22.582
1.00
38.43
6
C

ATOM
3540
OD1
ASP
B
235
−26.322
4.750
−23.090
1.00
39.46
8
O

ATOM
3541
OD2
ASP
B
235
−27.080
2.768
−22.764
1.00
40.23
8
O

ATOM
3542
C
ASP
B
235
−22.845
4.503
−21.028
1.00
37.73
6
C

ATOM
3543
O
ASP
B
235
−22.965
5.594
−20.473
1.00
38.05
8
O

ATOM
3544
N
LYS
B
236
−21.963
3.585
−20.630
1.00
38.45
7
N

ATOM
3545
CA
LYS
B
236
−21.037
3.841
−19.525
1.00
39.37
6
C

ATOM
3546
CB
LYS
B
236
−20.323
2.551
−19.090
1.00
39.23
6
C

ATOM
3547
CG
LYS
B
236
−21.217
1.548
−18.366
1.00
39.71
6
C

ATOM
3548
CD
LYS
B
236
−20.471
0.246
−18.082
1.00
41.45
6
C

ATOM
3549
CE
LYS
B
236
−21.307
−0.722
−17.248
1.00
42.36
6
C

ATOM
3550
NZ
LYS
B
236
−21.538
−0.227
−15.857
1.00
43.08
7
N

ATOM
3551
C
LYS
B
236
−20.023
4.945
−19.872
1.00
40.08
6
C

ATOM
3552
O
LYS
B
236
−19.752
5.822
−19.049
1.00
40.28
8
O

ATOM
3553
N
LEU
B
237
−19.472
4.909
−21.083
1.00
41.07
7
N

ATOM
3554
CA
LEU
B
237
−18.556
5.959
−21.531
1.00
42.34
6
C

ATOM
3555
CB
LEU
B
237
−18.019
5.659
−22.930
1.00
41.72
6
C

ATOM
3556
CG
LEU
B
237
−17.053
4.480
−23.116
1.00
41.42
6
C

ATOM
3557
CD1
LEU
B
237
−16.740
4.272
−24.589
1.00
40.13
6
C

ATOM
3558
CD2
LEU
B
237
−15.768
4.706
−22.325
1.00
40.29
6
C

ATOM
3559
C
LEU
B
237
−19.270
7.315
−21.526
1.00
43.87
6
C

ATOM
3560
O
LEU
B
237
−18.680
8.346
−21.188
1.00
43.65
8
O

ATOM
3561
N
LEU
B
238
−20.543
7.303
−21.909
1.00
45.67
7
N

ATOM
3562
CA
LEU
B
238
−21.360
8.516
−21.938
1.00
47.83
6
C

ATOM
3563
CB
LEU
B
238
−22.634
8.268
−22.739
1.00
47.56
6
C

ATOM
3564
CG
LEU
B
238
−22.472
8.426
−24.246
1.00
48.06
6
C

ATOM
3565
CD1
LEU
B
238
−23.576
7.688
−24.991
1.00
48.48
6
C

ATOM
3566
CD2
LEU
B
238
−22.452
9.905
−24.616
1.00
47.93
6
C

ATOM
3567
C
LEU
B
238
−21.725
9.030
−20.549
1.00
49.45
6
C

ATOM
3568
O
LEU
B
238
−21.847
10.240
−20.335
1.00
49.72
8
O

ATOM
3569
N
SER
B
239
−21.908
8.110
−19.609
1.00
51.38
7
N

ATOM
3570
CA
SER
B
239
−22.268
8.477
−18.245
1.00
53.19
6
C

ATOM
3571
CB
SER
B
239
−22.356
7.233
−17.361
1.00
53.24
6
C

ATOM
3572
OG
SER
B
239
−21.057
6.755
−17.034
1.00
54.23
8
O

ATOM
3573
C
SER
B
239
−21.252
9.437
−17.649
1.00
54.22
6
C

ATOM
3574
O
SER
B
239
−21.596
10.537
−17.217
1.00
54.39
8
O

ATOM
3575
N
SER
B
240
−19.996
9.006
−17.635
1.00
55.60
7
N

ATOM
3576
CA
SER
B
240
−18.910
9.784
−17.054
1.00
56.85
6
C

ATOM
3577
CB
SER
B
240
−18.060
8.884
−16.162
1.00
56.87
6
C

ATOM
3578
OG
SER
B
240
−17.359
7.932
−16.948
1.00
57.47
8
O

ATOM
3579
C
SER
B
240
−18.017
10.383
−18.128
1.00
57.56
6
C

ATOM
3580
O
SER
B
240
−16.807
10.141
−18.132
1.00
58.04
8
O

ATOM
3581
N
ALA
B
241
−18.605
11.155
−19.039
1.00
58.16
7
N

ATOM
3582
CA
ALA
B
241
−17.842
11.764
−20.125
1.00
58.82
6
C

ATOM
3583
CB
ALA
B
241
−18.769
12.239
−21.236
1.00
58.78
6
C

ATOM
3584
C
ALA
B
241
−16.964
12.912
−19.633
1.00
59.25
6
C

ATOM
3585
O
ALA
B
241
−17.366
14.082
−19.667
1.00
59.83
8
O

ATOM
3586
OXT
ALA
B
241
−15.832
12.686
−19.195
1.00
59.46
8
O

ATOM
3587
N
PRO
E
1
16.379
−7.591
9.788
1.00
40.91
7
N

ATOM
3588
CA
PRO
E
1
15.544
−7.800
8.572
1.00
40.69
6
C

ATOM
3589
CB
PRO
E
1
14.852
−6.442
8.381
1.00
40.92
6
C

ATOM
3590
CG
PRO
E
1
15.200
−5.629
9.591
1.00
41.09
6
C

ATOM
3591
CD
PRO
E
1
16.488
−6.166
10.134
1.00
41.02
6
C

ATOM
3592
C
PRO
E
1
16.423
−8.073
7.359
1.00
40.59
6
C

ATOM
3593
O
PRO
E
1
17.539
−7.559
7.287
1.00
40.41
8
O

ATOM
3594
N
MET
E
2
15.918
−8.856
6.411
1.00
40.05
7
N

ATOM
3595
CA
MET
E
2
16.683
−9.168
5.215
1.00
40.01
6
C

ATOM
3596
CB
MET
E
2
16.476
−10.634
4.812
1.00
41.09
6
C

ATOM
3597
CG
MET
E
2
17.149
−11.615
5.768
1.00
43.73
6
C

ATOM
3598
SD
MET
E
2
18.945
−11.390
5.805
1.00
51.91
16
S

ATOM
3599
CE
MET
E
2
19.424
−12.458
7.142
1.00
52.26
6
C

ATOM
3600
C
MET
E
2
16.326
−8.218
4.083
1.00
38.83
6
C

ATOM
3601
O
MET
E
2
16.804
−8.365
2.957
1.00
38.60
8
O

ATOM
3602
N
GLN
E
3
15.486
−7.233
4.394
1.00
37.38
7
N

ATOM
3603
CA
GLN
E
3
15.069
−6.232
3.427
1.00
36.61
6
C

ATOM
3604
CB
GLN
E
3
13.811
−6.680
2.676
1.00
37.52
6
C

ATOM
3605
CG
GLN
E
3
12.608
−7.020
3.564
1.00
39.75
6
C

ATOM
3606
CD
GLN
E
3
11.396
−7.467
2.757
1.00
44.85
6
C

ATOM
3607
OE1
GLN
E
3
11.509
−7.723
1.556
1.00
46.70
8
O

ATOM
3608
NE2
GLN
E
3
10.235
−7.553
3.411
1.00
46.10
7
N

ATOM
3609
C
GLN
E
3
14.793
−4.932
4.163
1.00
35.58
6
C

ATOM
3610
O
GLN
E
3
14.584
−4.938
5.377
1.00
34.86
8
O

ATOM
3611
N
SER
E
4
14.809
−3.821
3.437
1.00
34.23
7
N

ATOM
3612
CA
SER
E
4
14.508
−2.524
4.040
1.00
33.40
6
C

ATOM
3613
CB
SER
E
4
14.962
−1.393
3.125
1.00
32.85
6
C

ATOM
3614
OG
SER
E
4
14.259
−1.436
1.895
1.00
31.74
8
O

ATOM
3615
C
SER
E
4
13.003
−2.416
4.303
1.00
33.74
6
C

ATOM
3616
O
SER
E
4
12.262
−3.389
4.107
1.00
33.39
8
O

ATOM
3617
O3P
TPO
E
5
13.899
1.162
8.266
1.00
30.51
8
O

ATOM
3618
P
TPO
E
5
13.192
1.401
6.861
1.00
32.40
15
P

ATOM
3619
O1P
TPO
E
5
12.542
2.823
6.637
1.00
32.45
8
O

ATOM
3620
O2P
TPO
E
5
14.048
0.875
5.613
1.00
31.07
8
O

ATOM
3621
OG1
TPO
E
5
11.927
0.412
6.990
1.00
32.05
8
O

ATOM
3622
CB
TPO
E
5
11.038
0.274
5.883
1.00
33.24
6
C

ATOM
3623
CG2
TPO
E
5
9.631
0.611
6.355
1.00
34.92
6
C

ATOM
3624
CA
TPO
E
5
11.111
−1.171
5.347
1.00
33.88
6
C

ATOM
3625
N
TPO
E
5
12.470
−1.314
4.833
1.00
33.34
7
N

ATOM
3626
C
TPO
E
5
10.057
−1.420
4.285
1.00
34.38
6
C

ATOM
3627
O
TPO
E
5
10.147
−0.852
3.087
1.00
33.69
8
O

ATOM
3628
N
PRO
E
6
9.130
−2.342
4.537
1.00
38.71
7
N

ATOM
3629
CA
PRO
E
6
8.008
−2.757
3.643
1.00
40.41
6
C

ATOM
3630
CB
PRO
E
6
7.331
−3.894
4.422
1.00
40.16
6
C

ATOM
3631
CG
PRO
E
6
8.323
−4.323
5.457
1.00
40.63
6
C

ATOM
3632
CD
PRO
E
6
9.129
−3.091
5.804
1.00
39.21
6
C

ATOM
3633
C
PRO
E
6
6.999
−1.642
3.392
1.00
41.29
6
C

ATOM
3634
O
PRO
E
6
6.811
−0.739
4.215
1.00
41.42
8
O

ATOM
3635
N
LEU
E
7
6.338
−1.742
2.247
1.00
42.62
7
N

ATOM
3636
CA
LEU
E
7
5.340
−0.786
1.797
1.00
43.90
6
C

ATOM
3637
CB
LEU
E
7
4.866
−1.200
0.403
1.00
44.21
6
C

ATOM
3638
CG
LEU
E
7
4.188
−0.160
−0.479
1.00
45.75
6
C

ATOM
3639
CD1
LEU
E
7
4.942
1.161
−0.420
1.00
46.28
6
C

ATOM
3640
CD2
LEU
E
7
4.097
−0.682
−1.911
1.00
46.71
6
C

ATOM
3641
C
LEU
E
7
4.152
−0.676
2.758
1.00
44.39
6
C

ATOM
3642
O
LEU
E
7
3.923
−1.564
3.592
1.00
45.42
8
O

ATOM
3643
N
PRO
F
1
−7.373
−9.873
−15.860
1.00
63.61
7
N

ATOM
3644
CA
PRO
F
1
−6.089
−9.838
−16.612
1.00
63.48
6
C

ATOM
3645
CB
PRO
F
1
−6.509
−10.235
−18.031
1.00
63.69
6
C

ATOM
3646
CG
PRO
F
1
−7.815
−10.957
−17.863
1.00
63.62
6
C

ATOM
3647
CD
PRO
F
1
−8.500
−10.293
−16.711
1.00
63.72
6
C

ATOM
3648
C
PRO
F
1
−5.498
−8.433
−16.617
1.00
63.40
6
C

ATOM
3649
O
PRO
F
1
−6.217
−7.470
−16.871
1.00
63.50
8
O

ATOM
3650
N
MET
F
2
−4.204
−8.315
−16.343
1.00
63.09
7
N

ATOM
3651
CA
MET
F
2
−3.557
−7.008
−16.314
1.00
62.84
6
C

ATOM
3652
CB
MET
F
2
−2.636
−6.893
−15.099
1.00
63.11
6
C

ATOM
3653
CG
MET
F
2
−3.383
−6.837
−13.779
1.00
64.18
6
C

ATOM
3654
SD
MET
F
2
−4.393
−5.349
−13.628
1.00
66.44
16
S

ATOM
3655
CE
MET
F
2
−5.583
−5.865
−12.403
1.00
66.00
6
C

ATOM
3656
C
MET
F
2
−2.780
−6.745
−17.594
1.00
62.32
6
C

ATOM
3657
O
MET
F
2
−2.021
−5.781
−17.685
1.00
62.29
8
O

ATOM
3658
N
GLN
F
3
−2.984
−7.603
−18.585
1.00
61.72
7
N

ATOM
3659
CA
GLN
F
3
−2.304
−7.475
−19.864
1.00
61.27
6
C

ATOM
3660
CB
GLN
F
3
−0.896
−8.057
−19.762
1.00
61.46
6
C

ATOM
3661
CG
GLN
F
3
−0.859
−9.414
−19.086
1.00
62.29
6
C

ATOM
3662
CD
GLN
F
3
0.506
−10.055
−19.147
1.00
63.90
6
C

ATOM
3663
OE1
GLN
F
3
1.511
−9.373
−19.352
1.00
64.45
8
O

ATOM
3664
NE2
GLN
F
3
0.550
−11.371
−18.974
1.00
64.33
7
N

ATOM
3665
C
GLN
F
3
−3.078
−8.213
−20.951
1.00
60.64
6
C

ATOM
3666
O
GLN
F
3
−3.908
−9.072
−20.661
1.00
60.37
8
O

ATOM
3667
N
SER
F
4
−2.802
−7.874
−22.204
1.00
60.11
7
N

ATOM
3668
CA
SER
F
4
−3.463
−8.531
−23.324
1.00
59.74
6
C

ATOM
3669
CB
SER
F
4
−3.501
−7.620
−24.550
1.00
59.65
6
C

ATOM
3670
OG
SER
F
4
−2.201
−7.299
−25.014
1.00
59.69
8
O

ATOM
3671
C
SER
F
4
−2.765
−9.846
−23.654
1.00
59.29
6
C

ATOM
3672
O
SER
F
4
−2.174
−10.478
−22.776
1.00
59.28
8
O

ATOM
3673
O3P
TPO
F
5
−6.281
−11.938
−27.798
1.00
52.19
8
O

ATOM
3674
P
TPO
F
5
−6.257
−11.464
−26.261
1.00
51.54
15
P

ATOM
3675
O1P
TPO
F
5
−5.611
−10.011
−26.100
1.00
51.18
8
O

ATOM
3676
O2P
TPO
F
5
−7.603
−11.821
−25.481
1.00
49.40
8
O

ATOM
3677
OG1
TPO
F
5
−5.200
−12.467
−25.572
1.00
55.49
8
O

ATOM
3678
CB
TPO
F
5
−3.824
−12.505
−25.934
1.00
57.12
6
C

ATOM
3679
CG2
TPO
F
5
−3.469
−13.923
−26.369
1.00
57.14
6
C

ATOM
3680
CA
TPO
F
5
−2.991
−12.082
−24.729
1.00
57.42
6
C

ATOM
3681
N
TPO
F
5
−3.256
−10.658
−24.584
1.00
58.07
7
N

ATOM
3682
C
TPO
F
5
−1.523
−12.356
−24.980
1.00
58.27
6
C

ATOM
3683
O
TPO
F
5
−0.801
−11.544
−25.752
1.00
57.29
8
O

ATOM
3684
N
PRO
F
6
−1.153
−13.293
−24.105
1.00
61.36
7
N

ATOM
3685
CA
PRO
F
6
0.332
−13.349
−24.226
1.00
62.33
6
C

ATOM
3686
CB
PRO
F
6
0.746
−14.060
−22.935
1.00
62.12
6
C

ATOM
3687
CG
PRO
F
6
−0.506
−14.752
−22.491
1.00
62.02
6
C

ATOM
3688
CD
PRO
F
6
−1.596
−13.761
−22.781
1.00
61.75
6
C

ATOM
3689
C
PRO
F
6
0.781
−14.173
−25.426
1.00
62.85
6
C

ATOM
3690
O
PRO
F
6
0.027
−15.003
−25.931
1.00
63.05
8
O

ATOM
3691
N
LEU
F
7
2.012
−13.945
−25.866
1.00
63.66
7
N

ATOM
3692
CA
LEU
F
7
2.579
−14.672
−26.994
1.00
64.38
6
C

ATOM
3693
CB
LEU
F
7
3.904
−14.039
−27.415
1.00
64.68
6
C

ATOM
3694
CG
LEU
F
7
4.503
−14.555
−28.720
1.00
65.80
6
C

ATOM
3695
CD1
LEU
F
7
3.411
−14.781
−29.760
1.00
66.75
6
C

ATOM
3696
CD2
LEU
F
7
5.559
−13.587
−29.234
1.00
66.83
6
C

ATOM
3697
C
LEU
F
7
2.786
−16.146
−26.663
1.00
64.48
6
C

ATOM
3698
O
LEU
F
7
2.810
−16.535
−25.493
1.00
64.81
8
O

ATOM
3699
O
WAT
W
1
24.634
2.439
−7.629
1.00
30.47
8

ATOM
3700
O
WAT
W
2
17.166
2.736
2.573
1.00
32.38
8

ATOM
3701
O
WAT
W
3
27.595
14.681
23.241
1.00
29.83
8

ATOM
3702
O
WAT
W
4
−27.777
−6.869
−31.502
1.00
32.30
8

ATOM
3703
O
WAT
W
5
16.593
0.034
6.219
1.00
30.62
8

ATOM
3704
O
WAT
W
6
14.513
2.346
3.344
1.00
31.12
8

ATOM
3705
O
WAT
W
7
28.562
12.784
21.663
1.00
34.09
8

ATOM
3706
O
WAT
W
8
16.086
2.555
8.816
1.00
29.73
8

ATOM
3707
O
WAT
W
9
31.864
20.213
8.572
1.00
28.59
8

ATOM
3708
O
WAT
W
10
−30.992
−10.307
−32.072
1.00
29.68
8

ATOM
3709
O
WAT
W
11
−26.050
2.758
−38.362
1.00
32.55
8

ATOM
3710
O
WAT
W
12
27.489
−8.003
7.433
1.00
33.37
8

ATOM
3711
O
WAT
W
13
12.364
0.356
2.037
1.00
28.32
8

ATOM
3712
O
WAT
W
14
35.876
2.813
16.912
1.00
37.61
8

ATOM
3713
O
WAT
W
15
35.091
0.594
15.613
1.00
33.67
8

ATOM
3714
O
WAT
W
16
26.700
14.898
−0.416
1.00
31.60
8

ATOM
3715
O
WAT
W
17
33.877
−11.857
−3.921
1.00
36.78
8

ATOM
3716
O
WAT
W
18
33.521
9.338
18.361
1.00
36.77
8

ATOM
3717
O
WAT
W
19
10.619
5.795
4.943
1.00
38.47
8

ATOM
3718
O
WAT
W
20
12.148
9.034
4.695
1.00
33.68
8

ATOM
3719
O
WAT
W
21
21.930
2.694
−8.658
1.00
29.23
8

ATOM
3720
O
WAT
W
22
28.179
13.091
18.799
1.00
30.76
8

ATOM
3721
O
WAT
W
23
24.493
14.521
4.080
1.00
34.88
8

ATOM
3722
O
WAT
W
24
19.906
6.992
−13.260
1.00
32.20
8

ATOM
3723
O
WAT
W
25
7.557
−8.450
2.900
1.00
63.84
8

ATOM
3724
O
WAT
W
26
−27.367
1.228
−36.920
1.00
31.57
8

ATOM
3725
O
WAT
W
27
29.654
11.921
−1.388
1.00
29.35
8

ATOM
3726
O
WAT
W
28
21.217
−7.633
11.401
1.00
37.46
8

ATOM
3727
O
WAT
W
29
25.864
15.796
2.006
1.00
32.25
8

ATOM
3728
O
WAT
W
30
−24.053
1.535
−40.872
1.00
33.67
8

ATOM
3729
O
WAT
W
31
14.018
13.365
−22.746
1.00
62.02
8

ATOM
3730
O
WAT
W
32
9.697
5.905
9.452
1.00
42.01
8

ATOM
3731
O
WAT
W
33
18.932
3.541
17.552
1.00
58.68
8

ATOM
3732
O
WAT
W
34
25.616
10.543
−1.509
1.00
36.69
8

ATOM
3733
O
WAT
W
35
−29.998
−8.101
−30.511
1.00
33.22
8

ATOM
3734
O
WAT
W
36
35.706
−8.967
−12.207
1.00
33.88
8

ATOM
3735
O
WAT
W
37
−28.712
−14.686
−27.659
1.00
40.16
8

ATOM
3736
O
WAT
W
38
27.173
−7.960
−1.547
1.00
30.64
8

ATOM
3737
O
WAT
W
39
36.208
10.457
−1.144
1.00
58.59
8

ATOM
3738
O
WAT
W
40
25.892
25.852
25.176
1.00
36.79
8

ATOM
3739
O
WAT
W
41
−28.690
−12.596
−30.204
1.00
39.60
8

ATOM
3740
O
WAT
W
42
11.127
0.674
−6.719
1.00
36.86
8

ATOM
3741
O
WAT
W
43
12.634
7.055
6.478
1.00
34.24
8

ATOM
3742
O
WAT
W
44
26.064
−12.501
−16.222
1.00
39.99
8

ATOM
3743
O
WAT
W
45
−23.089
−17.237
−33.109
1.00
42.46
8

ATOM
3744
O
WAT
W
46
−21.850
−0.822
−47.719
1.00
35.95
8

ATOM
3745
O
WAT
W
47
33.872
−3.162
−16.877
1.00
34.13
8

ATOM
3746
O
WAT
W
48
24.365
34.040
18.694
1.00
41.11
8

ATOM
3747
O
WAT
W
49
−28.585
5.275
−23.685
1.00
47.83
8

ATOM
3748
O
WAT
W
50
27.720
11.812
−6.004
1.00
52.01
8

ATOM
3749
O
WAT
W
51
31.145
11.986
22.378
1.00
36.29
8

ATOM
3750
O
WAT
W
52
17.598
21.360
13.347
1.00
40.00
8

ATOM
3751
O
WAT
W
53
−27.143
−11.537
−24.167
1.00
42.92
8

ATOM
3752
O
WAT
W
54
21.250
3.872
18.777
1.00
62.89
8

ATOM
3753
O
WAT
W
55
−11.528
0.411
−15.116
1.00
43.89
8

ATOM
3754
O
WAT
W
56
−32.837
−6.837
−46.162
1.00
40.49
8

ATOM
3755
O
WAT
W
57
−13.041
3.240
−39.158
1.00
44.50
8

ATOM
3756
O
WAT
W
58
13.747
1.567
−9.380
1.00
38.07
8

ATOM
3757
O
WAT
W
59
40.365
3.671
12.390
1.00
40.05
8

ATOM
3758
O
WAT
W
60
34.206
3.404
18.995
1.00
35.50
8

ATOM
3759
O
WAT
W
61
−25.012
−9.493
−24.326
1.00
33.11
8

ATOM
3760
O
WAT
W
62
35.341
25.261
14.453
1.00
37.72
8

ATOM
3761
O
WAT
W
63
25.868
5.419
20.464
1.00
42.24
8

ATOM
3762
O
WAT
W
64
19.003
16.386
23.952
1.00
42.14
8

ATOM
3763
O
WAT
W
65
−37.060
−3.848
−30.377
1.00
41.53
8

ATOM
3764
O
WAT
W
66
20.233
10.551
−11.649
1.00
38.76
8

ATOM
3765
O
WAT
W
67
36.862
18.521
18.838
1.00
33.85
8

ATOM
3766
O
WAT
W
68
−1.871
6.647
−29.708
1.00
46.30
8

ATOM
3767
O
WAT
W
69
11.965
3.813
4.062
1.00
40.29
8

ATOM
3768
O
WAT
W
70
−27.733
−1.750
−21.747
1.00
39.35
8

ATOM
3769
O
WAT
W
71
35.651
−12.751
−6.391
1.00
43.81
8

ATOM
3770
O
WAT
W
72
20.746
16.973
−11.203
1.00
69.69
8

ATOM
3771
O
WAT
W
73
36.951
−3.167
13.673
1.00
46.35
8

ATOM
3772
O
WAT
W
74
33.452
18.001
12.333
1.00
35.28
8

ATOM
3773
O
WAT
W
75
39.836
−4.636
−17.377
1.00
41.94
8

ATOM
3774
O
WAT
W
76
−26.014
−7.701
−22.795
1.00
36.58
8

ATOM
3775
O
WAT
W
77
32.112
6.847
18.892
1.00
35.48
8

ATOM
3776
O
WAT
W
78
−24.158
−14.389
−45.202
1.00
49.56
8

ATOM
3777
O
WAT
W
79
12.359
7.284
9.231
1.00
35.29
8

ATOM
3778
O
WAT
W
80
−7.718
−11.828
−31.638
1.00
43.35
8

ATOM
3779
O
WAT
W
81
−4.433
−8.546
−27.834
1.00
46.46
8

ATOM
3780
O
WAT
W
82
12.662
11.800
4.986
1.00
37.04
8

ATOM
3781
O
WAT
W
83
18.628
4.051
−18.939
1.00
37.36
8

ATOM
3782
O
WAT
W
84
41.874
12.668
12.296
1.00
64.40
8

ATOM
3783
O
WAT
W
85
24.386
29.260
11.905
1.00
39.20
8

ATOM
3784
O
WAT
W
86
−35.916
−9.236
−35.846
1.00
44.00
8

ATOM
3785
O
WAT
W
87
24.932
26.384
21.522
1.00
45.38
8

ATOM
3786
O
WAT
W
88
−14.850
−1.218
−37.594
1.00
39.23
8

ATOM
3787
O
WAT
W
89
−28.949
−10.251
−28.386
1.00
34.73
8

ATOM
3788
O
WAT
W
90
−15.971
11.758
−33.481
1.00
66.16
8

ATOM
3789
O
WAT
W
91
29.015
−14.521
−18.294
1.00
50.73
8

ATOM
3790
O
WAT
W
92
−27.883
−3.366
−24.498
1.00
43.48
8

ATOM
3791
O
WAT
W
93
19.046
23.268
24.787
1.00
48.24
8

ATOM
3792
O
WAT
W
94
10.369
4.017
7.310
1.00
39.43
8

ATOM
3793
O
WAT
W
95
35.601
2.131
−10.164
1.00
44.36
8

ATOM
3794
O
WAT
W
96
17.848
24.939
17.240
1.00
43.72
8

ATOM
3795
O
WAT
W
97
19.195
−7.795
14.928
1.00
46.11
8

ATOM
3796
O
WAT
W
98
41.553
9.708
9.864
1.00
50.38
8

ATOM
3797
O
WAT
W
99
−20.658
12.851
−28.172
1.00
48.34
8

ATOM
3798
O
WAT
W
100
23.684
14.223
26.217
1.00
56.84
8

ATOM
3799
O
WAT
W
101
−9.687
−13.780
−31.210
1.00
41.65
8

ATOM
3800
O
WAT
W
102
−12.758
6.302
−35.847
1.00
44.53
8

ATOM
3801
O
WAT
W
103
34.728
8.879
−6.401
1.00
54.55
8

ATOM
3802
O
WAT
W
104
21.203
−8.624
20.819
1.00
49.17
8

ATOM
3803
O
WAT
W
105
−31.852
−0.946
−28.601
1.00
40.67
8

ATOM
3804
O
WAT
W
106
−9.893
3.411
−35.561
1.00
45.86
8

ATOM
3805
O
WAT
W
107
39.989
1.169
12.158
1.00
41.22
8

ATOM
3806
O
WAT
W
108
−29.350
−1.852
−27.097
1.00
41.79
8

ATOM
3807
O
WAT
W
109
32.061
6.131
−8.513
1.00
40.19
8

ATOM
3808
O
WAT
W
110
−13.807
11.289
−29.680
1.00
59.37
8

ATOM
3809
O
WAT
W
111
7.858
−11.260
0.049
1.00
60.48
8

ATOM
3810
O
WAT
W
112
16.627
1.411
−12.612
1.00
40.75
8

ATOM
3811
O
WAT
W
113
−19.983
14.426
−30.108
1.00
60.48
8

ATOM
3812
O
WAT
W
114
12.245
18.538
−24.003
1.00
82.55
8

ATOM
3813
O
WAT
W
115
39.571
15.004
13.995
1.00
37.84
8

ATOM
3814
O
WAT
W
116
33.463
−5.785
−20.431
1.00
58.24
8

ATOM
3815
O
WAT
W
117
−26.072
7.203
−21.426
1.00
55.49
8

ATOM
3816
O
WAT
W
118
18.188
20.859
6.035
1.00
42.20
8

ATOM
3817
O
WAT
W
119
5.384
−1.239
−31.530
1.00
59.18
8

ATOM
3818
O
WAT
W
120
20.262
−15.072
−13.492
1.00
51.73
8

ATOM
3819
O
WAT
W
121
30.189
11.922
24.851
1.00
49.55
8

ATOM
3820
O
WAT
W
122
10.788
−2.015
15.189
1.00
59.50
8

ATOM
3821
O
WAT
W
123
−7.050
−8.261
−24.625
1.00
45.64
8

ATOM
3822
O
WAT
W
124
18.191
23.083
19.249
1.00
41.19
8

ATOM
3823
O
WAT
W
125
43.545
−1.639
7.512
1.00
69.29
8

ATOM
3824
O
WAT
W
126
−14.472
0.948
−39.333
1.00
46.33
8

ATOM
3825
O
WAT
W
127
−31.621
−6.535
−29.094
1.00
39.11
8

ATOM
3826
O
WAT
W
128
−35.231
−1.122
−27.823
1.00
49.91
8

ATOM
3827
O
WAT
W
129
−19.094
13.991
−27.043
1.00
56.45
8

ATOM
3828
O
WAT
W
130
38.995
−6.826
−15.834
1.00
50.99
8

ATOM
3829
O
WAT
W
131
−11.364
6.658
−38.443
1.00
49.38
8

ATOM
3830
O
WAT
W
132
−10.858
−6.847
−15.161
1.00
45.27
8

ATOM
3831
O
WAT
W
133
33.263
13.504
27.027
1.00
49.51
8

ATOM
3832
O
WAT
W
134
−9.470
5.931
−46.144
1.00
79.22
8

ATOM
3833
O
WAT
W
135
17.824
26.088
20.282
1.00
56.19
8

ATOM
3834
O
WAT
W
136
14.973
20.317
5.379
1.00
60.66
8

ATOM
3835
O
WAT
W
137
9.146
7.236
3.036
1.00
47.76
8

ATOM
3836
O
WAT
W
138
25.903
13.234
−2.501
1.00
41.93
8

ATOM
3837
O
WAT
W
139
29.480
14.136
−11.457
1.00
62.93
8

ATOM
3838
O
WAT
W
140
40.320
6.951
3.255
1.00
42.71
8

ATOM
3839
O
WAT
W
141
−22.104
6.671
−41.503
1.00
54.26
8

ATOM
3840
O
WAT
W
142
14.225
−11.428
−3.778
1.00
70.87
8

ATOM
3841
O
WAT
W
143
20.799
−7.861
24.532
1.00
63.06
8

ATOM
3842
O
WAT
W
144
36.018
−1.336
−5.770
1.00
73.31
8

ATOM
3843
O
WAT
W
145
17.809
−8.084
11.994
1.00
63.01
8

ATOM
3844
O
WAT
W
146
31.942
−6.401
−22.214
1.00
62.79
8

ATOM
3845
O
WAT
W
147
−25.476
−5.436
−21.368
1.00
43.05
8

ATOM
3846
O
WAT
W
148
22.760
4.718
−22.884
1.00
54.95
8

ATOM
3847
O
WAT
W
149
13.421
−9.857
6.966
1.00
49.49
8

ATOM
3848
O
WAT
W
150
13.765
9.827
−16.866
1.00
74.60
8

ATOM
3849
O
WAT
W
151
−32.735
−9.192
−28.257
1.00
51.17
8

ATOM
3850
O
WAT
W
152
25.500
3.281
−22.705
1.00
53.30
8

ATOM
3851
O
WAT
W
153
18.235
−0.257
15.603
1.00
38.90
8

ATOM
3852
O
WAT
W
154
−6.061
−14.604
−28.731
1.00
54.75
8

ATOM
3853
O
WAT
W
155
40.951
8.546
0.629
1.00
62.07
8

ATOM
3854
O
WAT
W
156
32.698
22.571
7.376
1.00
53.61
8

ATOM
3855
O
WAT
W
157
−30.708
0.047
−42.456
1.00
58.10
8

ATOM
3856
O
WAT
W
158
−19.452
−17.048
−28.371
1.00
49.53
8

ATOM
3857
O
WAT
W
159
−34.314
−0.554
−35.382
1.00
48.91
8

ATOM
3858
O
WAT
W
160
6.903
3.894
−31.587
1.00
70.11
8

ATOM
3859
O
WAT
W
161
−30.049
2.673
−39.111
1.00
52.69
8

ATOM
3860
O
WAT
W
162
8.467
12.865
−17.494
1.00
65.54
8

ATOM
3861
O
WAT
W
163
33.864
18.806
10.016
1.00
44.82
8

ATOM
3862
O
WAT
W
164
10.938
−9.972
−8.363
1.00
54.44
8

ATOM
3863
O
WAT
W
165
−20.769
15.416
−25.966
1.00
61.44
8

ATOM
3864
O
WAT
W
166
29.110
−7.868
4.834
1.00
59.06
8

ATOM
3865
O
WAT
W
167
16.795
17.929
−7.581
1.00
62.10
8

ATOM
3866
O
WAT
W
168
−4.711
−12.245
−31.586
1.00
50.04
8

ATOM
3867
O
WAT
W
169
19.618
32.961
13.177
1.00
49.55
8

ATOM
3868
O
WAT
W
170
28.062
25.357
22.310
1.00
57.60
8

ATOM
3869
O
WAT
W
171
0.867
−2.085
3.050
1.00
65.30
8

ATOM
3870
O
WAT
W
172
−21.887
16.627
−31.029
1.00
70.31
8

ATOM
3871
O
WAT
W
173
25.330
−14.416
−19.149
1.00
44.85
8

ATOM
3872
O
WAT
W
174
13.027
−0.641
−7.690
1.00
40.87
8

ATOM
3873
O
WAT
W
175
29.491
15.933
−2.541
1.00
46.06
8

ATOM
3874
O
WAT
W
176
28.804
2.041
−24.955
1.00
50.03
8

ATOM
3875
O
WAT
W
177
−10.519
3.857
−38.349
1.00
56.56
8

ATOM
3876
O
WAT
W
178
−25.503
−15.400
−42.858
1.00
45.25
8

ATOM
3877
O
WAT
W
179
−37.796
−1.357
−30.241
1.00
63.19
8

ATOM
3878
O
WAT
W
180
−3.515
−12.703
−37.655
1.00
72.93
8

ATOM
3879
O
WAT
W
181
21.438
−3.901
24.689
1.00
64.51
8

ATOM
3880
O
WAT
W
182
−16.008
−13.906
−23.419
1.00
63.43
8

ATOM
3881
O
WAT
W
183
35.740
−8.463
−1.794
1.00
61.46
8

ATOM
3883
O
WAT
W
184
−10.500
2.764
−15.695
1.00
70.02
8

ATOM
3884
O
WAT
W
185
−9.860
−10.367
−26.412
1.00
47.62
8

ATOM
3885
O
WAT
W
186
−38.554
−6.542
−30.237
1.00
62.93
8

ATOM
3886
O
WAT
W
187
−32.714
−3.395
−28.295
1.00
48.28
8

ATOM
3887
O
WAT
W
188
31.369
12.845
7.044
1.00
56.30
8

ATOM
3888
O
WAT
W
189
−13.854
−17.255
−39.219
1.00
77.03
8

ATOM
3889
O
WAT
W
190
38.132
5.328
1.621
1.00
53.14
8

ATOM
3890
O
WAT
W
191
−29.743
−10.731
−22.814
1.00
56.88
8

ATOM
3891
O
WAT
W
192
16.319
−7.567
−19.734
1.00
68.76
8

ATOM
3892
O
WAT
W
193
20.905
5.870
21.460
1.00
58.69
8

ATOM
3893
O
WAT
W
194
−2.078
−9.343
−26.709
1.00
45.32
8

ATOM
3894
O
WAT
W
195
−27.973
−17.217
−34.661
1.00
50.54
8

ATOM
3895
O
WAT
W
196
8.090
−6.038
−12.667
1.00
72.72
8

ATOM
3896
O
WAT
W
197
5.456
−5.313
−29.937
1.00
64.35
8

ATOM
3897
O
WAT
W
198
−17.580
12.084
−27.772
1.00
61.72
8

ATOM
3898
O
WAT
W
199
39.310
5.354
15.767
1.00
65.14
8

ATOM
3899
O
WAT
W
200
27.072
−1.823
−7.016
1.00
56.78
8

ATOM
3900
O
WAT
W
201
−33.783
−9.367
−42.448
1.00
59.22
8

ATOM
3901
O
WAT
W
202
32.480
1.951
−21.517
1.00
51.48
8

ATOM
3902
O
WAT
W
203
27.509
7.904
−14.495
1.00
62.04
8

ATOM
3903
O
WAT
W
204
−6.762
−19.906
−37.936
1.00
67.41
8

ATOM
3904
O
WAT
W
205
10.151
2.040
−8.820
1.00
63.45
8

ATOM
3905
O
WAT
W
206
42.308
11.982
5.704
1.00
70.95
8

ATOM
3906
O
WAT
W
207
32.614
9.993
21.419
1.00
65.65
8

ATOM
3907
O
WAT
W
208
−19.924
−12.576
−42.478
1.00
64.81
8

ATOM
3908
O
WAT
W
209
−0.031
5.343
−30.924
1.00
61.37
8

ATOM
3909
O
WAT
W
210
7.595
4.610
−1.989
1.00
63.64
8

ATOM
3910
O
WAT
W
211
9.965
9.231
3.622
1.00
60.62
8

ATOM
3911
O
WAT
W
212
23.641
28.537
22.061
1.00
66.92
8

ATOM
3912
O
WAT
W
213
−4.088
−10.684
−29.666
1.00
66.92
8

ATOM
3913
O
WAT
W
214
4.345
−9.263
−30.965
1.00
66.64
8

ATOM
3914
O
WAT
W
215
26.160
29.184
20.917
1.00
71.88
8

ATOM
3915
O
WAT
W
216
37.856
8.051
−5.026
1.00
69.47
8

ATOM
3916
O
WAT
W
217
−27.466
0.737
−21.368
1.00
61.39
8

ATOM
3918
O
WAT
W
218
23.412
−16.483
−20.202
1.00
69.69
8

ATOM
3919
O
WAT
W
219
28.293
−13.330
−8.770
1.00
67.32
8

ATOM
3920
O
WAT
W
220
3.457
−0.665
5.706
1.00
75.41
8

ATOM
3921
O
WAT
W
221
21.431
−19.910
−19.507
1.00
66.40
8

ATOM
3922
O
WAT
W
222
35.336
2.654
−5.715
1.00
72.34
8

ATOM
3923
OW0
WAT
W
223
35.726
24.518
9.473
1.00
49.00
8

ATOM
3924
OW0
WAT
W
224
14.105
8.614
−1.895
1.00
69.00
8

ATOM
3925
OW0
WAT
W
225
−5.513
12.590
−37.262
1.00
69.00
8

ATOM
3926
OW0
WAT
W
226
28.752
28.494
20.210
1.00
70.00
8

ATOM
3927
OW0
WAT
W
227
20.227
15.904
−8.842
1.00
70.00
8

ATOM
3928
OW0
WAT
W
228
7.887
3.313
−4.421
1.00
71.00
8

ATOM
3929
OW0
WAT
W
229
18.680
0.000
18.315
1.00
71.00
8

ATOM
3930
OW0
WAT
W
230
−21.527
−17.229
−35.367
1.00
71.00
8

ATOM
3931
OW0
WAT
W
231
−32.631
−10.602
−30.315
1.00
72.00
8

ATOM
3932
OW0
WAT
W
232
−29.535
−7.952
−51.156
1.00
72.00
8

ATOM
3933
OW0
WAT
W
233
31.358
15.241
30.315
1.00
72.00
8

ATOM
3934
OW0
WAT
W
234
14.620
−11.265
−22.105
1.00
72.00
8

ATOM
3935
OW0
WAT
W
235
−31.286
7.289
−31.578
1.00
72.00
8

ATOM
3936
OW0
WAT
W
236
6.255
7.289
1.895
1.00
72.00
8

ATOM
3937
OW0
WAT
W
237
−3.231
23.193
−21.473
1.00
73.00
8

ATOM
3938
OW0
WAT
W
238
−6.016
−5.301
−28.420
1.00
73.00
8

ATOM
3939
OW0
WAT
W
239
12.255
−4.639
−25.262
1.00
73.00
8

ATOM
3940
OW0
WAT
W
240
8.824
−0.663
12.631
1.00
73.00
8

ATOM
3941
OW0
WAT
W
241
35.953
−5.301
−5.684
1.00
73.00
8

ATOM
3942
OW0
WAT
W
242
−20.326
2.651
−34.104
1.00
73.00
8

ATOM
3943
OW0
WAT
W
243
17.192
23.855
22.105
1.00
73.00
8

ATOM
3944
OW0
WAT
W
244
9.566
−10.602
−22.736
1.00
73.00
8

ATOM
3945
OW0
WAT
W
245
−16.351
−1.325
−14.526
1.00
73.00
8

ATOM
3946
OW0
WAT
W
246
26.578
26.506
2.526
1.00
74.00
8

ATOM
3947
OW0
WAT
W
247
−9.858
8.614
−39.157
1.00
74.00
8

Figures were produced with Ribbons (Carson, J. Appl. Crystallogr. 24:958-961, 1991) or SPOCK.

Plk1 PBD Binding to Cellular Substrates

HeLa cells were transfected with His/Xpress-tagged Plk1 (residues 326-603 or 326-506) or myc-tagged Plk1 (full-length). They were allowed to recover for 17 hours and then arrested in G2/M by treatment with nocodazole (50 ng/mL) for 14 hours. Cells were lysed in 25 mM Tris/HCl (pH7.5) containing 125 mM NaCl, 0.5% NP-40, 5 mM EDTA, 2 mM DTT, 4 μg/mL pepstatin, 4 μg/mL aprotinin, 4 μg/mL leupeptin, 1 mM Na₃VO₄, 50 mM NaF, and 1 μM microcystin. Lysates were incubated with 5 μL Ni²⁺ beads or 5 μl, α-myc-conjugated beads (Santa Cruz Biotechnology) for 90 minutes at 4° C. Beads were washed four times with lysis buffer. Precipitated proteins were eluted in sample buffer and detected by blotting with polyclonal anti-Cdc25C (Santa Cruz Biotechnology). Point mutations of Plk1 were constructed using the QuickChange site-directed mutagenesis system (Stratagene, La Jolla, Calif.) and verified by DNA sequencing.

Centrosomal Localization of the Plk1 PBD

U2OS cells were cultured in 8-well chamber slides and arrested in G2/M by treatment with nocodazole (50 ng/mL) for 14 hours. After rinsing with PBS, cells were incubated with 4 μM GST-Plk1 PBD (residues 326-603) and Streptolysin-O (1 U/ml) in permeabilization buffer (25 mM HEPES (pH 7.9), 100 mM KCl, 3 mM NaCl, 200 mM sucrose, 20 mM NaF, 1 mM NaOVO₄) for 20 minutes at 37° C. Cells were fixed in 3% paraformaldehyde/2% sucrose for 10 minutes at room temperature and extracted with a 0.5% Triton X-100 solution containing 20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 300 mM sucrose, and 3 mM MgCl₂for 10 minutes at Room temperature. Slides were stained with Alexa Fluor 488-conjugated anti-GST (Molecular Probes, Eugene, Oreg.) and monoclonal anti-γ-tubulin (Sigma) antibodies at 4° C. overnight, then stained with a Texas Red conjugated anti-mouse secondary antibody for 60 minutes at room temperature and counterstained with 4 μg/ml DAPI. Cells were examined using a Nikon Eclipse E600 fluorescence microscope equipped with a SPOT RT camera and software (Diagnostic Instruments Livingston, Scotland). Images were analyzed using NIH Image.

Cell Cycle Analysis

HeLa cells were transfected with wild-type and mutant forms of GFP-tagged Plk1 (residues 326-603) for 32 hours. Media containing floating cells was retained, and attached cells were released from plates by trypsinization. The two cell populations were combined, washed with PBS, and stained with Hoechst 33342 (10 μg/mL) for 30 minutes at 37° C. in DMEM/10% FBS (1×10⁶cells/mL). Dead cells were stained by incubation with propridium iodide (5 μg/mL) for 5 minutes at 4° C. GFP, Hoechst 33342, and propidium iodide fluorescent signals were quantitated on a FAC Star Plus (Becton Dickinson, Franklin Lakes, N.J.) cell sorting machine using Cell Quest software. Cell cycle analysis of the total live cell population (no propidium iodide staining) and live GFP-expressing cells (no propidium staining and GFP positive) was performed using Modfit 2.0.

Plk1 Kinase Assays

SF9 cells infected with baculoviral GST-Plk1 (full-length) were lysed in 20 mM Hepes/KOH (pH 7.5), 135 mM NaCl, 1% NP40, 5 mM EGTA, 5 μM 13-mercaptoethanol, 35 mM NaF, 0.5 mM Na₃VO₄, 20 mM β-glycerolphosphate, 3 μM microcystin, 1 μM okadaic acid, 10 μg/mL pepstatin, 10 μg/mL leupeptin, and 10 μg/mL aprotinin. Lysates were incubated for 2 hours at 4° C. with glutathione beads, which were subsequently washed five times with 20 mM Hepes/KOH (pH 7.5), 415 mM NaCl, 0.1% CHAPS, 5 mM EGTA, 5 μM β-mercaptoethanol, 35 mM NaF, and 0.5 mM Na₃VO₄at 4° C. Bound proteins were eluted with a buffer containing 30 mM glutathione, 50 mM Hepes/KOH (pH 8.0), 25 mM NaCl, 2 mM MgCl₂, 1 mM EGTA, and 5 μM β-mercaptoethanol and dialyzed against 10 mM Hepes, 10 mM NaCl, 1 mM EGTA, 1 mM DTT for 3 hours at 4° C. Kinase reactions were performed in 20 mM Hepes/KOH (pH7.5), 15 mM KCl, 10 mM MgCl₂, 1 mM EGTA, 100 M ATP, 5 μCi γ-[³²P]-ATP, 1 mM DTT, and 0.1 μg/μgL casein for 15 minutes at 30° C. Reaction aliquots were removed at various time points, added to sample buffer, and boiled to arrest phosphorylation. ³²P-incorporation into casein was determined by SDS-PAGE electrophoresis, autoradiography, and densitometry using ImageQuant software (Molecular Dynamics). For peptide activation experiments, 250 μM of the PBD optimal phosphopeptide (MAGPMQSpTPLNGAKK) (SEQ ID NO: 3) or its non-phosphorylated counterpart (MAGPMQSTPLNGAKK) (SEQ ID NO: 34) were pre-incubated with GST-Plk1 for 5 minutes at room temperature.

Molecular Modeling in Silico

The present invention provides an exemplary crystallized PBD-phosphopeptide complex and the atomic structural coordinates of this complex. The key structural features of the complex, particularly the shape of the substrate binding site, are useful in methods for designing or identifying selective inhibitors of a Polo-like kinase polypeptide, such as Plk-1, and in solving the structures of other proteins with similar features. The structure coordinates of this complex are encoded in a data storage medium, submitted herewith, for use with a computer for graphical three-dimensional representation of the structure and for computer-aided molecular design of new inhibitors. The differences in three-dimensional structure between PLK-1 and related proteins with known structures can be used to optimize selectivity of an inhibitor for PBD. In addition to the structural differences described herein, other differences between Plk-1 and other proteins can also be identified by a skilled artisan.

The three-dimensional atomic structures reported herein can be readily used as a template for selecting potent inhibitors, such as small molecules or peptidomimetics that are designed to “fit” into the binding interface. Methods for designing peptidomimetics using rational drug design are known to the skilled artisan, and are described, for example, in U.S. Pat. Nos. 6,225,076; 6,171,804; and in Han et al. (Bioorg Med. Chem. Lett, 10:39-43, 2000). Peptidomimetics capable of inhibiting complex formation can be identified, for example, through the use of computer modeling using a docking program such as GRAM, DOCK, or AUTODOCK (Dunbrack et al., Folding & Design, 2:27-42, 1997). This procedure can include computer fitting of candidate compounds to a the binding interface of a particular polypeptide to determine whether the shape and chemical structure of the potential ligand will allow it to bind within the structure of the polypeptide. Many methods can be used for this purpose such as, but not limited to, fast shape matching (Dock [Kuntz et al., J. Mol. Biol., 161:269-288, 1982]; Eudock [Perola et al., J. Med. Chem., 43:401-408, 2000]), incremental construction (FlexX [Rarey et al., J Mol Biol, 261, 470-89, 1996]; HAMMERHEAD [Welch et al., Chem. Biol., 3, 449-462, 1996]), TABU search (Pro_Leads [Baxter et al., Proteins 33:367-382, 1998]; SFDock [Hou et al., Protein Eng. 12:639-647, 1999]), genetic algorithms (GOLD [Gold et al., J. Mol. Biol. 267:727-748, 1997]; AutoDock 3.0 [Morris et al., J. Comput. Chem., 19:1639-1662, 1998]; Gambler [Charifson et al., J. Med. Chem., 42:5100-5109, 1999]), evolutionary programming [Gehlhaar et al., Chem. Biol., 2:317-324, 1995], simulated annealing (AutoDock 2.4 [Goodsell et al., Proteins, 8:195-202, 1990]), Monte Carlo simulations (MCDock [Liu et al., J. Comput.-Aided Mol. Des., 13:435-451, 1999]; QXP [McMartin et al., J. Comput.-Aided Mol. Des., 11:333-344, 1997]), and distance geometry (Dockit [Metaphorics LLC, Piemont, Calif. 94611 www.metaphorics.com]).

Those skilled in the art can readily identify many small molecules or fragments as hits. If desired, one can link the different functional groups or small molecules identified by the above procedure into a single, larger molecule. The resulting molecule is likely to be more potent and have higher specificity. The affinity and/or specificity of a hit can also be improved by adding more atoms or fragments that will interact with the target protein. The originally defined target site can be readily expanded to allow further necessary extension. Selected compounds may be systematically modified by computer modeling programs to identify peptidomimetics having the greatest therapeutic potential. Alternatively, candidate compounds are selected from chemical libraries, or are synthesized de novo.

The structural analysis disclosed herein in conjunction with computer modeling allows the selection of a finite number of rational chemical modifications. Thus, using the complex structure disclosed herein and computer modeling, a large number of candidate compounds can be rapidly screened in silico, and the most promising candidates can be identified. Candidate compounds, such as peptidomimetics, are then verified in vitro or in vivo, for example, by determining the effect of the candidate compound on PBD/phosphopeptide binding, Polo-like kinase biological activity, cell cycle regulation, apoptosis, or cell proliferation.

pSer/pThr-Binding Domains Function in The Cellular Response to Genotoxic Stress

Signal transduction by protein kinases in eukaryotes results in the directed assembly of multi-protein complexes at specific locations within the cell (Pawson et al., Science 300:445-52, 2003). This process is particularly evident following DNA damage, where activation of DNA damage kinases results in the formation of protein-protein complexes at discrete foci within the nucleus (Zhou et al., Nature 408:433-9, 2000).

In many cases, kinases directly control the formation of these multi-protein complexes by generating specific phosphorylated-motif sequences; modular binding domains then recognize these short phospho-motifs to mediate protein-protein interactions. The first phosphopeptide-binding modules that were recognized, SH2 and PTB domains, bind specificially to pTyr-containing sequences (Pawson et al., Science 278:2075-80, 1997; Kuriyan et al., Annu Rev Biophys Biomol Struct 26:259-88, 1997; Yaffe, Nat Rev Mol Cell Biol 3:177-86, 2002). As detailed above, a number of modular domains that specifically recognize short pSer/pThr-containing sequences have now been identified, including 14-3-3 proteins, WW domains, FHA domains, and the C-terminal domain of Polo-like kinases (Yaffe et al., Structure 9:R33-8, 2001; Yaffe et al., Curr Opin Cell Biol 13:131-8, 2001; Elia et al., Science 299:1228-31, 2003). All of these pSer/pThr-binding domains participate in cell cycle regulation and the cellular response to genotoxic stress.

The PTIP Tandem C-Terminal BRCT Pair is Necessary and Sufficient for Phospho-Specific Binding

Using the proteomic screening approach (Elia et al., Science 299:1228-31, 2003). described herein, we have now identified novel modular pSer/pThr-binding domains involved in the DNA damage response. Following γ-irradiation, phosphoinositide-like kinases including ATM/ATR and DNA-PK phosphorylate transcription factors, DNA repair proteins, protein kinases and scaffolds on Ser-Gln and Thr-Gln motifs (Abraham, Genes Dev 15:2177-96, 2001). We therefore constructed an oriented peptide library biased to resemble the (pSer or pThr)-Gln motif generated by ATM and ATR (Kim et al., J Biol Chem 274:37538-43, 1999; O'Neill et al., J Biol Chem 275:22719-27, 2000). (FIG. 17A legend). An immobilized form of this library was used in an interaction screen against a library of proteins produced by in vitro expression cloning (Lustig et al., Methods Enzymol 283:83-99, 1997). The amino acids Arg, Lys, and His were intentionally omitted from the degenerate positions in the peptide library to decrease the likelihood of identifying phosphopeptide-binding domains such as 14-3-3, which target basophilic motifs generated by kinases such as AKT, PKA, and PKCs. To control for phosphorylation-independent binding, an identical peptide library was constructed with (Ser or Thr)-Gln substituted for (pSer or pThr)-Gln.

The phosphorylated and non-phosphorylated peptide libraries were immobilized on streptavidin beads, and screened against approximately 96,000 in vitro translated (IVT) polypeptides (960 pools each encoding ˜100 transcripts) over a 10 week period using a high-throughput approach. The majority of IVT products either failed to bind to either of the immobilized peptide libraries or bound slightly better to the non-phosphorylated control (FIG. 17A). Several pools were found to contain cDNAs encoding proteins which bound preferentially to the (pSer or pThr)-Gln library. Pool EEl 1 contained the strongest phosphopeptide-binding clone, EE11-9, which when sib-selected, was found to encode the C-terminal 70% of the human Pax2 trans-activation domain-interacting protein (PTIP) (FIG. 17B) (Lechner et al., Nucleic Acids Res 28:2741-51, 2000; Cho et al., Mol Cell Biol 23:1666-73, 2003). Originally identified in a yeast 2-hybrid screen using Pax2 as bait (Lechner et al., Nucleic Acids Res 28:2741-51, 2000), PTIP appears to play a critical role in the DNA damage response pathway (Cho et al., Mol Cell Biol 23:1666-73, 2003), as well as in facilitating transcriptional responses downstream of TGF-β-Smad2 signaling (Shimizu et al., Mol Cell Biol 21:3901-12, 2001).

Full-length PTIP transcripts also displayed preferential binding to (pSer or pThr)-Gln peptides, though the differential binding was somewhat less pronounced, suggesting that the C-terminal fragment of PTIP likely contains a discrete phosphopeptide binding module. In addition to its Gln-rich region, human PTIP contains 4 BRCT domains, which are known protein-protein interaction modules present in many DNA damage response and cell cycle checkpoint proteins z (Huyton et al., Mutat Res 460:319-32, 2000). A series of deletion constructs was therefore generated and analyzed for phosphopeptide-specific binding (FIG. 17B). A construct containing only the tandem 3^rdand 4^thBRCT domains showed strong and specific binding to the (pSer or pThr)-Gln library. Constructs of PTIP lacking both of these domains failed to bind or lacked phospho-discrimination. Furthermore, neither the 3^rdor 4^thBRCT domains alone bound to phosphopeptides, suggesting that the PTIP tandem C-terminal BRCT pair functions as a single module that is necessary and sufficient for phospho-specific binding.

Tandem BRCT Domains Function as Single Unit to Mediate Phosphopeptide-Binding

BRCT domains are often found in tandem pairs, or multiple copies of tandem pairs. To investigate whether (pSer- or pThr)-binding is a general feature of these domains, we screened tandem BRCT pairs from a number of other DNA damage proteins (FIG. 18A). Like PTIP, the BRCA1 C-terminal BRCT domains also showed phospho-specific binding. Neither of the BRCA1 BRCT domains alone was sufficient for phospho-specific interactions, again suggesting that the tandem BRCT domains are functioning as a single unit. This observation is in excellent agreement with limited proteolysis and X-ray crystallography studies in which the tandem BRCA 1 BRCT domains together with the inter-domain linker behave as a single stable fragment (Williams et al., Nat Struct Biol 8:838-42, 2001). In contrast to PTIP and BRCA1, phospho-specific binding to the tandem BRCT domains of MDC1 or 53BP1 was not observed, and only a very low amount of phospho-specific binding for Rad9 was detected, suggesting that the phosphopeptide-binding function is present in only a subset of tandem BRCT domains.

Identification of Optimal Tandem BRCT Domain-Binding Peptide

Modular domains identified by binding to bead-immobilized phosphopeptide libraries are directly amenable to determination of their optimal binding motif by traditional peptide library screening (Yaffe et al., Methods Enzymol 328:157-70, 2000; Elia et al., Science 299:1228-31, 2003). We determined the optimal pSer/pThr binding motifs for the tandem C-terminal BRCTs in PTIP and BRCA1 using (pSer or pThr)-Gln, pSer- and pThr-containing peptide libraries (FIGS. 18B and 18C, Table 46). For the PTIP C-terminal BRCT, the binding motifs using (pSer or pThr)-Gln, pThr-, and two pSer-containing peptide libraries were identified as SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, and SEQ ID NO: 113, respectively (shown in Table 6). For the BRCA1 C-terminal BRCT, the binding motifs using (pSer or pThr)-Gln, pThr-, and two pSer-containing peptide libraries were identified as SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, respectively (shown in Table 6).

TABLE 6

Phosphoserine and phosphothreonine peptide motif selection

by PTIP and BRCA1 Tandem BRCT motifs

Phosphoserine and Phosphothreonine Peptide

Motif Selection by PTIP and BRCA1 Tandem BRCT Domains

−4
−3
−2
−1

+1
+2
+3
+4
+5

PTIP

X
Y (1.5)
G (2.3)
L (2.6)
pS/pT

Q

V (3.8)

F (7.0)

P (1.6)

I (2.9)

D (1.5)
I (2.5)

F (2.7)

E (1.4)
M (2.5)

L (2.4)

V (1.9)

I (2.8)

L (4.3)

V (2.0)

I (4.1)

Y (2.0)

X
X

E (1.3)
I (1.4)
PS

F (1.7)
V (1.8)

F

X

I (1.9)

M (1.4)

I (1.5)

T (1.5)

F (1.7)

V (1.4)

Q (1.5)

M (1.6)

L (1.3)

Y (1.3)

L (1.4)

G (1.6)
Y (1.1)
D (1.2)
L (1.2)
PS
Q (1.3)
V (2.1)

F (2.3)
P (1.2)
Y (1.3)

E (1.1)
I (1.2)

I (1.3)

I (1.7)

I (2.3)

M (1.2)

P (1.2)

V (1.8)

L (1.7)

Y (1.5)

X
X
X
I (2.1)
PT
Q (1.5)
Y (1.4)

I (1.4)
F (1.5)
A

L (1.8)

F (1.4)

L (1.3)
Y (1.4)

W (1.3)

I (1.3)

V (1.2)
P (1.3)

BRCA1

X

F (1.7)
D (1.2)
I (1.4)
pS/pT

Q

V (3.1)

F (7.5)

V (1.5)

F (4.5)

Y (1.6)

E (1.1)
V (1.3)

T (2.6)

Y (5.2)

P (1.4)
G (1.8)

L (1.2)

I (2.2)

M (1.2)

S (1.7)

X
R (1.5)

E (1.3)
V (1.4)
pS

F (2.1)

T (1.9)

F

X

F (1.6)

Y (1.4)
D (1.2)
I (1.3)

Y (1-6)
V (1.7)

M (1.4)

M (1.3)

I (1.4)

Y (1.3)

Q (1.4)

X
X
Y (1.2)
X
pS
Q (1.4)
V (1.2)

F (2.4)
I (1.2)
X

F (1.3)

I (1.2)
Y (1.5)

X

E (1.5)
D (1.9)
I (1.6)
pT
Q (1.5)
D (1.5)

F (1.9)
D (1.4)

A

E (1.5)
L (1.4)

E (1.4)
Y (1.3)
Y (1.2)
P (1.2)

F (1.3)

I (1.2)

A GST fusion of the PTIP or BRCA1 tandem BRCT domains was screened for binding to four phosphopeptide libraries, which contained the sequences GAXXXB(pS/pT)QJXXXAKKK (SEQ ID NO: 62), GAXXXXpSXXFXXAYKKK (SEQ ID NO: 59), MAXXXXpTXXXXAKKK (SEQ ID NO: 47), and MAXXXXpSpXXXXXAKKK (SEQ ID NO: 58), where X indicates all amino acids except Cys. In the library MAXXXB(pS/pT)QJXXXAKKK (SEQ ID NO: 62) B indicates A, I, L, M, N, P, S, T, V, and J represents a biased mixture of 25% E, 75% X, while X indicates all amino acids except Arg, Cys, His, Lys for all positions in this library. Residues showing strong enrichment are underlined.

Table 6 shows the results of a phosphoserine and phosphothreonine motif selection by PTIP and BRCA1 tandem BRCT domains. A GST fusion of the PTIP or BRCA 1 tandem BRCT domains was screened for binding to three phosphopeptide libraries, which contained the sequences MAXXXB(pS/pT)QJXXXAKKK SEQ ID NO: 57, MAXXXXpTXXXXAKKK SEQ ID NO: 47, and MAXXXXSpXXXXXAKKK SEQ ID NO: 58; where X indicates all amino acids except Cys. In the libraries MAXXXB(pS/pT)QJXXXAKKK (SEQ ID NO: 57) and GAXXXXpSXXFXXAYKKK (SEQ ID NO: 59), B indicates A, I, L, M, N, P, S, T, V; and J represents a biased mixture of 25% E, 75% X, while X indicates all amino acids except Arg, Cys, His, Lys. Residues showing very strong enrichment (ratio>3) are underlined.

PTIP and BRCA1 BRCTs displayed similar, but not identical motifs, with extremely strong selection for aromatic/aliphatic residues, and aromatic residues, respectively, in the (pSer or pThr)+3 position when screened with a (pSer or pThr)-Gln library. Prominent amino acid selection was also observed in the (pSer or pThr)+2 and +5 positions, in addition to more moderate selection at other positions. Because the BRCT domains were isolated in a screen for domains that bind to (pSer or pThr)-Gln motifs, we investigated the relative importance of Gln in the (pSer or pThr)+1 position using individual pThr- or pSer-oriented peptide libraries. This analysis revealed modest selection for Gln in the degenerate+1 position. Furthermore, the absence of a fixed Gln in the +1 position reduced the selection for aromatic and aliphatic residues in the +3 and +5 positions, suggesting that while Gln in the (pSer or pThr)+1 position was not essential, it was clearly a favored residue. In agreement with this finding, we observed considerably stronger binding of the tandem BRCT domains to bead-immobilized (pSer or pThr)-Gln libraries than to libraries containing only a fixed pSer motif (FIG. 18A).

On the basis of peptide library data, we defined an optimal tandem BRCT domain-binding peptide as Y-D-I-(pSer or pThr)-Q-V-F—P—F (SEQ ID NO: 60). Isothermal titration calorimetry (ITC) showed that the optimal phosphoserine-containing peptide bound to the tandem C-terminal BRCTs of PTIP with a dissociation constant of 280 nM, and to the BRCT domains of BRCA1 with a dissociation constant of 400 nM (Table 7). Binding affinity results for GAAYDI-pS-QVFPFAKKK (SEQ ID NO: 123), GAAYDI-pT-QVFPFAKKK (SEQ ID NO: 124), GAAYDI-S-QVFPFAKKK (SEQ ID NO: 125), GAAYDI-T-QVFPFAKKK (SEQ ID NO: 126), GAAYDI-pS-QVFPFAKKK (SEQ ID NO: 127), GAAYDI-S-QVFPFAKKK (SEQ ID NO: 128), and GAAYDI-T-QVFPFAKKK (SEQ ID NO: 129) are shown in Table 7.

TABLE 7

Peptide binding affinities

for the tandem BRCT domains

Table S2. Peptide Binding Affinities

for the Tandem BRCT Domains

(BRCT)₂

Peptide
Sequence
Domain
K_d

BRCTtide-7pS
GAAYDI-pS-QVFPFAKKK
PTIP
280 nM

BRCTtide-7pT
GAAYDI-pT-QVFPFAKKK
PTIP
14.3 μM

BRCTtide-7S
GAAYDI-S-QVFPFAKKK
PTIP
N.D.B.

BRCTtide-7T
GAAYDI-T-QVFPFAKKK
PTIP
N.D.B.

BRCTtide-7pS
GAAYDI-pS-QVFPFAKKK
BRCA1
400 nm

BRCTtide-7S
GAAYDI-S-QVFPFAKKK
BRCA1
N.D.B.

BRCTtide-7T
GAAYDI-T-QVFPFAKKK
BRCA1
N.D.B.

Isothermal titration calorimetry (ITC) was used to determine binding constants (K_d). All observed binding stoichiometries were consistent with a 1:1 complex of protein and phosphopeptide. N.D.B indicates no detectable binding by ITC fog a tandem BRCT domain with a concentration of at least 150 μM. pS and pT denote phosphosarine and phosphopthreonine, respectively.

PTIP and BRCA1 tandem BRCT domains were purified as GST-fusion proteins from E. coli and binding to individual peptides measured by isothermal titration calorimetry. Binding stoichiometries were consistent with a 1:1 complex of protein and phosphopeptide. Replacement of pThr for pSer reduced the affinity of the peptide for the PTIP BRCT domains, while substitution of Thr for pThr abrogated binding altogether.

To further verify motif selection, binding of the tandem BRCT domains to a solid-phase array of immobilized phosphopeptides was performed in which each amino acid flanking the pThr-Gln core (FIGS. 18D and 18E) or flanking the pSer (FIGS. 18F and 18G) in the optimal BRCTtide was varied. The resulting selectivities were generally consistent with the results obtained using oriented peptide libraries in solution. Substitution of pSer for pThr significantly enhanced binding for both PTIP and BRCA1, consistent with the ITC results for PTIP. Substitution of pTyr for pThr eliminated binding altogether, verifying that tandem BRCT domains are pSer/pThr-specific binding modules. As expected, replacement of pThr with Thr, Ser or Tyr abrogated tandem BRCT domain binding.

Tandem BRCT Domain Binding Eliminated by Pre-Incubation with (pSer or pThr)-Gln Peptide Library

To examine the role of tandem BRCT domains in binding to ATM/ATR/ATX-phosphorylated proteins after DNA damage, U2OS cell lysates, prior to and following 10 Gy of γ-irradiation, were incubated with GST-(BRCT)₂fusion proteins and blotted with an anti-(pSer or pThr)-Gln motif antibody raised against the phosphorylation motif generated by ATM and ATR (Cell Signaling Technologies) (FIGS. 19A-19D). Following γ-irradiation, both PTIP and BRCA1 tandem C-terminal BRCTs bound to numerous proteins recognized by the anti-ATM/ATR phosphoepitope motif antibody (FIG. 19A). This interaction could be inhibited by pre-incubating the tandem BRCT domains with a (pSer or pThr)-Gln peptide library, but not with a pThr-Pro library or with the non-phosphorylated (Ser or Thr)-Gln library. A time course analysis revealed optimal binding of both the PTIP and BRCA1 BRCT domains to (pSer or pThr)-Gln-containing proteins in irradiated cell lysates at 0.5 and 2 hours after DNA damage (FIGS. 19B and 19D). Binding was largely eliminated by the optimal BRCTtide (opt), but not by its non-phosphorylated analogue (7T), or by pre-treatment of the cells with caffeine to inhibit ATM and ATR prior to γ-irradiation. In both cases where the phospho-specific interaction was eliminated, we observed a ˜170 kDa immunoreactive band in the PTIP BRCT domain pulldowns, but not in the BRCA1 pulldowns; this band likely resulted from an interaction with the PTIP BRCT domains at a site distinct from its phosphopeptide-binding pocket.

Tandem C-Terminal BRCT Domains are Necessary and Sufficient for Nuclear Foci Formation Following DNA Damage

In response to γ-irradiation, the DNA damage protein 53BP1 undergoes phosphorylation by ATM and facilitates the ability of ATM to phosphorylate additional cellular substrates (Schultz et al., J Cell Biol 151:1381, 2000; Rappold et al., J Cell Biol 153:613-20, 2001; Anderson et al., Mol Cell Biol 21:1719-29, 2001; Abraham, Nat Cell Biol 4:E277-9, 2002; Wang et al., Science 298:1435-8, 2002; Fernandez-Capetillo et al., Nat Cell Biol 4:993-7, 2002; DiTullio, Jr. et al., Nat Cell Biol 4:998-1002, 2002). 53BP1 migrates at a similar Mr as one or more of the bands in FIGS. 19A and 19B and contains multiple potential Ser/Thr-Gln ATM/ATR phosphorylation sites that closely match the optimal PTIP tandem BRCT-binding motif. Endogenous 53BP1 from U2OS cell lysates bound to the tandem C-terminal BRCT domains of PTIP only following DNA damage (FIG. 19C). Similar to the results obtained with the (pSer or pThr)-Gln motif antibody, a time course of cells transfected with HA-tagged 53BP1 revealed optimal binding at 0.5 and 2 hours following γ-irradiation. This binding was inhibited by preincubation with optimal BRCTtide, but was not eliminated by pre-incubation with its non-phosphorylated counterpart. Binding was also eliminated by pre-incubation of the tandem BRCT domains with the (pSer or pThr)-Gln peptide library, but not by pre-incubation with a pThr-Pro library or the non-phosphorylated (Ser or Thr)-Gln library, as well as by treatment with caffeine prior to λ-irradiation or treatment of the lysates with k-phosphatase following irradiation.

Although PTIP was originally identified as a transcriptional control protein, recent data suggests that PTIP might also be involved in DNA damage signaling (Cho et al., Mol Cell Biol 23:1666-73, 2003). Mice homozygous for a PTIP null allele undergo embryonic lethality at E9.5, with evidence of extensive DNA damage and the presence of free DNA ends. Neither fibroblasts nor embryonic stem cells from PTIP null mice could be propagated in culture, and trophoblast cells, which showed decreased viability in general, showed an increased sensitivity to low doses of ionizing radiation (Cho et al., Mol Cell Biol 23:1666-73, 2003). This data, together with our finding that the tandem BRCT domains at the C-terminus of PTIP bind to ATM/ATR phosphorylated proteins, suggested that full-length PTIP might localize at sites of DNA damage in vivo.

To investigate this, U2OS cells were transfected with GFP fusions of full-length PTIP, PTIP lacking the last two C-terminal BRCT domains, or the isolated tandem C-terminal BRCT domains alone (FIGS. 20A-20C). In the absence of irradiation, PTIP was diffusely nuclear with a small amount of cytosolic staining. Two hours following DNA damage, PTIP re-localized into discrete nuclear foci that significantly co-localized with ATM/ATR phosphoepitopes, 53BP1 and phospho-H2AX (FIG. 20A). Deletion of the C-terminal BRCTs from PTIP resulted in its constitutive diffuse nuclear and cytoplasmic localization and an inability to form foci after DNA damage (FIG. 18B). The isolated PTIP C-terminal tandem BRCT domains, while predominantly diffusely nuclear in the absence of DNA damage, efficiently re-localized into the same punctate nuclear foci after γ-irradiation as full-length PTIP (FIG. 18C). Thus, the tandem C-terminal BRCT domains of PTIP, which are necessary and sufficient for binding to (pSer or pThr)-Gln peptides in solution, are necessary and sufficient for nuclear foci formation by full-length PTIP following DNA damage.

Caffeine attenuates recruitment of PTIP to DNA damage foci in response to ionizing radiation (FIGS. 21A and 21B). U2OS cells transfected with full-length PTIP-GFP cDNA were mock treated or pretreated with 10 mM caffeine for 70 minutes before exposure to 10Gy ionizing radiation. In reponse to IR ionizing radiation, mock-treated U2OS cells formed nuclear foci containing PTIP (in green) and H2AXp (in red); these two proteins co-localize at sites of DNA damage (merge). In response to IR, caffeine treated U2OS cells formed reduced numbers of nuclear foci; PTIP was mislocalized and did not form discrete nuclear foci (in green) and there were reduced numbers of H2AXp (in red) containing foci. These results demonstrate that pretreatment with caffeine effectively abolished co-localization of PT1P and H2AXp (merge).

Our identification of tandem BRCT domains as a new pSer/pThr-binding module targeting ATM and ATR phosphorylation motifs expands the range of functions subserved by this domain in response to DNA damage signaling. Only tandem pairs were observed to function in this capacity, and only a subset of BRCT domains, including those in PTIP and BRCA1, appear to show phospho-specific binding. The important role for tandem BRCT domains as phospho-binding modules is emphasized by the finding that ˜80% of germline mutations in BRCA1 result in C-terminal truncations involving the BRCT region, predisposing women to breast and ovarian cancer (Huyton et al., Mutat Res 460:319-32, 2000). Interestingly, a BRCA1 cancer-associated mutation in the (BRCT)2 module that ablates critical BRCA1 protein interactions, Met17753Arg (M1775R), fails to bind phosphopeptides (FIG. 2A), even though the M1775R crystal structure is nearly identical to that of the wild-type (BRCT)2. The finding that BRCT domains bind to pSer-containing peptides more strongly than to pThr-containing peptides is novel since WW domains, 14-3-3 proteins, FHA domains and Polobox domains either bind pThr-peptides better than pSer peptides, or do not bind to pSer-peptides at all (Verdecia et al., Nat Struct Biol 7:639-43, 2000; Durocher et al., Mol Cell, 6:1169-1182, 2000; Elia et al., Science 299:1228-31, 2003). Intriguingly, ATM and ATR preferentially phosphorylate Ser-Gln over Thr-Gln motifs (Kim et al., J Biol Chem 274:37538-43, 1999), suggesting functional convergence between the motifs generated by phosphoinositide-like kinases and the motifs recognized by BRCT domains. The observed BRCT domain selection for aromatic and aliphatic residues in the (pSer or pThr)+3 and +5 positions within their bound substrates exceeds their modest selection for Gln in the +1 position. Thus, only a subset of ATM/ATR phosphorylated substrates are likely to bind with high affinity. Kinases other than Gln-directed kinases might also generate potential BRCT domain-binding motifs. In addition, the results of our screen provide a molecular rationale for the early embryonic lethality of PTIP knock-out mice with extensive unrepaired DNA ends. The finding that the C-terminal tandem BRCT domains of PTIP bind to ATM/ATR-phosphorylated motifs and localize full-length PTIP to sites of DNA damage strongly suggests that PTIP functions as a key component of the DNA damage response. Interference with the normal process of DNA damage signaling is responsible not only for tumorigenesis but also for tumor cell death in the face of massive DNA damage induced by chemotherapeutic agents, depending on the remaining genetic background of the cancer cell (Scully et al., Nature 408:429-32, 2000). Agents that interfere with DNA damage signaling sensitize tumor cells to killing by radiation and chemotherapy. Thus, the phosphopeptide-binding pocket of tandem BRCT domains constitutes a promising target for anti-cancer drug development.\

ATM/ATR/ATX Phospho-Motif Screen for Phosphoserine/Threonine Binding Domains

An oriented (pSer/pThr) phosphopeptide library biased toward the phosphorylation motifs for ATM/ATR kinases and its non-phosphorylated counterpart were constructed as follows: biotin-Z-G-Z-G-G-A-X-X-X-B-(pS/pT)-QJ-X-X-X-A-K-K-K SEQ ID NO:35 and biotin-Z-G-Z-G-G-A-X-X-X-B-(S/T)-Q-J-X-X-X-A-K-K-K SEQ ID NO:61, where pS denotes phosphoserine; pT phosphothreonine; Z indicates aminohexanoic acid; B represents a biased mixture of the amino acids A, I, L, M, N, P, S, T, V; and J represents a biased mixture of 25% E and 75% X, where “X” denotes all amino acids except Arg, Cys, His, Lys. Streptavidin beads (Pierce, 75 pmol/μL gel) were incubated with a ten-fold molar excess of each biotinylated library in 50 mM Tris/HCl (pH7.6), 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 2 mM DTT and washed five times with the same buffer to remove unbound peptide. The bead-immobilized libraries (10 μL of gel) were added to 10 μL of an in vitro translated [³⁵S]-labeled protein pool in 150 μL binding buffer (50 mM Tris/HCl (pH7.6), 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 2 mM DTT, 8 μg/mL pepstatin, 8 μg/mL aprotinin, 8 μg/mL leupeptin, 800 μM Na₃VO4, 25 mM NaF). Each pool consisted of ˜100 radiolabeled proteins produced by the PROTEOLINK in vitro expression cloning system (Promega, Madison, Wis.). After incubation at 4° C. for 3 hours, the beads were rapidly washed three times 200 μL with binding buffer prior to SDS-PAGE (12.5%) and autoradiography. Positively scoring hits were identified as protein bands that interacted more strongly with the phosphorylated immobilized library than with the unphosphorylated counterpart. Pools containing positively scoring clones were progressively subdivided and re-screened for phosphobinding until single clones were isolated and identified by DNA sequencing.

Cloning, Expression, and Purification of PTIP and BRCA1

For deletion mapping of the PTIP and BRCA1 BRCT phospho-binding region and for expression of MDC 1, 53BP1 and Rad9 (FIG. 17-18), fragments were generated by PCR for in vitro transcription/translation and cloned into a pcDNA3.1 expression vector (Invitrogen, San Diego, Calif.). For production of recombinant GST-PTIP BRCT domains and GSTBRCA1 BRCT domains, residues 550-757 of PTIP and residues 1634-1863 of BRCA1 were ligated into the EcoRI and NotI sites of pGEX-4T1 (Pharmacia, Peapack, N.J.) and subsequently transformed into DH5a E. Coli. Protein induction occurred at 37° C. for 4 hours or at 25° C. for 16 hours in the presence of 0.4 mM IPTG. For peptide filter blot analysis and measurements of peptide binding affinity by ITC, GSTPTIP BRCT domains (residues 550-757) and GST-BRCA1 BRCT domains (residues 1634-1863) were isolated from bacterial lysates using glutathione agarose, eluted with 40 mM glutathione, and dialyzed into 50 mM Tris/HCl (pH 8.1), 300 mM NaCl. The GFP-PTIP constructs FL (residues 1-757), !BRCT (residues 1-550), or (BRCT)2 (residues 550-757) were cloned into the EcoRI and SalI sites of the pEGFP-C2 (BD Biosciences Clontech Franklin Lakes, N.J.) expression vector.

Peptide Library Screening

Phosphoserine and phosphothreonine oriented degenerate peptide libraries consisting of the sequences Gly-Ala-X-X-X-B-(pSer/pThr)-Gln-J-X-X-X-Ala-Lys-Lys-Lys SEQ ID NO: 62, Met-Ala-X-X-X-X-pThr-X-X-X-X-Ala-Lys-Lys-Lys SEQ ID NO: 47, and Met-Ala-X-X-X-XpSer-X-X-X-X-X-Ala-Lys-Lys-Lys SEQ ID NO: 58; where pS is phosphoserine, pT is phosphothreonine; and X denotes all amino acids except Cys. In the (pSer/pThr)-Gln library, B is a biased mixture of the amino acids A, I, L, M, N, P, S, T, V, and J represents a biased mixture of 25% E, 75% X, where X denotes all amino acids except Arg, Cys, His, Lys. Peptides were synthesized using N-α-FMOC-protected amino acids and standard BOP/HOBt coupling chemistry. Peptide library screening was performed using 125 μl of glutathione beads containing saturating amounts of GST-PTIP BRCT or GST-BRCA1 BRCT domains (1-1.5 mg) as described by Yaffe and Cantley (Methods Enzymol 328:157-70, 2000). Beads were packed in a 1 mL column and incubated with 0.45 mg of the peptide library mixture for 10 minutes at room temperature in PBS (150 mM NaCl, 3 mM KCl, 10 mM Na₂HPO₄, 2 mm KH2PO4, pH 7.6). Unbound peptides were removed from the column by two washes with PBS containing 1.0% NP-40 followed by two washes with PBS. Bound peptides were eluted with 30% acetic acid for 10 minutes at room temperature, lyophilized, resuspended in H2O, and sequenced by automated Edman degradation on a PROCISE protein microsequencer (Perkin-Elmer Corporation, Norwalk Conn.). Selectivity values for each amino acid were determined by comparing the relative abundance (mole percentage) of each amino acid at a particular sequencing cycle in the recovered peptides to that of each amino acid in the original peptide library mixture at the same position.

Isothermal Titration Calorimetry

Peptides were synthesized by solid phase technique with three C-terminal lysines to enhance solubility. The peptides were then purified by reverse phase HPLC following deprotection and confirmed by MALDI-TOF mass spectrometry. Calorimetry measurements were performed using a VP-ITC microcalorimeter (MicroCal Inc., Studio City, Calif.). Experiments involved serial 10 μL injections of peptide solutions (20 μM-150 μM) into a sample cell containing 15 μM GST-PTIP BRCT domains (residues 550-757) or 15 μM GST-BRCA1 BRCT domains (residues 1634-1863) in 50 mM Tris/HCl (pH 8.1), 300 mM NaCl. Twenty injections were performed with 240 second intervals between injections and a reference power of 25 μCal/s. Binding isotherms were plotted and analyzed using ORIGIN Software (MicroCal Inc. Studio City, Calif.).

Peptide Filter Array

An ABIMED peptide arrayer with a computer controlled Gilson diluter and liquid handling robot (Abimed GmbH, Dusseldorf, Germany) was used to synthesize peptides onto an amino-PEG cellulose membrane using N-α-FMOC-protected amino acids and DIC/HOBT coupling chemistry. The membranes were blocked in 5% milk/TBS-T (0.1%) for lhour at room temperature, incubated with 0.05 μM GST-PTIP BRCT domains (residues 550-757) or GST-BRCA1 BRCT domains (residues 1634-1863) in 5% milk, 50 mM Tris/HCl (pH 7.6), 150 mM NaCl, 2 mM EDTA, 2 mM DTT for 1 hour at room temperature and washed four times with TBS-T (0.1%). The membranes were then incubated with anti-GST conjugated HRP (Amersham) in 5% milk/TBS-T (0.1%) for 1 hour at room temperature, washed five times with TBS-T (0.1%), and subjected to chemiluminescence.

PTIP BRCT Domains and BRCA1 BRCT Domains Binding to Cellular Substrates

U2OS cells were either treated with 10 Gy of ionizing radiation or mock irradiated and allowed to recover for 30-120 minutes. Cells were subsequently lysed in 50 mM Tris/HCl (pH7.6), 150 mM NaCl, 1.0% NP-40, 5 mM EDTA, 2 mM DTT, 8 μg/mL pepstatin, 8 μg/mL aprotinin, 8 μg/mL leupeptin, 2 mM Na3VO4, 10 mM NaF, 1 μM microcystin. The lysates (0.5-2 mg) were incubated with 20 μL glutathione beads containing 10-20 μg of GST-PTIP BRCT domains (residues 550-757), GST-BRCA1 BRCT domains (residues 1634-1863), or GST for 120 minutes at 4° C. Beads were washed three times with lysis buffer. Precipitated proteins were eluted in sample buffer and detected by blotting with anti-ATM/ATR substrate (pSer/pThr)Gln antibody (CELL SIGNALING TECHNOLOGY, Inc Beverly, Mass.), polyclonal anti-53BP1 (ONCOGENE RESEARCH PRODUCTS, San Diego, Calif. 92121), or monoclonal anti-HA (COVANCE Inc, Princeton, N.J.). For peptide competition experiments, GST-PTIP BRCT domains or GST-BRCA1 BRCT domains were immobilized on glutathionine beads and preincubated with 350 μM of BRCTtide-optimal, 7pT, 7T, pSQ-library, SQ-library, or pTP-library for 1 hour at 4° C. and washed three times with lysis buffer.

Immunofluorescence and Microscopy

U2OS cells were seeded onto 18 mm2 coverslips and transfected with GFP-PTIP constructs FL (residues 1-757), !BRCT (residues 1-550), or (BRCT)2 (residues 550-757) using FUGENE6 transfection reagent (Roche, Base1, Switzerland) according to manufacture's protocol. Twenty-four hours following transfection, the cells were either treated with 10 Gy of ionizing radiation or mock irradiated and allowed to recover for 120 minutes. Cells were fixed in 3% paraformaldehyde/2% sucrose for 15 minutes at room temperature and extracted with a 0.5% Triton X-100 solution containing 20 mM Tris-HCl (pH 7.8), 75 mM NaCl, 300 mM sucrose, and 3 mM MgC12 for 15 minutes at room temperature. Slides were stained with primary antibodies at 4° C. overnight, then stained with a Texas Red conjugated anti-mouse or anti-rabbit secondary antibody for 60 minutes (Molecular Probes, Eugene, Oreg.) at room temperature. Primary antibodies used were rabbit anti-53BP1 (Oncogene Research Products, San Diego, Calif.), mouse anti-g-H2AX (Upstate, Charlottesville, Va.), and rabbit anti-(pS/pT)Q (Cell Signaling Technology, Inc., Beverly, Mass.). Images were collected on a Deltavision microscope (Carl Zeiss, Thornwood, N.Y.) and digitally deconvolved using SOFTWORX graphics processing software (SGI, CSIF, Stanford, Calif.).

Peptidomimetics

Peptide derivatives (e.g. peptidomimetics) include cyclic peptides, peptides obtained by substitution of a natural amino acid residue by the corresponding D-stereoisomer, or by a unnatural amino acid residue, chemical derivatives of the peptides, dual peptides, multimers of the peptides, and peptides fused to other proteins or carriers. A cyclic derivative of a peptide of the invention is one having two or more additional amino acid residues suitable for cyclization. These residues are often added at the carboxyl terminus and at the amino terminus. A peptide derivative may have one or more amino acid residues replaced by the corresponding D-amino acid residue. In one example, a peptide or peptide derivative of the invention is all-L, all-D, or a mixed D,L-peptide. In another example, an amino acid residue is replaced by a unnatural amino acid residue. Examples of unnatural or derivatized unnatural amino acids include Na-methyl amino acids, Cα-methyl amino acids, and β-methyl amino acids.

A chemical derivative of a peptide of the invention includes, but is not limited to, a derivative containing additional chemical moieties not normally a part of the peptide. Examples of such derivatives include: (a) N-acyl derivatives of the amino terminal or of another free amino group, where the acyl group may be either an alkanoyl group, e.g., acetyl, hexanoyl, octanoyl, an aroyl group, e.g., benzoyl, or a blocking group such as Fmoc (fluorenylmethyl-O—CO—), carbobenzoxy (benzyl-O—CO—), monomethoxysuccinyl, naphthyl-NH—CO—, acetylamino-caproyl, adamantyl-NH—CO—; (b) esters of the carboxyl terminal or of another free carboxyl or hydroxy groups; (c) amides of the carboxyl terminal or of another free carboxyl groups produced by reaction with ammonia or with a suitable amine; (d) glycosylated derivatives; (e) phosphorylated derivatives; (f) derivatives conjugated to lipophilic moieties, e.g., caproyl, lauryl, stearoyl; and (g) derivatives conjugated to an antibody or other biological ligand. Also included among the chemical derivatives are those derivatives obtained by modification of the peptide bond—CO—NH—, for example, by: (a) reduction to —CH₂—NH—; (b) alkylation to —CO—N(alkyl)—; and (c) inversion to —NH—CO—.

A dual peptide of the invention consists of two of the same, or two different, peptides of the invention covalently linked to one another, either directly or through a spacer.

Multimers of the invention consist of polymer molecules formed from a number of the same or different peptides or derivatives thereof.

In one example, a peptide derivative is more resistant to proteolytic degradation than the corresponding non-derivatized peptide. For example, a peptide derivative having D-amino acid substitution(s) in place of one or more L-amino acid residue(s) resists proteolytic cleavage.

In another example, the peptide derivative has increased permeability across a cell membrane as compared to the corresponding non-derivatized peptide. For example, a peptide derivative may have a lipophilic moiety coupled at the amino terminus and/or carboxyl terminus and/or an internal site. Such derivatives are highly preferred when targeting intracellular protein-protein interactions, provided they retain the desired functional activity.

In another example, a peptide derivative binds with increased affinity to a ligand (e.g., a Polo box domain).

The peptides or peptide derivatives of the invention are obtained by any method of peptide synthesis known to those skilled in the art, including synthetic and recombinant techniques. For example, the peptides or peptide derivatives can be obtained by solid phase peptide synthesis which, in brief, consists of coupling the carboxyl group of the C-terminal amino acid to a resin and successively adding N-alpha protected amino acids. The protecting groups may be any such groups known in the art. Before each new amino acid is added to the growing chain, the protecting group of the previous amino acid added to the chain is removed. The coupling of amino acids to appropriate resins has been described by Rivier et al. (U.S. Pat. No. 4,244,946). Such solid phase syntheses have been described, for example, by Merrifield, J. Am. Chem. Soc. 85:2149, 1964; Vale et al., Science 213:1394-1397, 1984; Marki et al., J. Am. Chem. Soc. 10:3178, 1981, and in U.S. Pat. Nos. 4,305,872 and 4,316,891. In a preferred aspect, an automated peptide synthesizer is employed.

Purification of the synthesized peptides or peptide derivatives is carried out by standard methods, including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, hydrophobicity, or by any other standard technique for the purification of proteins. In one embodiment, thin layer chromatography is employed. In another embodiment, reverse phase HPLC (high performance liquid chromatography) is employed.

Finally, structure-function relationships determined from the peptides, peptide derivatives, and other small molecules of the invention may also be used to prepare analogous molecular structures having similar properties. Thus, the invention is contemplated to include molecules in addition to those expressly disclosed that share the structure, hydrophobicity, charge characteristics and side chain properties of the specific embodiments exemplified herein.

In one example, such derivatives or analogs that have the desired binding activity can be used for binding to a molecule or other target of interest, such as any Polo-box domain. Derivatives or analogs that retain, or alternatively lack or inhibit, a desired property-of-interest (e.g., inhibit PBD binding to a natural ligand), can be used to inhibit the biological activity of a Polo-like kinase (e.g., Plk-1, 2, or 3).

In particular, peptide derivatives are made by altering amino acid sequences by substitutions, additions, or deletions that provide for functionally equivalent molecules, or for functionally enhanced or diminished molecules, as desired. Due to the degeneracy of the genetic code, other nucleic acid sequences that encode substantially the same amino acid sequence may be used for the production of recombinant peptides. These include, but are not limited to, nucleotide sequences comprising all or portions of a peptide of the invention that is altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.

The derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations that result in their production can occur at the gene or protein level. For example, a cloned nucleic acid sequence can be modified by any of numerous strategies known in the art (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.

Modified Phosphopeptides

A phosphopeptide of the invention may include, but it is not limited to, an unnatural N-terminal amino acid of the formula (III):

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where A¹is an amino acid or peptide chain linked via an α-amino group; R¹and R³are independently hydrogen, C_1-5branched or linear C_1-5alkyl, C_1-5alkaryl, heteroaryl, and aryl, each of which are unsubstituted or substituted with a substitutent selected from: 1 to 3 of C_1-5alkyl, 1 to 3 of halogen, 1 to 2 of —OR⁵, N(R⁵)(R⁶), SR⁵, N—C(NR⁵)NR⁶R⁷, methylenedioxy, —S(O)_mR⁵, 1 to 2 of —CF₃, —OCF₃, nitro, —N(R⁵)C(O)(R⁶), —C(O)OR⁵, —C(O)N(R⁵)(R⁶), -1H-tetrazol-5-yl, —SO₂N(R⁵)(R⁶), —N(R⁵)SO₂aryl, or —N(R⁵)SO₂R⁶; R⁵, R⁶and R⁷are independently selected from hydrogen, C_1-5linear or branched alkyl, C_1-5alkaryl, aryl, heteroaryl, and C_3-7cycloalkyl, and where two C_1-5alkyl groups are present on one atom, they optionally are joined to form a C_3-8cyclic ring, optionally including oxygen, sulfur or NR⁷, where R⁷is hydrogen, or C_1-5alkyl, optionally substituted by hydroxyl; R²is hydrogen, F, C_1-5linear or branched alkyl, C_1-5alkaryl; or R²and R¹are joined to form a C_3-8cyclic ring, optionally including oxygen, sulfur, or NR⁷, where R⁷is hydrogen, or C_1-5alkyl, optionally substituted by hydroxyl, or R²and R³are joined to form a C_3-8cyclic ring, optionally substituted by hydroxyl and optionally including oxygen, sulfur or NR⁷, where R⁷is hydrogen, or C_1-5alkyl; R²is hydrogen, F, C_1-5linear or branched alkyl, C_1-5alkaryl; and R⁴is hydrogen, C_1-5branched or linear C_1-5alkyl, C_1-5alkaryl, heteroaryl, and aryl, each of which are unsubstituted or substituted with a substitutent selected from: 1 to 3 of C_1-5alkyl, 1 to 3 of halogen, 1 to 2 of —OR⁵, N(R⁵)(R⁶), N—C(NR⁵)NR⁶R⁷, methylenedioxy, —S(O)_mR⁵(where m is 0-2), 1 to 2 of —CF₃, —OCF₃, nitro, —N(R⁵)C(O)(R⁶), —N(R⁵)C(O)(OR⁶), —C(O)OR⁵, —C(O)N(R⁵)(R⁶), -1H-tetrazol-5-yl, —SO₂N(R⁵)(R⁶), —N(R⁵)SO₂aryl, or —N(R⁵)SO₂R⁶, R⁵, R⁶and R⁷are independently selected from hydrogen, C_1-5linear or branched alkyl, C_1-5alkaryl, aryl, heteroaryl, and C_3-7cycloalkyl, and where two C_1-5alkyl groups are present on one atom, they optionally are joined to form a C_3-8cyclic ring, optionally including oxygen, sulfur or NR⁷, where R⁷is hydrogen, or C_1-5alkyl, optionally substituted by hydroxyl.

The phosphopeptides of the invention may also include an internal unnatural internal amino acid of the formula:

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where A²is an amino acid or peptide chain linked via an α-carboxy group; A¹is an amino acid or peptide chain linked via an α-amino group; R¹and R³are independently hydrogen, C_1-5branched or linear C_1-5alkyl, C_1-5alkaryl, heteroaryl, and aryl, each of which are unsubstituted or substituted with a substitutent selected from: 1 to 3 of C_1-5alkyl, 1 to 3 of halogen, 1 to 2 of —OR⁵, N(R⁵)(R⁶), SR⁵, N—C(NR⁵)NR⁶R⁷, methylenedioxy, —S(O)_mR⁵(m is 1-2), 1 to 2 of —CF₃, —OCF₃, nitro, —N(R⁵)C(O)(R⁶), —C(O)OR⁵, —C(O)N(R⁵)(R⁶), -1H-tetrazol-5-yl, —SO₂N(R⁵)(R⁶), —N(R⁵)SO₂aryl, or —N(R⁵)SO₂R⁶; R⁵, R⁶and R⁷are independently selected from hydrogen, C_1-5linear or branched alkyl, C_1-5alkaryl, aryl, heteroaryl, and C_3-7cycloalkyl, and where two C_1-5alkyl groups are present on one atom, they optionally are joined to form a C_3-8cyclic ring, optionally including oxygen, sulfur or NR⁷, where R⁷is hydrogen, or C_1-5alkyl, optionally substituted by hydroxyl; and R²is hydrogen, F, C_1-5linear or branched alkyl, C_1-5alkaryl; or R²and R¹are joined to form a C_3-8cyclic ring, optionally including oxygen, sulfur or NR⁷, where R⁷is hydrogen, or C_1-5alkyl, optionally substituted by hydroxyl, or R²and R³are joined to form a C_3-8cyclic ring, optionally substituted by hydroxyl and optionally including oxygen, sulfur or NR⁷, where R⁷is hydrogen, or C_1-5alkyl.

The invention also includes modifications of the phosphopeptides of the invention, wherein an internal unnatural internal amino acid of the formula:

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is present, where A²is an amino acid or peptide chain linked via an α-carboxy group; A¹is an amino acid or peptide chain linked via an α-amino group; R¹and R³are independently hydrogen, C_1-5branched or linear C_1-5alkyl, and C_1-5alkaryl; R²is hydrogen, F, C_1-5linear or branched alkyl, C_1-5alkaryl; or R²and R¹are joined to form a C_3-8cyclic ring, optionally including oxygen, sulfur or NR⁷, where R⁷is hydrogen, or C_1-5alkyl, optionally substituted by hydroxyl; X is O or S; and R⁵and R⁶are independently selected from hydrogen, C_1-5linear or branched alkyl, C_1-5alkaryl, aryl, heteroaryl, and C_3-7cycloalkyl, and where two C_1-5alkyl groups are present on one atom, they optionally are joined to form a C_3-8cyclic ring, optionally including oxygen, sulfur or NR⁷, where R⁷is hydrogen, or C_1-5alkyl, optionally substituted by hydroxyl; or R⁵and R⁶are joined to form a C_3-8cyclic ring, optionally including oxygen, sulfur or NR⁷, where R⁷is hydrogen, or C_1-5alkyl, optionally substituted by hydroxyl.

The phosphopeptides of the invention may also include a C-terminal unnatural internal amino acid of the formula:

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where A²is an amino acid or peptide chain linked via an α-carboxy group; R¹and R³are independently hydrogen, C_1-5branched or linear C_1-5alkyl, C_1-5alkaryl, heteroaryl, and aryl, each of which are unsubstituted or substituted with a substitutent selected from: 1 to 3 of C_1-5alkyl, 1 to 3 of halogen, 1 to 2 of —OR⁵, N(R⁵)(R⁶), SR^S, N—C(NR⁵)NR⁶R⁷, methylenedioxy, —S(O)_mR⁵, 1 to 2 of —CF₃, -OCF₃, nitro, —N(R⁵)C(O)(R⁶), —C(O)OR⁵, —C(O)N(R⁵)(R⁶), -1H-tetrazol-5-yl, —SO₂N(R⁵)(R⁶), —N(R⁵)SO₂aryl, or —N(R⁵)SO₂R⁶; R⁵, R⁶and R⁷are independently selected from hydrogen, C_1-5linear or branched alkyl, C_1-5alkaryl, aryl, heteroaryl, and C_3-7cycloalkyl, and where two C_1-5alkyl groups are present on one atom, they optionally are joined to form a C_3-8cyclic ring, optionally including oxygen, sulfur or NR⁷, where R⁷is hydrogen, or C_1-5alkyl, optionally substituted by hydroxyl; R²is hydrogen, F, C_1-5linear or branched alkyl, C_1-5alkaryl; or R²and R¹are joined to form a C_3-8cyclic ring, optionally including oxygen, sulfur or NR⁷, where R⁷is hydrogen, or C_1-5alkyl, optionally substituted by hydroxyl; or R²and R³are joined to form a C_3-8cyclic ring, optionally substituted by hydroxyl and optionally including oxygen, sulfur or NR⁷, where R⁷is hydrogen, or C_1-5alkyl; R²is hydrogen, F, C_1-5linear or branched alkyl, C_1-5alkaryl; and Q is OH, OR^S, or NR⁵R⁶, where R⁵, R⁶are independently selected from hydrogen, C_1-5linear or branched alkyl, C_1-5alkaryl, aryl, heteroaryl, and C_3-7cycloalkyl, and where two C_1-5alkyl groups are present on one atom, they optionally are joined to form a C_3-8cyclic ring, optionally including oxygen, sulfur or NR⁷, where R⁷is hydrogen, or C_1-5alkyl, optionally substituted by hydroxyl. Methods well known in the art for modifying peptides are found, for example, in “Remington: The Science and Practice of Pharmacy” (20th ed., ed. A. R. Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia).

Therapeutic Uses

Peptide Synthesis and Conjugation

Phosphopeptides of the invention are prepared as detailed above. Alternatively, phosphopeptides can be prepared using standard FMOC chemistry on 2-chlorotrityl chloride resin (Int. J. Pept. Prot. Res. 38, 1991, 555-61). Cleavage from the resin is performed using 20% acetic acid in dichloromehane (DCM), which leaves the side chain still blocked. Free terminal carboxylate peptide is then coupled to 4′(aminomethy)-fluorescein (Molecular Probes, A-1351; Eugene, Oreg.) using excess diisopropylcarbodiimide (DIC) in dimethylformamide (DMF) at room temperature. The fluorescent N—C blocked peptide is purified by silica gel chromatography (10% methanol in DCM). The N terminal FMOC group is then removed using piperidine (20%) in DMF, and the N-free peptide, purified by silica gel chromatography (20% methanol in DCM, 0.5% HOAc). Finally, any t-butyl side chain protective groups are removed using 95% trifluoroacetic acid containing 2.5% water and 2.5% triisopropyl silane. The peptide obtained in such a manner should give a single peak by HPLC and is sufficiently pure for carrying on with the assay described below.

Phosphopeptide Modifications

It is understood that modifications can be made to the amino acid residues of the phosphopeptides of the invention, to enhance or prolong the therapeutic efficacy and/or bioavailability of the phosphopeptide. Accordingly, α-amino acids having the following general formula (I):

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where R defines the specific amino acid residue, may undergo various modifications. Exemplary modifications of α-amino acids, include, but are not limited to, the following formula (II):

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R₁, R₂, R₃, R₄, and R₅, are independently hydrogen, hydroxy, nitro, halo, C_1-5branched or linear alkyl, C_1-5alkaryl, heteroaryl, and aryl; wherein the alkyl, alkaryl, heteroaryl, and aryl may be unsubstituted or substituted by one or more substituents selected from the group consisting of C_1-5alkyl, hydroxy, halo, nitro, C_1-5alkoxy, C_1-5alkylthio, trihalomethyl, C_1-5acyl, arylcarbonyl, heteroarylcarbonyl, nitrile, C_1-5alkoxycarbonyl, oxo, arylalkyl (wherein the alkyl group has from 1 to 5 carbon atoms) and heteroarylalkyl (wherein the alkyl group has from 1 to 5-carbon atoms); alternatively, R₁and R₂are joined to form a C_3-8cyclic ring, optionally including oxygen, sulfur or hydrogen, or C_1-5alkyl, optionally substituted by hydroxyl; or R₂and R₃are joined to form a C_3-8cyclic ring, optionally substituted by hydroxyl and optionally including oxygen, sulfur, C_1-5aminoalkyl, or C_1-5alkyl. Methods well known in the art for making modifications are found, for example, in “Remington: The Science and Practice of Pharmacy” (20th ed., ed. A. R. Gennaro, 2000, Lippincott Williams & Wilkins), hereby incorporated by reference.

Assays and High Throughput Assays

Fluorescence polarization assays can be used in displacement assays to identify small molecule peptidomimetics. The following is an exemplary method for use of fluorescence polarization, and should not be viewed as limiting in any way. For screening, all reagents are diluted at the appropriate concentration and the working solution, kept on ice. The working stock concentration for GST and GST fusion proteins are ˜4 ng/μL, Fluorescein-labeled phosphopeptides can be used at a concentration of 1.56 fmol/μL, while cold phosphopeptides and peptides at 25 pmol/μL. Samples are incubated at a total volume of 200 μL per well in black flat bottom plates, Biocoat, #359135 low binding (BD BioSciences; Bedford, Mass.). Assays are started with the successive addition using a Labsystem Multi-prop 96/384 device (Labsystem; Franklin, Mass.) of 50 μL test compounds, diluted in 10% DMSO (average concentration of 28 μM), 50 μL of 50 mM MES-pH 6.5, 50 μL of Fluorescein-phosphopeptide, 50 μL of GST-Plk-1 PBD, 50 μL of unlabeled phosphopeptide, or unphosphorylated peptide can be used as a negative control. Once added, all the plates are placed at 4° C. Following overnight incubation at 4° C., the fluorescence polarization is measured using a Polarion plate reader (Tecan, Research Triangle Park, N.C.). A xenon flash lamp equipped with an excitation filter of 485 nm and an emission filter of 535 nm. The number of flashes is set at 30. Raw data can then be converted into a percentage of total interaction(s). All further analysis can be performed using SPOTFIRE data analysis software (SPOTFIRE, Somerville, Mass.)

Upon selection of active compounds, auto-fluorescence of the hits is measured as well as the fluorescein quenching effect, where a measurement of 2000 or more units indicates auto-fluorescence, while a measurement of 50 units indicates a quenching effect. Confirmed hits can then be analyzed in dose-response curves (IC₅₀) for reconfirmation. Best hits in dose—response curves can then be assessed by isothermal titration calorimetry using GST-Plk-1 PBD.

Alternate Binding and Displacement Assays

Fluorescence polarization assays are but one means to measure phosphopeptide-protein interactions in a screening strategy. Alternate methods for measuring phosphopeptide-protein interactions are known to the skilled artisan. Such methods include, but are not limited to mass spectrometry (Nelson and Krone, J. Mol. Recognit., 12:77-93, 1999), surface plasmon resonance (Spiga et al., FEBS Lett., 511:33-35, 2002; Rich and Mizka, J. Mol. Recognit., 14:223-8, 2001; Abrantes et al., Anal. Chem., 73:2828-35, 2001), fluorescence resonance energy transfer (FRET) (Bader et al., J. Biomol. Screen, 6:255-64, 2001; Song et al., Anal. Biochem. 291:133-41, 2001; Brockhoff et al., Cytometry, 44:338-48, 2001), bioluminescence resonance energy transfer (BRET) (Angers et al., Proc. Natl. Acad. Sci. USA, 97:3684-9, 2000; Xu et al., Proc. Natl. Acad. Sci. USA, 96:151-6, 1999), fluorescence quenching (Engelborghs, Spectrochim. Acta A. Mol. Biomol. Spectrosc., 57:2255-70, 70; Geoghegan et al., Bioconjug. Chem. 11:71-7, 2000), fluorescence activated cell scanning/sorting (Barth et al., J. Mol. Biol., 301:751-7, 2000), ELISA, and radioimmunoassay (RIA).

Test Extracts and Compounds

In general, peptidomimetic compounds that affect phosphopeptide-protein interactions are identified from large libraries of both natural products, synthetic (or semi-synthetic) extracts or chemical libraries, according to methods known in the art.

Those skilled in the art will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modifications of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from, for example, Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.)

Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including, but not limited to, Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art (e.g., by combinatorial chemistry methods or standard extraction and fractionation methods). Furthermore, if desired, any library or compound may be readily modified using standard chemical, physical, or biochemical methods.

Administration of Phosphopeptides and Peptidomimetic Small Molecules

By selectively disrupting or preventing a phosphoprotein from binding to its natural partner(s) through its binding site, the phosphopeptides of the invention, or derivatives, or peptidomimetics thereof, can significantly alter the biological activity or the biological function of a polo-like kinase. Therefore, the phosphopeptides, or derivatives thereof, of the invention can be used for the treatment of a disease or disorder characterized by inappropriate cell cycle regulation or apoptosis.

Diseases or disorders characterized by inappropriate cell cycle regulation, include hyperproliferative disorders, such as neoplasias. Examples of neoplasms include, without limitation, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenriglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

Cells undergoing inappropriate apoptosis include neurons in a patient who has a neurodegenerative disease (e.g., Parkinson's disease, Alzheimer's disease, or stroke), and cardiomyocytes (e.g., after myocardial infarction or over the course of congestive heart failure). Compositions of the invention, i.e., inhibitors of Plk-3, may be useful in treating a cell undergoing inappropriate apoptosis.

A Plk-1 PBD-binding phosphopeptide or peptidomimetic small molecule may be administered within a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to patients suffering from a disease that is caused by excessive cell proliferation. Administration may begin before the patient is symptomatic. Any appropriate route of administration may be employed, for example, administration may be parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, suppository, or oral administration. For example, therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.

The pharmaceutical compositions of the present invention are prepared in a manner known per se, for example by means of conventional dissolving, lyophilising, mixing, granulating or confectioning processes. Methods well known in the art for making formulations are found, for example, in “Remington: The Science and Practice of Pharmacy” (20th ed., ed. A. R. Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia).

Solutions of the active ingredient, and also suspensions, and especially isotonic aqueous solutions or suspensions, are preferably used, it being possible, for example in the case of lyophilized compositions that comprise the active ingredient alone or together with a carrier, for example mannitol, for such solutions or suspensions to be produced prior to use. The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilisers, wetting and/or emulsifying agents, solubilisers, salts for regulating the osmotic pressure and/or buffers, and are prepared in a manner known per se, for example by means of conventional dissolving or lyophilising processes. The said solutions or suspensions may comprise viscosity-increasing substances, such as sodium carboxymethylcellulose, carboxymethylcellulose, dextran, poly vinylpyrrolidone or gelatin.

Suspensions in oil comprise as the oil component the vegetable, synthetic or semi-synthetic oils customary for injection purposes. There may be mentioned as such especially liquid fatty acid esters that contain as the acid component a long-chained fatty acid having from 8 to 22, especially from 12 to 22, carbon atoms, for example lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, arachidic acid, behenic acid or corresponding unsaturated acids, for example oleic acid, elaidic acid, erucic acid, brasidic acid or linoleic acid, if desired with the addition of anti oxidants, for example, vitamins E, β-carotene, or 3,5-di-tert-butyl-4-hydroxytoluene. The alcohol component of those fatty acid esters has a maximum of 6 carbon atoms and is a mono- or poly-hydroxy, for example a mono-, di- or tri-hydroxy, alcohol, for example methanol, ethanol, propanol, butanol or pentanol or the isomers thereof, but especially glycol and glycerol. The following examples of fatty acid esters are there fore to be mentioned: ethyl oleate, isopropyl myristate, isopropyl palmitate, “Labrafil M 2375” (poly oxyethylene glycerol trioleate, Gattefoss, Paris), “Miglyol 812” (triglyceride of saturated fatty acids with a chain length of C_gto C₁₂, Huls AG, Germany), but especially vegetable oils, such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and more especially groundnut oil.

The injection compositions are prepared in customary manner under sterile conditions; the same applies also to introducing the compositions into ampoules or vials and sealing the containers.

Pharmaceutical compositions for oral administration can be obtained by combining the active ingredient with solid carriers, if desired granulating a resulting mixture, and processing the mixture, if desired or necessary, after the addition of appropriate excipients, into tablets, drage cores or capsules. It is also possible for them to be incorporated into plastics carriers that allow the active ingredients to diffuse or be released in measured amounts.

Suitable carriers are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and binders, such as starch pastes using for example corn, wheat, rice or potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinyl-pyrrolidone, and/or, if desired, disintegrates, such as the above-mentioned starches, also carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, such as sodium alginate. Excipients are especially flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol. Drage cores are provided with suitable, optionally enteric, coatings, there being used, inter alia, concentrated sugar solutions which may comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as ethylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Capsules are dry-filled capsules made of gelatin and soft sealed capsules made of gelatin and a plasticiser, such as glycerol or sorbitol. The dry-filled capsules may comprise the active ingredient in the form of granules, for example with fillers, such as lactose, binders, such as starches, and/or glidants, such as talc or magnesium stearate, and if desired with stabilisers. In soft capsules the active ingredient is preferably dissolved or suspended in suitable oily excipients, such as fatty oils, paraffin oil or liquid polyethylene glycols, it being possible also for stabilisers and/or antibacterial agents to be added. Dyes or pigments may be added to the tablets or drage coatings or the capsule casings, for example for identification purposes or to indicate different doses of active ingredient.

The pharmaceutical compositions comprise from approximately 1% to approximately 95%, preferably from approximately 20% to approximately 90%, active ingredient. Pharmaceutical compositions according to the invention may be, for example, in unit dose form, such as in the form of ampoules, vials, suppositories, drages, tablets or capsules.

The formulations can be administered to human patients in a therapeutically effective amount (e.g., an amount that decreases, suppresses, attenuates, diminishes, arrests, or stabilizes the development or progression of a disease, disorder, or infection in a eukaryotic host organism). The preferred dosage of therapeutic agent to be administered is likely to depend on such variables as the type and extent of the disorder, the overall health status of the particular patient, the formulation of the compound excipients, and its route of administration.

For any of the methods of application described above, a Plk-1 PBD-interacting small molecule may be applied to the site of the needed therapeutic event (for example, by injection), or to tissue in the vicinity of the predicted therapeutic event or to a blood vessel supplying the cells predicted to require enhanced therapy.

The dosages of Plk-1 PBD-interacting small molecule(s) depends on a number of factors, including the size and health of the individual patient, but, generally, between 0.1 mg and 1000 mg inclusive are administered per day to an adult in any pharmaceutically acceptable formulation. In addition, treatment by any of the approaches described herein may be combined with more traditional therapies.

Combination Therapy

If desired, treatment with Plk-1 PBD-interacting small molecule may be combined with more traditional therapies for the proliferative disease such as surgery or administration of chemotherapeutics or other anti-cancer agents, including, for example, γ-radiation, alkylating agents (e.g., nitrogen mustards such as cyclophosphamide, ifosfamide, trofosfamide, and chlorambucil; nitrosoureas such as carmustine, and lomustine; alkylsulphonates such as bisulfan and treosulfan; triazenes such as dacarbazine; platinum-containing compounds such as cisplatin and carboplatin), plant alkaloids (e.g., vincristine, vinblastine, anhydrovinblastine, vindesine, vinorelbine, paclitaxel, and docetaxol), DNA topoisomerase inhibitors (e.g., etoposide, teniposide, topotecan, 9-aminocamptothecin, (campto) irinotecan, and crisnatol), mytomycins (e.g., mytomicin C), antifolates (e.g., methotrexate, trimetrexate, mycophenolic acid, tiazofurin, ribavirin, EICAR, hydroxyurea, and deferoxamine), uracil analogs (5-fluorouracil, floxuridine, doxifluridine, and ratitrexed), cytosine analogs (cytarbine, cytosine arabinoside, and fludarabine), purine analogs (e.g., mercaptopurine, and thioguanine), hormonal therapies (e.g., tamoxifen, raloxifene, megestrol, goserelin, leuprolide acetate, flutamide, and bicalutamide), vitamin D3 analogs (EB 1089, CB 1093, and KH 1060), vertoporfin, phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A, interferon-α, interferon-γ, tumor necrosis factor, lovastatin, 1-methyl-4-phenylpyridinium ion, staurosporine, actinomycin D, dactinomycin, bleomycin A2, bleomycin B2, adriamycin, peplomycin, daunorubican, idarubican, epirubican, pirarubican, zorubican, mitoxantrone, and verapamil.

Other Embodiments

From the foregoing description, it is apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

All patents and publications mentioned in this specification are hereby incorporated by reference to the same extent as if each independent publication or patent application, including 60/426,132, was specifically and individually indicated to be incorporated by reference.

Number	Date	Country
60487899	Jul 2003	US
60485641	Jul 2003	US
60426132	Nov 2002	US

	Number	Date	Country
Parent	10713978	Nov 2003	US
Child	12229797		US

	Number	Date	Country
Parent	12229797	Aug 2008	US
Child	12893957		US

PRODUCTS AND PROCESSES FOR MODULATING PEPTIDE-PEPTIDE BINDING DOMAIN INTERACTIONS

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