Compositions and methods for regulating the kinase domain of receptor tyrosine kinases

[0001] A portion of the disclosure of this patent document contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

FIELD OF THE INVENTION

[0002] The present invention relates to binding pockets of receptor tyrosine kinases (RTKs). The binding pockets may regulate the kinase domain of the receptor tyrosine kinases. In particular, the invention relates to a crystal comprising a binding pocket of a receptor tyrosine kinase that regulates the kinase domain of the receptor tyrosine kinase. The crystal may be useful for modeling and/or synthesizing mimetics of a binding pocket or ligands that associate with the binding pocket. Such mimetics or ligands may be capable of acting as modulators of receptor tyrosine kinase receptor activity, and they may be useful for treating, inhibiting, or preventing diseases modulated by such receptors.

[0003] Methods are also provided for regulating the kinase domain of an RTK by changing a binding pocket of the RTK that regulates the kinase domain from an autoinhibited state to an active state or from an active state to an autoinhibited state.

BACKGROUND

[0004] Cell surface receptors with protein-tyrosine kinase activity mediate the biological effects of many extracellular signaling proteins, and thereby regulate aspects of normal cellular behavior such as growth and differentiation, movement, metabolism and survival (van der Geer and Hunter, 1994). The profound consequences of phosphotyrosine signaling on cellular function are emphasized by the effects of mutations that deregulate receptor tyrosine kinase activity, which are frequently associated with malignant transformation or developmental abnormalities. Under normal circumstances, the activation of receptor tyrosine kinases (RTK) requires binding of the appropriate extracellular ligand, which induces either receptor oligomerization or a spatial re-organization of pre-associated receptor chains (Heldin, 1995; Remy et al., 1999; Schlessinger, 2000). As a result, the receptor undergoes autophosphorylation through an intermolecular reaction, both on tyrosine residues which regulate kinase activity, and on residues within non-catalytic regions of the receptor which form binding sites for cytoplasmic targets with SH2 or PTB domains (Pawson and Scott, 1997; Kuriyan and Cowburn, 1997).

[0005] The catalytic activity of tyrosine kinases is frequently stimulated by autophosphorylation within a region of the kinase domain termed the activation segment (Weinnmaster et al., 1984), and indeed this has been viewed as the principal mechanism through which RTKs are activated (Hubbard and Till, 2000; Hubbard, 1997). Structural analysis of the isolated kinase domains of several receptors has revealed how the activation segment represses kinase activity, and the means by which phosphorylation releases this autoinhibition. In the case of the inactive insulin receptor, Tyr 1162 in the activation segment protrudes into the active site, and the activation segment blocks access to the ATP-binding site (Hubbard et al., 1994). Autophosphorylation of Tyr 1162 and two adjacent tyrosine residues repositions the activation segment, thereby freeing the active site to engage exogenous substrates and reorganizing the residues required for catalysis into a functional conformation (Hubbard, 1997). In contrast, the activation segment of the fibroblast growth factor (FGF) receptor is relatively mobile and the tyrosines which become phosphorylated upon receptor activation do not occupy the active site. However, the C-terminal end of the FGFR1 activation segment appears to block access to substrate (Mohammadi et al., 1996).

[0006] Despite the evident importance of the kinase domain activation segment, it remains possible that other mechanisms are important in regulating RTK activity, which might have been missed through an exclusive focus on the kinase domain itself. In particular, recent biochemical and mutational analysis has suggested that Eph receptors may be regulated through a more complex mechanism, involving the juxtamembrane region (Binns et al., 2000; Zisch et al., 1998; Zisch et al., 2000).

[0007] There is only a single Eph receptor tyrosine kinase encoded by the C. elegans genome (VAB-1) (George et al., 1998; Wang et al., 1999a), but the subfamily has undergone a remarkable expansion during metazoan evolution to include at least 14 mammalian members, which therefore represent the largest class of vertebrate RTKs (Holder and Klein, 1999). These Eph receptors fall into two groups, A and B, based on their ability to bind ligands (ephrins), which are themselves cell surface proteins anchored to the plasma membrane either through a GPI linkage (A-type ephrins) or a transmembrane region (B-type) (Eph Nomenclature Committee, 1997; Gale et al., 1996). Signaling between Eph receptors and ephrins generally involves direct cell-cell interactions (Holland et al., 1996; Bruckner et al., 1997), and frequently results in the repulsion of these cells one from another (Drescher et al., 1995; Wang and Anderson, 1997; Mellitzer et al., 1999). Eph receptors are implicated in morphogenetic cell movements (Wang et al., 1999a; Chin-Sang et al., 1999), in defining cell boundaries in structures such as the rhombomeres of the embryonic hindbrain (Xu et al., 1999), in controlling axon guidance and the establishment of topographic maps in the central nervous system (Nakamoto et al., 1996; Brown et al., 2000), and in determining the trajectories of migrating neural crest cells (Krull et al., 1997). Signaling between ephrin and Eph receptor-expressing cells is also essential for angiogenesis, and in conferring distinct arterial and venous identities to developing blood vessels (Wang et al., 1999b; Adams et al., 1999; Gerety et al., 1999).

[0008] The extracellular region of Eph receptors contains an N-terminal ephrin-binding domain (Labrador et al., 1997), that folds into a jellyroll β-sandwich (Himanen et al., 1998), followed by a cysteine-rich region and two fibronectin type III repeats (Pasquale, 1991; Henkemeyer et al., 1994). A single membrane-spanning sequence is followed by a relatively lengthy juxtamembrane region, an uninterrupted kinase domain, an o-helical sterile alpha motif (SAM) domain implicated in receptor oligomerization (Stapleton et al., 1999; Thanos et al., 1999), and a C-terminal motif capable of binding PDZ domain proteins (Hock et al., 1998; Torres et al., 1998). Activation of receptors such as EphB2 or EphA4 is accompanied by autophosphorylation on multiple residues, most notably on two tyrosines within a highly conserved juxtamembrane motif (YIDPFTYEDP in EphB2) and on a tyrosine within the activation segment of the kinase domain (Holland et al., 1997; Choi and Park, 1999; Ellis et al., 1996; Kalo and Pasquale, 1999; Zisch et al., 1998; Binns et al., 2000). By analogy with other RTKs, it might be expected that autophosphorylation of the activation segment tyrosine would stimulate kinase activity, while the juxtamembrane phosphotyrosine sites would recruit cytoplasmic targets. Indeed, the juxtamembrane phosphotyrosine motifs do bind SH2 domain signaling proteins, including p120-RasGAP, Nck, phosphatidylinositol 3′-kinase, SHEP-1 and Src family kinases among others, which can potentially direct cellular responses to ephrin stimulation (Dodelet et al., 1999; Ellis et al., 1996; Holland et al., 1997; Holland et al., 1998; Zisch et al., 1998).

[0009] Consistent with the possibility that phosphorylation of the conserved juxtamembrane tyrosines is important for signaling, substitution of these residues in EphB2 with phenylalanine abrogates EphB2-mediated growth cone collapse upon stimulation of NG108 neuronal cells with ephrin B1. However, this loss of biological activity is apparently not due solely to a failure to engage SH2-containing targets, since substitution of the juxtamembrane tyrosines in EphB2 and EphA4 with phenylalanine leads to a severe loss of ephrin-induced kinase activity (Binns et al., 2000).

SUMMARY OF THE INVENTION

[0010] Applicants have solved the x-ray crystal structure of an Eph receptor tyrosine kinase domain and juxtamembrane region in an autoinhibited state. The results show that in its unphosphorylated state, the juxtamembrane region adopts a helical structure that distorts the conformation of the small lobe of the kinase domain, thereby disrupting the active site. These results indicate a novel mechanism for the regulation of RTKs.

[0011] Solving the crystal structure has enabled the determination of key structural features of the kinase domain and juxtamembrane region, particularly the shape of binding pockets, or parts thereof, that permit the juxtamembrane region and kinase domain to associate resulting in an autoinhibited state. The crystal structure has also enabled the determination of key structural features in molecules or ligands that interact or associate (e.g. nucleotides, cofactors, inhibitors, and substrates) with the binding pockets.

[0012] Knowledge of the autoinhibited conformation of binding pockets of RTKs that regulate the kinase domain is of significant utility in drug discovery. The association of natural ligands and substrates with the binding pockets of RTKs is the basis of many biological mechanisms. In addition, many drugs exert their effects through association with the binding pockets of RTKs. The associations may occur with all or any parts of a binding pocket. An understanding of the association of a drug with the active and autoinhibited conformations of binding pockets of RTKs, will lead to the design and optimization of drugs having more favorable associations with their target RTKs and thus provide improved biological effects. Therefore, information about the shape and structure of binding pockets of RTKs in their autoinhibited and activated states, is invaluable in designing potential modulators of the receptors for use in treating diseases and conditions associated with or modulated by the receptors.

[0013] The present invention relates to a binding pocket of a receptor tyrosine kinase (RTK). In an aspect of the invention, the binding pocket regulates the kinase domain of the receptor tyrosine kinase or is involved in maintaining an autoinhibited state or active state of an RTK.

[0014] The invention also relates to a crystal comprising a binding pocket of an RTK that regulates the kinase domain of the RTK. The binding pocket may be in an autoinhibited state, or active state. Thus, a binding pocket may be involved in maintaining an autoinhibited state or active state of an RTK.

[0015] In an embodiment, the invention, provides a crystal comprising a juxtamembrane region and/or kinase domain of an RTK, or part thereof. The invention contemplates a crystal formed by a juxtamembrane region and a kinase domain of an RTK in an autoinhibited state or active state.

[0016] The invention also contemplates a crystal comprising a binding pocket of a receptor tyrosine kinase that regulates the kinase domain of the receptor tyrosine kinase in association with a ligand.

[0017] The present invention also contemplates molecules or molecular complexes that comprise all or parts of either one or more binding pockets of the invention, or homologs of these binding pockets that have similar structure and shape.

[0018] The present invention also provides a crystal comprising a binding pocket of an RTK of the invention and at least one ligand. A ligand may be complexed or associated with a binding pocket. Ligands include a nucleotide or analogue or part thereof, a substrate or analogue thereof, a cofactor, and/or heavy metal atom. A ligand may be a modulator of the activity of an RTK.

[0019] In an aspect the invention contemplates a crystal comprising a binding pocket of an RTK of the invention complexed with a nucleotide or analogue thereof from which it is possible to derive structural data for the nucleotide or analogue thereof.

[0020] The shape and structure of a binding pocket may be defined by selected atomic contacts in the pocket. In an embodiment, the binding pocket is defined by one or more atomic interactions or enzyme atomic contacts as set forth in Table 2. Each of the atomic interactions is defined in Table 2 by an atomic contact (more preferably, a specific atom where indicated) on the juxtamembrane region and by an atomic contact (more preferably a specific atom where indicated) on the kinase domain, juxtamembrane region, or ligand.

[0021] An isolated polypeptide comprising a binding pocket with the shape and structure of a binding pocket described herein is also within the scope of the invention.

[0022] The invention also provides a method for preparing a crystal of the invention, preferably a crystal of a binding pocket of an Eph receptor, or a complex of such a binding pocket and a ligand.

[0023] Crystal structures of the invention enable a model to be produced for a binding pocket of the invention, or complexes or parts thereof. The models will provide structural information about the autoinhibited or active state of a binding pocket of a RTK or a ligand and its interactions with a binding pocket. Models may also be produced for ligands. A model and/or the crystal structure of the present invention may be stored on a computer-readable medium.

[0024] The present invention includes a model of a binding pocket of the present invention that substantially represents the structural coordinates specified in Table 3. The invention also includes a model that comprises modifications of the model substantially represented by the structural coordinates specified in Table 3. A modification may represent a binding pocket that is involved in maintaining an autoinhibited state or active state of an RTK or regulates the kinase domain of an RTK. A model is a representation or image that predicts the actual structure of the binding pocket. As such, a model is a tool that can be used to probe the relationship between a binding pocket's structure and function at the atomic level and to design molecules that can modulate the binding site and accordingly RTK activity.

[0025] Thus, the invention provides a model of: (a) a binding pocket of an RTK that is involved in maintaining an autoinhibited state or active state of an RTK or regulates the kinase domain of an RTK; and (b) a modification of the model of (a).

[0026] A method is also provided for producing a model of the invention representing a binding pocket of an RTK that is involved in maintaining an autoinhibited state or active state of an RTK or regulates the kinase domain of an RTK, comprising representing amino acids of the binding pocket at substantially the structural coordinates specified in Table 3.

[0027] A crystal and/or model of the invention may be used in a method of determining the secondary and/or tertiary structures of a polypeptide or binding pocket with incompletely characterised structure. Thus, a method is provided for determining at least a portion of the secondary and/or tertiary structure of molecules or molecular complexes which contain at least some structurally similar features to a binding pocket of the invention. This is achieved by using at least some of the structural coordinates set out in Table 3.

[0028] A crystal of the invention may be useful for designing, modeling, identifying, evaluating, and/or synthesizing mimetics of a binding pocket or ligands that associate with a binding pocket. Such mimetics or ligands may be capable of acting as modulators of receptor tyrosine kinase activity, and they may be useful for treating, inhibiting, or preventing diseases modulated by such receptors.

[0029] Thus, the present invention contemplates a method of identifying a modulator of an RTK comprising the step of applying the structural coordinates of a binding pocket, or atomic interactions, or atomic contacts of a binding pocket, to computationally evaluate a test ligand for its ability to associate with the binding pocket, or part thereof. Use of the structural coordinates of a binding pocket, or atomic interactions, or atomic contacts of a binding pocket to design or identify a modulator is also provided.

[0030] In an embodiment, the invention contemplates a method of identifying a modulator of an RTK comprising determining if a test agent inhibits or potentiates an autoinhibited state or active state of a kinase domain of the RTK.

[0031] The invention further contemplates classes of modulators of RTKs based on the shape and structure of a ligand defined in relation to the molecule's spatial association with a binding pocket of the invention. Generally, a method is provided for designing potential inhibitors of RTKs comprising the step of applying the structural coordinates of a ligand defined in relation to its spatial association with a binding pocket, or a part thereof, to generate a compound that is capable of associating with the binding pocket.

[0032] It will be appreciated that a modulator of an RTK may be identified by generating an actual secondary or three-dimensional model of a binding pocket, synthesizing a compound, and examining the components to find whether the required interaction occurs.

[0033] A potential modulator of an RTK identified by a method of the present invention may be confirmed as a modulator by synthesizing the compound, and testing its effect on the RTK in an assay for that receptor's enzymatic activity. Such assays are known in the art (e.g. phosphorylation assays).

[0034] A modulator of the invention may be converted using customary methods into pharmaceutical compositions. A modulator may be formulated into a pharmaceutical composition containing a modulator either alone or together with other active substances.

[0035] Therefore, the methods of the invention for identifying modulators may comprise one or more of the following additional steps:

[0036] (a) testing whether the modulator is a modulator of the activity of a RTK, preferably testing the activity of the modulator in cellular assays and animal model assays;

[0037] (b) modifying the modulator;

[0038] (c) optionally rerunning steps (a) or (b); and (d) preparing a pharmaceutical composition comprising the modulator.

[0039] Steps (a), (b) (c) and (d) may be carried out in any order, at different points in time, and they need not be sequential.

[0040] Still another aspect of the present invention provides a method of conducting a drug discovery business comprising:

[0041] (a) providing one or more systems employing the atomic interactions, atomic contacts, or structural coordinates of a binding pocket of an RTK, for identifying agents by their ability to inhibit or potentiate the atomic interactions or atomic contacts of a binding pocket; and

[0042] (b) conducting therapeutic profiling of agents identified in step (a), or further analogs thereof, for efficacy and toxicity in animals; and

[0043] (c) formulating a pharmaceutical preparation including one or more agents identified in step (b) as having an acceptable therapeutic profile.

[0044] A further aspect of the present invention provides a method of conducting a drug discovery business comprising:

[0045] (a) providing one or more systems for identifying agents by their ability to inhibit or potentiate an autoinhibited state or active state of a kinase domain of an RTK; and

[0046] (b) conducting therapeutic profiling of agents identified in step (a), or further analogs thereof, for efficacy and toxicity in animals; and

[0047] (c) formulating a pharmaceutical preparation including one or more agents identified in step (b) as having an acceptable therapeutic profile.

[0048] In certain embodiments, the subject methods can also include a step of establishing a distribution system for distributing the pharmaceutical preparation for sale, and may optionally include establishing a sales group for marketing the pharmaceutical preparation.

[0049] Yet another aspect of the invention provides a method of conducting a target discovery business comprising:

[0050] (a) providing one or more systems employing the atomic interactions, atomic contacts, or structural coordinates of a binding pocket of an RTK, for identifying agents by their ability to inhibit or potentiate the atomic interactions or atomic contacts, or providing one or more systems for identifying agents by their ability to inhibit or potentiate an autoinhibited state or active state of a kinase domain of an RTK;

[0051] (b) (optionally) conducting therapeutic profiling of agents identified in step (a) for efficacy and toxicity in animals; and

[0052] (c) licensing, to a third party, the rights for further drug development and/or sales for agents identified in step (a), or analogs thereof.

[0053] Methods are also provided for regulating the kinase domain of an RTK by changing a binding domain or pocket of a RTK that regulates the kinase domain from an autoinhibited state to an active state or from an active state to an autoinhibited state. A binding domain or pocket of a RTK may be changed from an autoinhibited state by altering amino acid residues forming the binding pocket (e.g. introducing mutations) or using a modulator.

[0054] In an aspect the invention provides a method for inhibiting kinase activity of an RTK comprising maintaining the RTK or a binding pocket thereof involved in regulating the kinase domain in an autoinhibited state, or potentiating an autoinhibited state for the RTK or binding pocket thereof involved in regulating the kinase domain. An autoinhibited state may be maintained or potentiated by inhibiting phosphorylation of phosphoregulatory sites of the juxtamembrane segment and/or kinase domain (e.g. activation segment). Inhibition may be accomplished using modulators, or altering the structure of a binding pocket of the RTK comprising the phosphoregulatory sites, to prevent phosphorylation of the sites.

[0055] The invention contemplates a method for altering the stability of an autoinhibited state of an RTK comprising phosphorylating phosphoregulatory sites of a juxtamembrane region of the RTK.

[0056] In an aspect the invention relates to a method for changing an RTK from an autoinhibited state to an active state comprising phosphorylating phosphoregulatory sites of a juxtamembrane region of the RTK.

[0057] In another aspect the invention provides a method for activating kinase activity of an RTK comprising phosphorylating phosphoregulatory sites of a juxtamembrane region and kinase domain (e.g. activation segment) of the RTK involved in maintaining the RTK in an autoinhibited state.

[0058] The invention also contemplates a method of treating or preventing a condition or disease associated with an RTK in a cellular organism, comprising:

[0059] (a) administering a modulator of the invention in an acceptable pharmaceutical preparation; and

[0060] (b) activating or inhibiting the RTK to treat or prevent the disease.

[0061] In an aspect the invention provides a method for treating or preventing a condition or disease involving increased RTK activity comprising maintaining the RTK or a binding pocket thereof involved in regulating the kinase domain of the RTK in an autoinhibited state. An autoinhibited state may be maintained as described herein. In an embodiment the condition or disease is cancer.

[0062] The invention provides for the use of a modulator identified by the methods of the invention in the preparation of a medicament to treat or prevent a disease in a cellular organism. Use of modulators of the invention to manufacture a medicament is also provided.

[0063] These and other aspects of the present invention will become evident upon reference to the following detailed description and Tables, and attached drawings.

DESCRIPTION OF THE DRAWINGS AND TABLES

[0064] The present invention will now be described only by way of example, in which reference will be made to the following Figures:

[0065]
FIG. 1. Structure-based sequence alignment of the juxtamembrane segments and kinase domains of murine and human EphB2, murine EphA4 and cAPK, and human IRK, FGFR1, Hck, Kit, PDGFRβ, and Flt3. The secondary structure elements of murine EphB2 are indicated, with the juxtamembrane segment, the N-terminal kinase, the g-loop, and the C-terminal lobe coloured red, green, orange, and blue, respectively. Residues Phe 620 and Tyr 750 and those marked with a star are involved in the juxtamembrane/kinase domain interface. The two juxtamembrane tyrosines (604 and 610) that were mutated to phenylalanine are highlighted in light blue. Additional tyrosines identified by Kalo and Pasquale (1999) as in vivo phosphorylation sites are highlighted in purple. The solid triangle indicates the site of a 16 amino acid insertion in chicken EphB2 resulting from alternate RNA processing (Connor and Pasquale, 1995). For Kit and PDGFRβ, tyrosines highlighted in yellow denote autophosphorylation sites, while sites of activating point mutations and deletions are shaded gray (Tsujimura et al., 1996; Irusta and DiMaio, 1998; Kitayama et al., 1995; Hirota et al., 1998). The locations and regions of duplicated sequence for activating Flt3 mutations are indicated by solid black triangles and underlining (Hayakawa et al, 2000).

[0066]
FIG. 2. Overview of the autoinhibited EphB2 structure. (a) Ribbon diagram of the EphB2 crystal structure in complex with AMP-PNP. The juxtamembrane region, N-terminal kinase lobe, C-terminal kinase lobe, and g-loop are coloured red, green, blue and orange, respectively. Phosphoregulatory residues Tyr/Phe 604 and Tyr/Phe 610 are coloured light blue, Tyr667 is coloured purple, and the adenine moiety of AMP-PNP is coloured red. (b) Ribbon representation of EphB2 colored as in (a), rotated 90° about the vertical axis. (c) and (d) The juxtamembrane regions in (a) and (b), respectively, have been magnified to detail the interactions between the juxtamembrane region and helix αC of the N-terminal kinase lobe. Carbon, oxygen, nitrogen and sulfur atoms are shown in yellow, red, blue, and green, respectively. Residues involved in the juxtamembrane/kinase domain interface but not shown include Ala616, Ala621, Leu676, Leu693, and Val696. All ribbon diagrams were prepared with RIBBONS (Carson, 1991b).

[0067]
FIG. 3. Comparison of autoinhibited EphB2 RTK with the active insulin receptor kinase. (a) Superposition of EphB2 with active insulin receptor kinase (Protein Data Bank ID code 1ir3). The backbone of the juxtamembrane region of EphB2 is shown in red, with the side chains of Tyr/Phe 604 and Tyr/Phe 610 coloured light blue. The EphB2 kinase domain, g-loop and bound adenine moiety are colored blue, orange and red, respectively. The backbone of active IRK is coloured dark green with its activation segment, g loop, and bound nucleotide shown in purple, pink, and light green respectively. The two receptors were aligned using all elements of the C-terminal lobes except the kinase insert region, the activation segment, helix αJ, and the C-terminal tail (rms fit=1.91 Å). (b) Stereo view of the boxed region in (a), with EphB2 phosphorylation sites shown in purple, other EphB2 side chain atoms coloured as in FIGS. 2c and 2d and IRK side chains shown in green and pink. (c) Stereo view of the boxed region in A), highlighting the kinase catalytic region. This panel is colored as in (b). (d) Stereo view of boxed region in A) highlighting switch region 1. Inactive IRK (Protein Data Band ID code 1irk), shown in yellow, is also superimposed. All side chains are colored according to their respective backbones. IRK residue labeled Thr776 corresponds to Ser776 in EphB2.

[0068]
FIG. 4. Electrostatic surface representation of EphB2. Blue and red regions indicate positive and negative potential, respectively (10 to −10 kBT). Phosphoregulatory residues Tyr/Phe 604 and Tyr/Phe 610 are coloured light blue. The molecular surface of EphB2 is oriented as in FIG. 2a and was generated using GRASP (Nicholls et al., 1991)

[0069]
FIG. 5. Comparison of the kinase activities of EphA4 and EphB2 wild-type and mutant proteins. (a) GST-EphA4 proteins were expressed in E. coli, and cell lysates were subjected to immunoblot analysis with anti-pTyr antibody (top panel) and anti-GST antibody (lower panel). (b) Equal quantities of GST-EphA4 proteins bound to glutathione sepharose were assessed for their ability to autophosphorylate and phosphorylate enolase by an in vitro kinase assay (top panel). Immunblot analysis of GST-EphA4 proteins with anti-GST antibody (lower panel). (c) Histogram of the specific activities of EphA4 wild-type and mutant proteins as measured by the spectrophotometric coupling assay at 1 mM S-1 peptide and 0.5 μM EphA4 proteins. The velocities represent the mean of triplicate reactions and have been normalized to the specific activity of wild-type EphA4 (top panel). Coomassie stained SDS-PAGE analysis of EphA4 proteins (lower panel). (d) EphB2 and its mutants were expressed in COS-1 cells and immunoprecipitated. The immunoprecipitates were resolved by SDS-PAGE, immunoblotted with anti-pTyr (top panel) or anti-EphB2 (middle panel) antibodies, and assessed for their ability to autophosphorylate and phosphorylate enolase by an in vitro kinase assay (bottom panel).

[0070]
FIG. 6. Schematic diagram highlighting differences between the autoinhibited (left) and active (right) states of the Eph receptor family of tyrosine kinases. The active configuration is based on the crystal structure of active IRK (Protein Data Bank ID code 1ir3). Dashed lines indicate regions of activation segment disorder. The numbering scheme corresponds to murine EphB2.

[0071] The present invention will now be described only by way of example, in which reference will be made to the following Tables:

[0072] Table 1 shows the data collection, structure determination and refinement statistics

[0073] Table 2 shows intermolecular contacts in a binding pocket of the invention.

[0074] Table 3 shows the structural coordinates of the juxtamembrane region and kinase domain of an EphB2 receptor.

[0075] In Table 3, from the left, the second column identifies the atom number; the third identifies the atom type; the fourth identifies the amino acid type; the sixth identifies the residue number; the seventh identifies the x coordinates; the eighth identifies the y coordinates; the ninth identifies the z coordinates; the tenth identifies the occupancy; and the eleventh identifies the temperature factor.

DETAILED DESCRIPTION OF THE INVENTION

[0076] Unless otherwise indicated, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Current Protocols in Molecular Biology (Ansubel) for definitions and terms of the art.

[0077] In accordance with the present invention there may be employed conventional biochemistry, enzymology, molecular biology, crystallography, bioinformatics, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See for example, Sambrook, Fritsch, & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization B. D. Hames & S. J. Higgins eds. (1985); Transcription and Translation B. D. Hames & S. J. Higgins eds (1984); Animal Cell Culture R. I. Freshney, ed. (1986); Immobilized Cells and enzymes IRL Press, (1986); and B. Perbal, A Practical Guide to Molecular Cloning (1984).

[0078] For ease of reference the murine numbering scheme for EphB2 is employed herein to describe specific amino acid residues in aspects of the invention. However, a person skilled in the art could readily determine the corresponding amino acid residues in other RTKs, more particularly in Eph receptors.

[0079] Receptor Tyrosine Kinases (RTKs)

[0080] The invention generally relates to RTKs. RTKs mediate pathways involving multiple extracellular and intracellular signals, integration and amplification of these signals by second messengers, and the activation of cellular processes including cell proliferation, cell division, cell growth, the cell cycle, cell differentiation, cell migration, axonogenesis, nerve cell interactions, and regeneration. Signaling pathways mediated by receptor tyrosine kinases may be initiated by growth factors binding to specific RTKs on cell surfaces. The binding of a growth factor to its receptor activates RTK signaling pathways. The RTKs have an extracellular N-terminal domain that binds the growth factor and a cytoplasmic C-terminal domain containing a protein tyrosine kinase that is capable of autophosphorylation, and the phosphorylation of other protein substrates. Autophosphorylation takes place within a region of the kinase domain of the RTK termed the “activation segment” (Weinnmaster et al., 1984). The binding of a growth factor to its receptor activates the tyrosine kinase which phosphorylates a variety of signaling molecules thereby initiating signaling pathways that can lead to DNA replication, RNA and protein synthesis, and cell division.

[0081] Receptor tyrosine kinases within the scope of the present invention include but are not limited to epidermal growth factor receptor (EGFR), PDGF receptor, insulin receptor tyrosme kinase (IRK), Met receptor tyrosine kinase, fibroblast growth factor (FGF) receptor, insulin receptor, insulin growth factor (IGF-1) receptor, TrkA receptor, IL-3 receptor, B cell receptor, TIE-1, Tek/Tie2, Flt-1, Flk, VEGFR3, EFGR/Erbb, Erb2/neu, Erb3, Ret, Kit, Alk, Ax1, FGFR1, FGFR2, FGFR3, Hck, cAPK, keratinocyte growth factor (KGF) receptor, and Eph receptors.

[0082] The invention preferably contemplates Eph receptors, more preferably EphB2 receptors.

[0083] The term “Eph receptor” refers to a subfamily of closely related transmembrane receptor tyrosine kinases related to Eph, a receptor named for its expression in an erythropoietin-producing human hepatocellular carcinomas cell line. The receptors contain cell adhesion-like domains on their extracellular surface. The N-terminal extracellular region of all Eph family members contains a domain necessary for ligand binding and specificity, followed by a cysteine-rich domain and two fibronectin type II repeats. The cytoplasmic region has a centrally located tyrosine kinase domain. C-terminal to the catalytic region is a sterile alpha motif (SAM) domain, which forms dimers of oligomers in solution and may contribute to regulation of receptor clustering. Localization of clustering of Eph proteins may also be influenced by PDZ domain effectors which potentially interact with specific C-terminal receptor motifs.

[0084] N-terminal to the kinase domain is the juxtamembrane domain. Two invariant tyrosine residues (tyrosines 596 and 602 of EphA4; tyrosines 604 and 610 of EphB2) in the juxtamembrane domain are embedded in a characteristic and highly conserved ˜10 amino acid sequence motif. These tyrosine residues are major sites for autophosphorylation and they have been found to associate with a number of SH2 domain-containing cytoplasmic proteins such as Ras GTPase-activating protein (RasGAP), the p85 subunit of phosphatidylinositol 3′ kinase, Src family kinases, the adapter protein Nck, and SHEP-1 which binds the R-Ras and Rap1A GTPases. Signaling mediated by such SH2 domain-containing proteins may contribute to the physiological effects of Eph receptor stimulation on cell adhesion and cytoskeletal structures.

[0085] There are currently 14 related vertebrate members of the Eph receptor family including receptors in Caenorhabditis elegans and Drosophila. Eph receptors are activated by ephrins. Ephrins are attached to the plasma membrane either via a glycosylphosphatidylinositol linkage (A class) or a transmembrane sequence (B class). Eph receptors are also divided into A and B classes corresponding to their ligand binding specificities and phylogenetic relationships. Class A receptors generally bind A class ephrins, whereas B class ephrins stimulate B class receptors. However, EphA4 is an exception in that it binds and responds to B as well as A class ephrins.

[0086] The group that includes receptors interacting preferentially with ephrin A proteins is called EphA and includes EphA1 (also known as Eph and Esk), EphA2 (also known as Eck, Myk2, Sek2), EphA3 (also known as Cek4, Mek4, Hek, Tyro4, Hek4), EphA4 (also known as Sek, Sek1, Cek8, Hek8, Tyro1), EphA5 (also known as Ehk1, Bsk, Cek7, Hek7, and Rek7), EphA6 (Ehk2, and Hek12) EphA7 (also known as Mdk1, Hek11, Ehk3, Ebk, Cek11), and EphA8 (also known as Eek, Hek3). The group that includes receptors interacting preferentially with ephrin B proteins is called Eph B and includes EphB1 (also known as Elk Cek6, Net, Hek6), EphB2 (also known as Cek5, Nuk, Erk, Qek5, Tyro5, Sek3, hek5, Drt), EphB3 (also known as Cek10, Hek2, Mdks, Tyro6, and Sek4), EphB4 (also known as Htk, Myk1, Tyrol 1, Mdk2), EphB5 (also known as Cek9, Hek9), and EphB6 (also known as Mep).

[0087] “Ephrin” refers to a class of ligands which are anchored to the cell membrane through a transmembrane domain, and bind to the extracellular domain of an Eph receptor, facilitating dimerization and autophosphorylation of the receptor and autophosphorylation of the ligand. The ephrin-A ligands (GPI-anchored ligands) are ephrin-A (also known as B61, LERK1, EFL-1), ephrin-A2 (also known as LERK6, Elf1, mCek7-L, cElf1), ephrin-A3 (also known as LERK3, Ehk1-L, and EFL-2), ephrin-A4 (also known as LERK4, EFL-4, mLERK4), ephrin-A5 (AL1, LERK7, EFL-5, mAL1, [rLERK7], RAGS). The ephrin-B ligands (transmembrane ligands) are ephrin-B1 (also known as LEKR2, ELK-L, EFL-3, Cek5-L, Stra1, [LERK2]), ephrin-B2 (also known as LERK5, HTK-L, NLERK1, Elf2, Htk-L), and ephrin-B3 (also known as LERK8, ELK-L3, NLERK2, EFL-6, Elf3, [rELK-L3]).

[0088] RTKs may be derivable from a variety of sources, including viruses, bacteria, fungi, plants and animals. In a preferred embodiment an RTK is derivable from a mammal, for example, a human.

[0089] An RTK in the present invention may be a wild type enzyme, or part thereof, or a mutant, variant or homolog of such an enzyme.

[0090] The term “wild type” refers to a polypeptide having a primary amino acid sequence which is identical with the native enzyme (for example, the human enzyme).

[0091] The term “mutant” refers to a polypeptide having a primary amino acid sequence which differs from the wild type sequence by one or more amino acid additions, substitutions or deletions. Preferably, the mutant has at least 90% sequence identity with the wild type sequence. Preferably, the mutant has 20 mutations or less over the whole wild-type sequence. More preferably the mutant has 10 mutations or less, most preferably 5 mutations or less over the whole wild-type sequence.

[0092] The term “variant” refers to a naturally occurring polypeptide which differs from a wild-type sequence. A variant may be found within the same species (i.e. if there is more than one isoform of the enzyme) or may be found within a different species. Preferably the variant has at least 90% sequence identity with the wild type sequence. Preferably, the variant has 20 mutations or less over the whole wild-type sequence. More preferably, the variant has 10 mutations or less, most preferably 5 mutations or less over the whole wild-type sequence.

[0093] The term “part” indicates that the polypeptide comprises a fraction of the wild-type amino acid sequence. It may comprise one or more large contiguous sections of sequence or a plurality of small sections. The “part” may comprise a binding pocket as described herein. The polypeptide may also comprise other elements of sequence, for example, it may be a fusion protein with another protein (such as one which aids isolation or crystallisation of the polypeptide). Preferably the polypeptide comprises at least 50%, more preferably at least 65%, most preferably at least 80% of the wild-type sequence.

[0094] The term “homolog” means a polypeptide having a degree of homology with the wild-type amino acid sequence. The term “homology” refers to a degree of complementarity. There may be partial homology or complete homology. In an embodiment of the invention a RTK is substantially homologous to a wild type enzyme. A sequence that is “substantially homologous” refers to a partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid. Inhibition of hybridization of a completely complementary sequence to the target sequence may be examined using a hybridization assay (e.g. Southern or northern blot, solution hybridization, etc.) under conditions of reduced stringency. A sequence that is substantially homologous or a hybridization probe will compete for and inhibit the binding of a completely homologous sequence to the target sequence under conditions of reduced stringency. However, conditions of reduced stringency can be such that non-specific binding is permitted, as reduced stringency conditions require that the binding of two sequences to one another be a specific (i.e., a selective) interaction. The absence of non-specific binding may be tested using a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% homology or identity). The substantially homologous sequence or probe will not hybridize to the second non-complementary target sequence in the absence of non-specific binding.

[0095] A sequence of an RTK may have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity. The phrase “percent identity” or “% identity” refers to the percentage of sequence similarity found in a comparison of two or more amino acid sequences. Percent identity can be determined electronically using conventional programs, e.g., by using the MEGALIGN program (LASERGENE software package, DNASTAR). The MEGALIGN program can create alignments between two or more amino acid sequences according to different methods, e.g., the Clustal Method. (Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.) Gaps of low or of no homology between the two amino acid sequences are not included in determining percentage similarity.

[0096] In the present context, a homologous sequence is taken to include an amino acid sequence which may have at least 75, 85 or 90% identity, preferably at least 95 or 98% identity to the wild-type sequence. The homologs will comprise the same sites (for example, binding pocket) as the subject amino acid sequence.

[0097] A sequence may have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent enzyme. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0098] The polypeptide may also have a homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as 0), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.

[0099] Binding Pocket “Binding pocket” refers to a region or site of a RTK or molecular complex thereof that as a result of its shape, associates with another region of the RTK or with a ligand or a part thereof. A binding pocket may regulate the kinase domain of the RTK. A binding pocket may be involved in maintaining an autoinhibited state or active state of an RTK For example, a binding pocket may comprise part of a juxtamembrane region of an RTK that associates with a kinase domain of the RTK (e.g. strand segment Ex1), a site formed by interacting amino acid residues in the juxtamembrane region (e.g. switch region 2), a site formed by interacting amino acid residues in the juxtamembrane region and kinase domain (switch region 1), or a region responsible for binding a ligand.

[0100] The invention contemplates a binding pocket of an RTK in an autoinhibited state or an active state.

[0101] A “ligand” refers to a compound or entity that associates with a binding pocket including nucleotides or analogues or parts thereof, substrates or analogues or parts thereof, or modulators of RTKs, including inhibitors. A ligand may be designed rationally by using a model according to the present invention.

[0102] In an aspect of the invention a binding pocket comprises one or more of the residues involved in coordination of a nucleotide or analog thereof, in particular the amino acid residues involved in coordinating the sugar and phosphate groups of the nucleotide.

[0103] In an aspect of the invention the binding pocket comprises phosphoregulatory sites of a juxtamembrane region or kinase domain. Phosphoregulatory sites are sites that are autophosphorylated following ligand binding of an RTK and that potentiate binding of cytoplasmic signalling targets such as SH2 or SH3 domain signalling proteins. In a specific aspect the binding pocket comprises invariant tyrosine residues (e.g. tyrosines 596 and 602 of EphA4; tyrosines 604 and 610 of EphB2) within a conserved amino acid sequence (e.g. YIDPFTYEPD in EphB2) in the juxtamembrane region A binding pocket may comprise one or more of the amino acid residues for an Eph receptor crystal identified as numbers 1 through 49 shown in Table 2. In an aspect the binding pocket comprises the atomic contacts of atomic interactions 1 to 24 (juxtamembrane-kinase interactions) or interactions 25 to 49 (juxtamembrane-juxtamembrane interactions) identified in Table 2. In a preferred embodiment the binding pocket comprises atomic interactions or atomic contacts 27, 28, 29, and 38; 39 and 40; or 9, 13, 14, 16, 18, 19, 32, 39, 40, and 42 in Table 2. In an aspect of the invention the binding pocket comprises all of the amino acid residues identified in Table 2.

[0104] A binding pocket may be involved in coordination of a ligand or substrate. For example a binding pocket may be involved in coordination of a nucleotide, or part or analog thereof. Therefore, a binding pocket may comprise two or more of the amino acid residues Phe 709, Met 710 Glu 708, Thr 707, Leu 761, Gly 713, (Lys 661), Ala 659, Ile 691, and (Ser 771) of an RTK structure as described herein, that are capable of associating with or coordinating a nucleotide as described herein.

[0105] The term “binding pocket” (BP) also includes a homolog of the binding pocket or a portion thereof. As used herein, the term “homolog” in reference to a binding pocket refers to a binding pocket or a portion thereof which may have deletions, insertions or substitutions of amino acid residues as long as the binding specificity is retained. In this regard, deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the binding specificity of the binding pocket is retained.

[0106] As used herein, the term “portion thereof” means the structural coordinates corresponding to a sufficient number of amino acid residues of a binding pocket (or homologs thereof) that are capable of providing an autoinhibited or active state or for associating with a ligand For example, the structural coordinates provided in a crystal structure may contain a subset of the amino acid residues in a binding pocket which may be useful in the modelling and design of compounds that bind to the binding pocket.

[0107] Autoinhbited/Active State

[0108] An RTK or a binding pocket thereof may be in an autoinhibited state or active state. An “autoinhibited state” refers to the state of a RTK or a binding pocket that results in disruption of the activation segment of the kinase domain and effective coordination of bound nucleotide. The autoinhibited state results in perturbed catalytic function of an RTK. An autoinhibited state typically occurs in the absence of phosphorylation of the RTK.

[0109] An “active state” refers to the state of a RTK or a binding pocket that does not result in disruption of the activation segment of the kinase domain and effective coordination of bound nucleotide. In the active state the RTK is catalytically active and the juxtamembrane segment is free to bind to signalling proteins such as SH2 domain containing proteins, including p120-RasGAP, Nck, phosphatidylinositol 3′-kinase, SHEP-1, Src family kinases, and the adapter protein Nck. An active state typically occurs in the presence of phosphorylation of the RTK.

[0110] Crystal

[0111] The invention provides crystal structures. As used herein, the term “crystap” or “crystalline” means a structure (such as a three dimensional (3D) solid aggregate) in which the plane faces intersect at definite angles and in which there is a regular structure (such as internal structure) of the constituent chemical species. Thus, the term “crystal” can include any one of: a solid physical crystal form such as an experimentally prepared crystal, a crystal structure derivable from the crystal (including secondary and/or tertiary and/or quaternary structural elements), a 2D and/or 3D model based on the crystal structure, a representation thereof such as a schematic representation thereof or a diagrammatic representation thereof, or a data set thereof for a computer.

[0112] In one aspect the crystal is usable in X-ray crystallography techniques. Here, the crystals used can withstand exposure to X-ray beams used to produce a diffraction pattern data necessary to solve the X-ray crystallographic structure. A crystal may be characterized as being capable of diffracting x-rays in a pattern defined by one of the crystal forms depicted in Blundel et al 1976, Protein Crystallography, Academic Press.

[0113] A crystal of the invention is generally produced in a laboratory; that is, it is an isolated crystal produced by an individual.

[0114] The invention contemplates a crystal comprising a binding pocket of the invention, in particular a binding pocket that regulates the kinase domain of the receptor tyrosine kinase. The binding pocket may be of an autoinhibited state RTK or an active RTK.

[0115] In an aspect of the invention a crystal is provided that comprises the juxtamembrane region and kinase domain of an RTK. In an embodiment the RTK is an Eph Receptor, preferably an EphB receptor. In a preferred embodiment the crystal comprises the juxtamembrane region and the catalytic domain (amino acid residues 595 to 906) of EphB2. The juxtamembrane region and the catalytic domain may be in an autoinhibited state.

[0116] A crystal of the invention may be characterized by one or more of the following characteristics:

[0117] (a) an N-terminal lobe for binding and coordinating ATP for transfer of an α-phosphate to a substrate, comprising a twisted 5-strand β-sheet (denoted β1 to β5) and a single helix αC; and optionally further characterized by (i) a flexible loop that interacts with the adenine base, ribose sugar and the non-hydrolyzable phosphate groups of ATP which loop is formed by β-strands 1 and 2 and a connecting glycine rich segment (g-loop) and

[0118] (ii) an invariant salt bridge between a lysine side chain in 3 strand 3 and a glutamic acid side chain in helix αC that coordinates the position of the P-phosphate of ATP; and

[0119] (b) a C-terminal lobe comprising two β-strands (β7 and β8) and a series of α-helices (αD to αI which is further characterized by (i) strands β7 and β8 in the cleft region between the N- and C-terminal lobes where they contribute side chains that participate in catalysis and the binding of magnesium for the coordination of ATP phosphate groups, (ii) an activation segment flanked by the sequence Asp-Phe-Gly of sub-domain VII and Pro-Ile-Arg of sub-domain VIII, and (iii) a helix αI adjacent to helix αH.

[0120] A crystal of the invention comprising a juxtamembrane region of an RTK, in particular an Eph receptor, more particularly an EphB receptor, most particularly an EphB2 receptor, may be characterized as comprising a single-turn helix αA+ (i.e. a 3/10 helix), and a four-turn helix αB′ from the amino terminus of an extended strand segment Ex1. The crystal may also comprise this juxtamemembrane region in association with interacting amino acid residues on the N- and C-terminal lobes of the RTK. (See FIGS. 2-4, and Table 2.)

[0121] A crystal of the invention may comprise a juxtamembrane strand segment Ex1 comprising amino acid residues Lys 602 to Ile 605 which strand extends along the cleft region between the N-and C-terminal lobes of an RTK. The strand is stabilized by hydrogen bonding interactions involving the amide group of Phe 604 with the carbonyl group of Met 748 and the Gln 684 side chain with the backbone amide and carbonyl groups of Ile 605.

[0122] In a further aspect of the invention a crystal is provided comprising a hydrophobic interface site (referred to herein as switch region 1) comprising side chains of Met 748 and Tyr 750 of the C-terminal kinase lobe; Phe 685 and Ile 681 from helix αC, and Pro 607 from the juxtamembrane helix αAt, and the phosphoregulatory site or residue Phe 604 which orients into the site.

[0123] A crystal of the invention may comprise helix aA′ which is more particularly characterized by one or more of the following characteristics:

[0124] (a) it is composed of a single rigid turn initiated by Asp 606 and Pro 607 and terminated by Thr 609;

[0125] (b) it is stabilized by the conformational regidity of Pro 607 and the capping interactions involving Asp 606 and Thr 609 with the free backbone amino group and carbonly groups of Phe 608 and Asp 606.

[0126] A crystal of the invention may comprise helix αB1 which is more particularly characterized by one or more of the following characteristics:

[0127] (a) it is initiated by an Asp Pro sequence (residues 612 and 613); and

[0128] (b) Asp 612 makes capping interactions with the backbone amino and side chain of Asn 614.

[0129] A crystal of the invention may comprise helices αA′ and αB′ of a juxtamembrane region of an RTK and the portion of the N-terminal lobe of the kinase domain centering on helix αC of the RTK which forms an interface with helices αA′ and αB′ and is further characterized as follows:

[0130] (a) hydrophobic side chains projecting from αA′ and αB′ include Pro 607, Phe 608, Pro 613, Val 617, Phe620 and Ala 621 which residues associate intimately with the side chains of Arg 673, Leu 676, and Ile 681 from helix αC and the side chains of Leu 693 and Val 696 from 1-strand 4;

[0131] (b) a hydrogen bond interaction (2.9 Å) between Asn 614 and Arg 672; and

[0132] (c) the small side chains at positions 616 (Ala), 677 (Ser) and 680 (Ser) facilitate the close packing of helices αA′, αB′ and αC.

[0133] A crystal of the invention may comprise a hydrophobic interface site (also referred to herein as “switch region 2”) formed by association of helix αC, strand Ex1 and helices αA′ and αB′ of the juxtamembrane region of an RTK. The interface is characterized as follows:

[0134] (a) projection of the side chain of the phosphoregulatory residue Tyr/Phe 610 onto the surface of the site;

[0135] (b) composed of the side chains of Ile 605 from strand Ex1 and the side chains of Ala 616 and Phe 620 from helix αB′; and

[0136] (c) an electrostatic environment dominated by Asp 606, Glu 611, Asp 612, Glu 615, and Glu 619.

[0137] A crystal of the invention may comprise the following amino acids residues:

[0138] (a) Arg 672, Phe Arg 672, Phe 675, and Leu 676 from helix αC, Tyr 667 from the 13/αC linker and Leu 663, Val 696, Thr 698, Val 703, and Ile 705; or

[0139] (b) Met 748, Tyr 750, Phe 685, Ile 681, Pro 607, and Phe 604; or

[0140] (c) Phe 709, Met 710, Glu 708, Thr 707, Leu 761, Gly 713, Ala 659, Ile 691, Lys 661, and Ser 771; or

[0141] (d) Asp 606, Pro 607, Thr 609, Phe 608 and Asp 606; or

[0142] (e) Asp 612, Pro 613, Asp 612, and Asn 614; or

[0143] (f) Pro 607, Phe 608, Pro 613, Val 617, Phe 620, Als 621, Arg 673, Leu 676, Ile 681, Leu 693, Val 696, Asn 614, Arg 672, Ala 616, Ser 677, and Ser 680; or

[0144] (g) Tyr/Phe 610, Ile 605, Ala 616, Phe 620, Asp 606, Glu 611, Asp 615, and Glu 619.

[0145] Preferably the atoms of the amino acid residues in (a) to (g) have the structural coordinates as set out in Table 3.

[0146] In an embodiment, a crystal of a Eph receptor of the invention belongs to space group P21 or P1. The term “space group” refers to the lattice and symmetry of the crystal. In a space group designation the capital letter indicates the lattice type and the other symbols represent symmetry operations that can be carried out on the contents of the asymmetric unit without changing its appearance.

[0147] A crystal of the invention may comprise a unit cell having the following unit dimensions: a=47.05 (±0.05) Å, b=57.62 (±0.05) Å, c=67.74 (±0.05) Å, or a=47.86 (±0.05) Å, b=98.09 (±0.05) Å, c=68.18 (±0.05) Å. The term “unit cell” refers to the smallest and simplest volume element (i.e. parallelpiped-shaped block) of a crystal that is completely representative of the unit of pattern of the crystal. The unit cell axial lengths are represented by a, b, and c. Those of skill in the art understand that a set of atomic coordinates determined by X-ray crystallography is not without standard error.

[0148] In a preferred embodiment, a crystal of the invention has the structural coordinates as shown in Table 3. As used herein, the term “structural coordinates” refers to a set of values that define the position of one or more amino acid residues with reference to a system of axes. The term refers to a data set that defines the three dimensional structure of a molecule or molecules (e.g. Cartesian coordinates, temperature factors, and occupancies). Structural coordinates can be slightly modified and still render nearly identical three dimensional structures. A measure of a unique set of structural coordinates is the root-mean-square deviation of the resulting structure. Structural coordinates that render three dimensional structures (in particular a three dimensional structure of a ligand binding pocket) that deviate from one another by a root-mean-square deviation of less than 5 Å, 4 Å, 3 Å, 2 Å, 1.5 Å. 1.0 Å, or 0.5 Å may be viewed by a person of ordinary skill in the art as very similar.

[0149] Variations in structural coordinates may be generated because of mathematical manipulations of the structural coordinates of a glycosyltransferase described herein. For example, the structural coordinates of Table 3 may be manipulated by crystallographic permutations of the structural coordinates, fractionalization of the structural coordinates, integer additions or substractions to sets of the structural coordinates, inversion of the structural coordinates or any combination of the above.

[0150] Variations in the crystal structure due to mutations, additions, substitutions, and/or deletions of the amino acids, or other changes in any of the components that make up the crystal may also account for modifications in structural coordinates. If such modifications are within an acceptable standard error as compared to the original structural coordinates, the resulting structure may be the same. Therefore, a ligand that bound to a binding pocket of an RTK, in particular an Eph receptor, would also be expected to bind to another binding pocket whose structural coordinates defined a shape that fell within the acceptable error. Such modified structures of a binding pocket thereof are also within the scope of the invention.

[0151] Various computational analyses may be used to determine whether a molecule or the binding pocket thereof is sufficiently similar to all or parts of an RTK or a binding pocket thereof. Such analyses may be carried out using conventional software applications and methods as described herein.

[0152] A crystal of the invention may also be specifically characterised by the parameters, diffraction statistics and/or refinement statistics set out in Tables 1.

[0153] With reference to a crystal of the present invention, residues in a binding pocket may be defined by their spatial proximity to a ligand in the crystal structure. For example, a binding pocket may be defined by its proximity to a nucleotide, substrate molecule, or modulator.

[0154] A crystal of the invention may comprise a binding pocket that is involved in coordination of a nucleotide, or part or analog thereof. Therefore, a crystal may comprise a binding pocket comprising two or more of the amino acid residues Phe 709, Met 710 Glu 708, Thr 707, Leu 761, Gly 713, (Lys 661), Ala 659, Ile 691, and (Ser 771) of an RTK structure as described herein, that are capable of associating with or coordinating a nucleotide as described herein.

[0155] A crystal or secondary or three-dimensional structure of a binding pocket of an RTK, in particular an EphB2 receptor, may be specifically defined by one or more of the atomic contacts of the atomic interactions identified in Table 2. The atomic interactions in Table 2 are defined therein by an atomic contact (more preferably, a specific atom of an amino acid residue where indicated) on the juxtamembrane region, and an atomic contact (more preferably, a specific atom of an amino acid residue where indicated) on the kinase domain, juxtamembrane region, or ligand. In certain embodiments, a crystal of the invention comprises the atomic contacts of atomic interactions 1 to 24 (juxtamembrane-kinase interactions) or atomic interactions 25 to 49 (juxtamembrane-juxtamembrane interactions) identified in Table 2. In certain particular embodiments a crystal is provided comprising the atomic contacts of atomic interactions 27, 28, 29, and 38; 39 and 40; or 9, 13, 14, 16, 18, 19, 32, 39, 40, and 42.

[0156] Preferably, a crystal is defined by the atoms of the atomic contacts in the binding pocket having the structural coordinates for the atoms listed in Table 3.

[0157] A crystal of the invention includes a binding pocket in association with one or more moieties, including heavy-metal atoms i.e. a derivative crystal, or one or more ligands or molecules i.e. a co-crystal.

[0158] The term “associate”, “association” or “associating” refers to a condition of proximity between a moiety (i.e. chemical entity or compound or portions or fragments thereof), and a binding pocket. The association may be non-covalent i.e. where the juxtaposition is energetically favored by for example, hydrogen-bonding, van der Waals, or electrostatic or hydrophobic interactions, or it may be covalent.

[0159] The term “heavy-metal atoms” refers to an atom that can be used to solve an x-ray crystallography phase problem, including but not limited to a transition element a lanthamide metal, or an actinide metal. Lanthamide metals include elements with atomic numbers between 57 and 71, inclusive. Actinide metals include elements with atomic numbers between 89 and 103, inclusive.

[0160] Multiwavelength anomalous diffraction (MAD) phasing may be used to solve protein structures using selenomethionyl (SeMet) proteins. Therefore, a complex of the invention may comprise a crystalline binding pocket with selenium on the methionine residues of the protein.

[0161] A crystal may comprise a complex between a binding pocket and one or more ligands or molecules. In other words the binding pocket may be associated with one or more ligands or molecules in the crystal. The ligand may be any compound that is capable of stably and specifically associating with the binding pocket. A ligand may, for example, be a modulator of an Eph receptor, or a nucleotide or substrate or analogue thereof.

[0162] In an embodiment of the invention, a binding pocket is in association with a cofactor in the crystal. A “cofactor” refers to a molecule required for RTK enzyme activity and/or stability. For example, the cofactor may be a metal ion, including magnesium and other similar atoms or metals.

[0163] In an embodiment, a crystal of the invention comprises a complex between a binding pocket, and a nucleotide or analogue thereof and/or a substrate or analogue thereof. A “nucleotide” includes ATP, ADP, AMP, or analogues thereof, for example, β,γ-imidoadenosine-5′-triphosphate (AMP-PNP, STI-571, and quercetin. A substrate may be for example, a signalling protein, or another portion of the same RTK (e.g juxtamembrane-kinase domain complex). An analog of a nucleotide or substrate is one which mimics the nucleotide or substrate molecule, binding in the binding pocket, but which is incapable (or has a significantly reduced capacity) to take part in a kinase reaction.

[0164] Therefore, the present invention also provides:

[0165] (a) a crystal comprising a binding pocket of an RTK and a nucleotide or analogue thereof;

[0166] (b) a crystal comprising a binding pocket of an RTK and a substrate or analogue thereof;

[0167] (c) a crystal comprising a binding pocket of an RTK and a nucleotide or analogue thereof, and a substrate or analogue thereof.

[0168] A complex may comprise one or more of the intermolecular interactions identified in Table 2. A structure of a complex of the invention may be defined by selected intermolecular contacts, preferably the structural coordinates of the intermolecular contacts as defined in Table 3.

[0169] A crystal of the invention may enable the determination of structural data for a ligand. In order to be able to derive structural data for a ligand, it is necessary for the molecule to have sufficiently strong electron density to enable a model of the molecule to be built using standard techniques. For example, there should be sufficient electron density to allow a model to be built using XTALVIEW (McRee 1992 J. Mol. Graphics. 10 44-46).

[0170] Illustrations of particular crystals of the invention are shown in FIGS. 2, 3, and 4.

[0171] Method of Making a Crystal

[0172] The present invention also provides a method of making a crystal according to the invention. The crystal may be formed from an aqueous solution comprising a purified polypeptide comprising an RTK, in particular an Eph receptor including a variant, part, homolog, or fragment thereof (e.g. a binding pocket). A method may utilize a purified polypeptide comprising a binding pocket to form a crystal. A method may utilize a purified polypeptide comprising a juxtamembrane region and kinase domain of an RTK, in particular an Eph receptor, preferably an EphB receptor, or more preferably an EphB2 receptor.

[0173] The term “purified” in reference to a polypeptide, does not require absolute purity such as a homogenous preparation rather it represents an indication that the polypeptide is relatively purer than in the natural environment. Generally, a purified polypeptide is substantially free of other proteins, lipids, carbohydrates, or other materials with which it is naturally associated, preferably at a functionally significant level for example at least 85% pure, more preferably at least 95% pure, most preferably at least 99% pure. A skilled artisan can purify a polypeptide comprising using standard techniques for protein purification. A substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel. Purity of the polypeptide can also be determined by amino-terminal amino acid sequence analysis.

[0174] A polypeptide used in the method may be chemically synthesized in whole or in part using techniques that are well-known in the art. Alternatively, methods are well known to the skilled artisan to construct expression vectors containing a native or mutated RTK coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic recombination. See for example the techniques described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks. (See also Sarker et al, Glycoconjugate J. 7:380, 1990; Sarker et al, Proc. Natl. Acad, Sci. USA 88:234-238, 1991, Sarker et al, Glycoconjugate J. 11: 204-209, 1994; Hull et al, Biochem Biophys Res Commun 176:608, 1991 and Pownall et al, Genomics 12:699-704, 1992).

[0175] Crystals may be grown from an aqueous solution containing the purified polypeptide by a variety of conventional processes. These processes include batch, liquid, bridge, dialysis, vapor diffusion, and hanging drop methods. (See for example, McPherson, 1982 John Wiley, New York; McPherson, 1990, Eur. J. Biochem. 189: 1-23; Webber. 1991, Adv. Protein Chem. 41:1-36). Generally, native crystals of the invention are grown by adding precipitants to the concentrated solution of the polypeptide. The precipitants are added at a concentration just below that necessary to precipitate the protein. Water is removed by controlled evaporation to produce precipitating conditions, which are maintained until crystal growth ceases.

[0176] Derivative crystals of the invention can be obtained by soaking native crystals in a solution containing salts of heavy metal atoms. A complex of the invention can be obtained by soaking a native crystal in a solution containing a compound that binds the polypeptide, or they can be obtained by co-crystallizing the polypeptide in the presence of one or more compounds. In order to obtain co-crystals with a compound which binds deep within the tertiary structure of the polypeptide it is necessary to use the second method.

[0177] In a preferred embodiment, the polypeptide is co-crystallised with a compound which stabilises the polypeptide (e.g. AMP-PNP).

[0178] Once the crystal is grown it can be placed in a glass capillary tube and mounted onto a holding device connected to an X-ray generator and an X-ray detection device. Collection of X-ray diffraction patterns are well documented by those skilled in the art (See for example, Ducruix and Geige, 1992, IRL Press, Oxford, England). A beam of X-rays enter the crystal and diffract from the crystal. An X-ray detection device can be utilized to record the diffraction patterns emanating from the crystal. Suitable devices include the Marr 345 imaging plate detector system with an RU200 rotating anode generator.

[0179] Multiwavelength anomalous diffraction (MAD) phasing using selenomethionyl (SeMet) proteins may be used to determine a crystal of the invention. Thus, the invention contemplates a method for determining a crystal structure of the invention using a selenomethionyl derivative of an RTK, including a variant, part, homolog or fragement thereof.

[0180] Methods for obtaining the three dimensional structure of the crystalline form of a molecule or complex are described herein and known to those skilled in the art (see Ducruix and Geige 1992, IRL Press, Oxford, England). Generally, the x-ray crystal structure is given by the diffraction patterns. Each diffraction pattern reflection is characterized as a vector and the data collected at this stage determines the amplitude of each vector. The phases of the vectors may be determined by the isomorphous replacement method where heavy atoms soaked into the crystal are used as reference points in the X-ray analysis (see for example, Otwinowski, 1991, Daresbury, United Kingdom, 80-86). The phases of the vectors may also be determined by molecular replacement (see for example, Naraza, 1994, Proteins 11:281-296). The amplitudes and phases of vectors from the crystalline form determined in accordance with these methods can be used to analyze other related crystalline polypeptides.

[0181] The unit cell dimensions and symmetry, and vector amplitude and phase information can be used in a Fourier transform function to calculate the electron density in the unit cell i.e. to generate an experimental electron density map. This may be accomplished using the PHASES package (Furey, 1990). Amino acid sequence structures are fit to the experimental electron density map (i.e. model building) using computer programs (e.g. Jones, TA. et al, Acta Crystallogr A47, 100-119, 1991). This structure can also be used to calculate a theoretical electron density map. The theoretical and experimental electron density maps can be compared and the agreement between the maps can be described by a parameter referred to as R-factor. A high degree of overlap in the maps is represented by a low value R-factor. The R-factor can be minimized by using computer programs that refine the structure to achieve agreement between the theoretical and observed electron density map. For example, the XPLOR program, developed by Brunger (1992, Nature 355:472-475) can be used for model refinement A three dimensional structure of the molecule or complex may be described by atoms that fit the theoretical electron density characterized by a minimum R value. Files can be created for the structure that defines each atom by coordinates in three dimensions.

[0182] Model

[0183] A crystal structure of the present invention may be used to make a model of a binding pocket of an RTK, preferably an Eph receptor, more preferably an EphB receptor. A model may, for example, be a structural model or a computer model. A model may represent the secondary, tertiary and/or quaternary structure of the binding pocket. The model itself may be in two or three dimensions. It is possible for a computer model to be in three dimensions despite the constraints imposed by a conventional computer screen, if it is possible to scroll along at least a pair of axes, causing “rotation” of the image.

[0184] As used herein, the term “modelling” includes the quantitative and qualitative analysis of molecular structure and/or function based on atomic structural information and interaction models. The term “modelling” includes conventional numeric-based molecular dynamic and energy minimization models, interactive computer graphic models, modified molecular mechanics models, distance geometry and other structure-based constraint models.

[0185] Preferably, modelling is performed using a computer and may be further optimized using known methods. This is called modelling optimisation.

[0186] An integral step to an approach of the invention for designing modulators (e.g. inhibitors) of a subject receptor involves construction of computer graphics models of the binding pocket of a receptor which can be used to design pharmacophores by rational drug design. For instance, for an inhibitor to interact optimally with the subject binding pocket, it will generally be desirable that it have a shape which is at least partly complimentary to that of a particular binding pocket of the receptor, as for example those binding pockets of the receptor which are involved in recognition of a ligand, regulating the kinase domain, or regulating signal transduction. Additionally, other factors, including electrostatic interactions, hydrogen bonding, hydrophobic interactions, desolvation effects, and cooperative motions of ligand and receptor, all influence the binding effect and should be taken into account in attempts to design bioactive modulators (e.g. inhibitors).

[0187] As described herein, a computer-generated molecular model of the subject receptors can be created. In preferred embodiments, at least the Ca-carbon positions of the RTK sequence of interest are mapped to a particular coordinate pattern, such as the coordinates for a binding pocket of an EphB2 shown in Table 3, by homology modeling, and the structure of the protein and velocities of each atom are calculated at a simulation temperature (To) at which the docking simulation is to be determined. Typically, such a protocol involves primarily the prediction of side-chain conformations in the modeled protein, while assuming a main-chain trace taken from a tertiary structure such as provided in Table 3 and the Figures. Computer programs for performing energy minimization routines are commonly used to generate molecular models. For example, both the CHARMM (Brooks et al. (1983) J Comput Chem 4:187-217) and AMBER (Weiner et al (1981) J. Comput. Chem. 106: 765) algorithms handle all of the molecular system setup, force field calculation, and analysis (see also, Eisenfield et al. (1991) Am J Physiol 261:C376-386; Lybrand (1991) J Pharm Belg 46:49-54; Froimowitz (1990) Biotechniques 8:640-644; Burbam et al. (1990) Proteins 7:99-111; Pedersen (1985) Environ Health Perspect 61:185-190; and Kini et al. (1991) J Biomol Struct Dyn 9:475-488). At the heart of these programs is a set of subroutines that, given the position of every atom in the model, calculate the total potential energy of the system and the force on each atom. These programs may utilize a starting set of atomic coordinates, such as the coordinates provided in Table 3, the parameters for the various terms of the potential energy function, and a description of the molecular topology (the covalent structure). Common features of such molecular modeling methods include: provisions for handling hydrogen bonds and other constraint forces; the use of periodic boundary conditions; and provisions for occasionally adjusting positions, velocities, or other parameters in order to maintain or change temperature, pressure, volume, forces of constraint, or other externally controlled conditions.

[0188] Most conventional energy minimization methods use the input data described above and the fact that the potential energy function is an explicit, differentiable function of Cartesian coordinates, to calculate the potential energy and its gradient (which gives the force on each atom) for any set of atomic positions. This information can be used to generate a new set of coordinates in an effort to reduce the total potential energy and, by repeating this process over and over, to optimize the molecular structure under a given set of external conditions. These energy minimization methods are routinely applied to molecules similar to the subject RTK proteins as well as nucleic acids, polymers and zeolites.

[0189] In general, energy minimization methods can be carried out for a given temperature, Ti, which may be different than the docking simulation temperature, To. Upon energy minimization of the molecule at Ti, coordinates and velocities of all the atoms in the system are computed. Additionally, the normal modes of the system are calculated. It will be appreciated by those skilled in the art that each normal mode is a collective, periodic motion, with all parts of the system moving in phase with each other, and that the motion of the molecule is the superposition of all normal modes. For a given temperature, the mean square amplitude of motion in a particular mode is inversely proportional to the effective force constant for that mode, so that the motion of the molecule will often be dominated by the low frequency vibrations.

[0190] After the molecular model has been energy minimized at Ti, the system is “heated” or “cooled” to the simulation temperature, To, by carrying out an equilibration run where the velocities of the atoms are scaled in a step-wise manner until the desired temperature, To, is reached. The system is further equilibrated for a specified period of time until certain properties of the system, such as average kinetic energy, remain constant. The coordinates and velocities of each atom are then obtained from the equilibrated system.

[0191] Further energy minimization routines can also be carried out. For example, a second class of methods involves calculating approximate solutions to the constrained EOM for the protein. These methods use an iterative approach to solve for the Lagrange multipliers and, typically, only need a few iterations if the corrections required are small. The most popular method of this type, SHAKE (Ryckaert et al. (1977) J Comput Phys 23:327; and Van Gunsteren et al. (1977) Mol Phys 34:1311) is easy to implement and scales as O(N) as the number of constraints increases. Therefore, the method is applicable to macromolecules such as the RTK proteins of the present invention. An alternative method, RATTLE (Anderson (1983) J Comput Phys 52:24) is based on the velocity version of the Verlet algorithm. Like SHAKE, RATTLE is an iterative algorithm and can be used to energy minimize the model of the subject protein.

[0192] Overlays and super positioning with a three dimensional model of a binding pocket of the invention may be used for modelling optimisation. Additionally alignment and/or modelling can be used as a guide for the placement of mutations on a binding pocket to characterize the nature of the site in the context of a cell.

[0193] The three dimensional structure of a new crystal may be modelled using molecular replacement The term “molecular replacement” refers to a method that involves generating a preliminary model of a molecule or complex whose structural coordinates are unknown, by orienting and positioning a molecule whose structural coordinates are known within the unit cell of the unknown crystal, so as best to account for the observed diffraction pattern of the unknown crystal. Phases can then be calculated from this model and combined with the observed amplitudes to give an approximate Fourier synthesis of the structure whose coordinates are unknown. This, in turn, can be subject to any of the several forms of refinement to provide a final, accurate structure of the unknown crystal. Lattman, E., “Use of the Rotation and Translation Functions”, in Methods in Enzymology, 115, pp. 55-77 (1985); M. G. Rossmann, ed., “The Molecular Replacement Method”, Int. Sci. Rev. Ser., No. 13, Gordon & Breach, New York, (1972).

[0194] Commonly used computer software packages for molecular replacement are X-PLOR (Brunger 1992, Nature 355: 472-475), AMoRE (Navaza, 1994, Acta Crystallogr. A50:157-163), the CCP4 package (Collaborative Computational Project, Number 4, “The CCP4 Suite: Programs for Protein Crystallography”, Acta Cryst., Vol. D50, pp. 760-763, 1994), the MERLOT package (P. M. D. Fitzgerald, J. Appl. Cryst., Vol. 21, pp. 273-278, 1988) and XTALVIEW (McCree et al (1992) J. Mol. Graphics 10: 44-46. It is preferable that the resulting structure not exhibit a root-mean-square deviation of more than 3 Å.

[0195] Molecular replacement computer programs generally involve the following steps: (1) determining the number of molecules in the unit cell and defining the angles between them (self rotation function); (2) rotating the known structure against diffraction data to define the orientation of the molecules in the unit cell (rotation function); (3) translating the known structure in three dimensions to correctly position the molecules in the unit cell (translation function); (4) determining the phases of the X-ray diffraction data and calculating an R-factor calculated from the reference data set and from the new data wherein an R-factor between 30-50% indicates that the orientations of the atoms in the unit cell have been reasonably determined by the method; and (5) optionally, decreasing the R-factor to about 20% by refining the new electron density map using iterative refinement techniques known to those skilled in the art (refinement).

[0196] The quality of the model may be analysed using a program such as PROCHECK or 3D-Profiler [Laskowski et al 1993 J. Appl. Cryst. 26:283-291; Luthy R et al, Nature 356: 83-85, 1992; and Bowie, J. U. et al, Science 253: 164-170, 1991]. Once any irregularities have been resolved, the entire structure may be further refined.

[0197] Other molecular modelling techniques may also be employed in accordance with this invention. See, e.g., Cohen, N. C. et al, “Molecular Modelling Software and Methods for Medicinal Chemistry”, J. Med. Chem., 33, pp. 883-894 (1990). See also, Navia, M. A. and M. A. Murcko, “The Use of Structural Information in Drug Design”, Current Opinions in Structural Biology, 2, pp. 202-210 (1992).

[0198] Using the structural coordinates of crystal provided by the invention, molecular modelling may be used to determine the structural coordinates of a crystalline mutant or homolog of an RTK binding pocket By the same token a crystal of the invention can be used to provide a model of a ligand. Modelling techniques can then be used to approximate the three dimensional structure of ligand derivatives and other components which may be able to mimic the atomic contacts between a ligand and binding pocket.

[0199] Computer Format of Crystals/Models

[0200] Information derivable from a crystal of the present invention (for example the structural coordinates) and/or the model of the present invention may be provided in a computer-readable format Therefore, the invention provides a computer readable medium or a machine readable storage medium which comprises the structural coordinates of a binding pocket of an RTK including all or any parts thereof, or ligands including portions thereof. Such storage medium or storage medium encoded with these data are capable of displaying on a computer screen or similar viewing device, a three-dimensional graphical representation of a molecule or molecular complex which comprises such binding pockets or similarly shaped homologous binding pockets. Thus, the invention also provides computerized representations of the secondary or three-dimensional structures of a binding pocket of the invention, including any electronic, magnetic, or electromagnetic storage forms of the data needed to define the structures such that the data will be computer readable for purposes of display and/or manipulation.

[0201] In an aspect the invention provides a computer for producing a three-dimensional representation of a molecule or molecular complex, wherein said molecule or molecular complex comprises a binding pocket defined by structural coordinates of a binding pocket or structural coordinates of atoms of a ligand, or a three-dimensional representation of a homolog of said molecule or molecular complex, wherein said homolog comprises a binding pocket or ligand that has a root mean square deviation from the backbone atoms not more than 1.5 angstroms wherein said computer comprises:

[0202] (a) a machine-readable data storage medium comprising a data storage material encoded with machine readable data wherein said data comprises the structural coordinates of a binding pocket of an RTK or a ligand according to Table 3;

[0203] (b) a working memory for storing instructions for processing said machine-readable data;

[0204] (c) a central-processing unit coupled to said working memory and to said machine-readable data storage medium for processing said machine readable data into said three-dimensional representation; and

[0205] (d) a display coupled to said central-processing unit for displaying said three-dimensional representation.

[0206] The invention also provides a computer for determining at least a portion of the structural coordinates corresponding to an X-ray diffraction pattern of a molecule or molecular complex wherein said computer comprises:

[0207] (a) a machine-readable data storage medium comprising a data storage material encoded with machine readable data wherein said data comprises the structural coordinates according to Table 3;

[0208] (b) a machine-readable data storage medium comprising a data storage material encoded with machine readable data wherein said data comprises an X-ray diffraction pattern of said molecule or molecular complex;

[0209] (c) a working memory for storing instructions for processing said machine-readable data of (a) and (b);

[0210] (d) a central-processing unit coupled to said working memory and to said machine-readable data storage medium of (a) and (b) for performing a Fourier transform of the machine readable data of (a) and for processing said machine readable data of (b) into structural coordinates; and

[0211] (e) a display coupled to said central-processing unit for displaying said structural coordinates of said molecule or molecular complex.

[0212] Structural Studies

[0213] The present invention also provides a method for determining the secondary and/or tertiary structures of a polypeptide or part thereof by using a crystal, or a model according to the present invention. The polypeptide or part thereof may be any polypeptide or part thereof for which the secondary and or tertiary structure is uncharacterised or incompletely characterised. In a preferred embodiment the polypeptide shares (or is predicted to share) some structural or functional homology to a crystal of the present invention. For example, the polypeptide may show a degree of structural homology over some or all parts of the primary amino acid sequence.

[0214] The polypeptide may be an RTK, preferably an Eph receptor with a different specificity for a nucleotide, or substrate. The polypeptide may be an RTK preferably an Eph receptor which requires a different metal cofactor. Alternatively (or in addition) the polypeptide may be an RTK, preferably an Eph receptor from a different species.

[0215] The polypeptide may be a mutant of a wild-type RTK, in particular an Eph receptor. A mutant may arise naturally, or may be made artificially (for example using molecular biology techniques). The mutant may also not be “made” at all in the conventional sense, but merely tested theoretically using the model of the present invention. A mutant may or may not be functional.

[0216] Thus, using a model of the present invention, the effect of a particular mutation on the overall two and/or three dimensional structure of an RTK, in particular an Eph receptor, the autoinhibited state or active state, and/or the interaction between a binding pocket of the enzyme and a ligand can be investigated.

[0217] Alternatively, the polypeptide may perform an analogous function or be suspected to show a similar catalytic mechanism to an RTK, in particular an Eph receptor.

[0218] The polypeptide may also be the same as the polypeptide of the crystal, but in association with a different ligand (for example, modulator or inhibitor) or cofactor. In this way it is possible to investigate the effect of altering the ligand or compound with which the polypeptide is associated on the structure of the binding pocket.

[0219] Secondary or tertiary structure may be determined by applying the structural coordinates of the crystal or model of the present invention to other data such as an amino acid sequence, X-ray crystallographic diffraction data, or nuclear magnetic resonance (NMR) data. Homology modeling, molecular replacement, and nuclear magnetic resonance methods using these other data sets are described below.

[0220] Homology modeling (also known as comparative modeling or knowledge-based modeling) methods develop a three dimensional model from a polypeptide sequence based on the structures of known proteins (i.e. an RTK, in particular an Eph receptor, of the crystal). The method utilizes a computer model of a crystal of the present invention (the “known structure”), a computer representation of the amino acid sequence of the polypeptide with an unknown structure, and standard computer representations of the structures of amino acids. The method in particular comprises the steps of; (a) identifying structurally conserved and variable regions in the known structure; (b) aligning the amino acid sequences of the known structure and unknown structure (c) generating co-ordinates of main chain atoms and side chain atoms in structurally conserved and variable regions of the unknown structure based on the coordinates of the known structure thereby obtaining a homology model; and (d) refining the homology model to obtain a three dimensional structure for the unknown structure. This method is well known to those skilled in the art (Greer, 1985, Science 228, 1055; Bundell et al 1988, Eur. J. Biochem. 172, 513; Knighton et al., 1992, Science 258:130-135, http://biochem.vtedu/courses/modeling/homology.htn). Computer programs that can be used in homology modelling are Quanta and the Homology module in the Insight II modelling package distributed by Molecular Simulations Inc, or MODELLER (Rockefeller University, www.iucr.ac.uk/sinris-top/logical/prg-modeller.html).

[0221] In step (a) of the homology modelling method, a known structure is examined to identify the structurally conserved regions (SCRs) from which an average structure, or framework, can be constructed for these regions of the protein. Variable regions (VRs), in which known structures may differ in conformation, also must be identified. SCRs generally correspond to the elements of secondary structure, such as alpha-helices and beta-sheets, and to ligand- and substrate-binding sites (e.g. nucleotide binding sites). The VRs usually lie on the surface of the proteins and form the loops where the main chain turns.

[0222] Many methods are available for sequence alignment of known structures and unknown structures. Sequence alignments generally are based on the dynamic programming algorithm of Needleman and Wunsch [J. Mol. Biol. 48: 442-453, 1970]. Current methods include FASTA, Smith-Waterman, and BLASTP, with the BLASTP method differing from the other two in not allowing gaps. Scoring of alignments typically involves construction of a 20×20 matrix in which identical amino acids and those of similar character (i.e., conservative substitutions) may be scored higher than those of different character. Substitution schemes which may be used to score alignments include the scoring matrices PAM (Dayhoff et al., Meth. Enzymol. 91: 524-545, 1983), and BLOSUM (Henikoff and Henikoff, Proc. Nat. Acad. Sci. USA 89: 10915-10919, 1992), and the matrices based on alignments derived from three-dimensional structures including that of Johnson and Overington (JO matrices) (J. Mol. Biol. 233: 716-738,1993).

[0223] Alignment based solely on sequence may be used; however, other structural features also may be taken into account. In Quanta, multiple sequence alignment algorithms are available that may be used when aligning a sequence of the unknown with the known structures. Four scoring systems (i.e. sequence homology, secondary structure homology, residue accessibility homology, CA-CA distance homology) are available, each of which may be evaluated during an alignment so that relative statistical weights may be assigned.

[0224] When generating coordinates for the unknown structure, main chain atoms and side chain atoms, both in SCRs and VRs need to be modelled A variety of approaches known to those skilled in the art may be used to assign co-ordinates to the unknown. In particular, the co-ordinates of the main chain atoms of SCRs will be transferred to the unknown structure. VRs correspond most often to the loops on the surface of the polypeptide and if a loop in the known structure is a good model for the unknown, then the main chain co-ordinates of the known structure may be copied. Side chain coordinates of SCRs and VRs are copied if the residue type in the unknown is identical to or very similar to that in the known structure. For other side chain coordinates, a side chain rotamer library may be used to define the side chain coordinates. When a good model for a loop cannot be found fragment databases may be searched for loops in other proteins that may provide a suitable model for the unknown. If desired, the loop may then be subjected to conformational searching to identify low energy conformers if desired.

[0225] Once a homology model has been generated it is analyzed to determine its correctness. A computer program available to assist in this analysis is the Protein Health module in Quanta which provides a variety of tests. Other programs that provide structure analysis along with output include PROCHECK and 3D-Profiler [Luthy R et al, Nature 356: 83-85, 1992; and Bowie, J. U. et al, Science 253: 164-170, 1991]. Once any irregularities have been resolved, the entire structure may be further refined. Refinement may consist of energy minimization with restraints, especially for the SCRs. Restraints may be gradually removed for subsequent minimizations. Molecular dynamics may also be applied in conjunction with energy minimization.

[0226] Molecular replacement involves applying a known structure to solve the X-ray crystallographic data set of a polypeptide of unknown structure. The method can be used to define the phases describing the X-ray diffraction data of a polypeptide of unknown structure when only the amplitudes are known. Thus in an embodiment of the invention, a method is provided for determining three dimensional structures of polypeptides with unknown structure by applying the structural coordinates of a crystal of the present invention to provide an X-ray crystallographic data set for a polypeptide of unknown structure, and (b) determining a low energy conformation of the resulting structure.

[0227] The structural coordinates of a crystal of the present invention may be applied to nuclear magnetic resonance (NMR) data to determine the three dimensional structures of polypeptides with uncharacterised or incompletely characterised sturcture. (See for example, Wuthrich, 1986, John Wiley and Sons, New York: 176-199; Pflugrath et al., 1986, J. Molecular Biology 189: 383-386; Kline et al., 1986 J. Molecular Biology 189:377-382). While the secondary structure of a polypeptide may often be determined by NMR data, the spatial connections between individual pieces of secondary structure are not as readily determined. The structural coordinates of a polypeptide defined by X-ray crystallography can guide the NMR spectroscopist to an understanding of the spatial interactions between secondary structural elements in a polypeptide of related structure. Information on spatial interactions between secondary structural elements can greatly simplify Nuclear Overhauser Effect (NOE) data from two-dimensional NMR experiments. In addition, applying the structural coordinates after the determination of secondary structure by NMR techniques simplifies the assignment of NOE's relating to particular amino acids in the polypeptide sequence and does not greatly bias the NMR analysis of polypeptide structure.

[0228] In an embodiment, the invention relates to a method of determining three dimensional structures of polypeptides with unknown structures, by applying the structural coordinates of a crystal of the present invention to nuclear magnetic resonance (NMR) data of the unknown structure. This method comprises the steps of: (a) determining the secondary structure of an unknown structure using NMR data; and (b) simplifying the assignment of through-space interactions of amino acids. The term “through-space interactions” defines the orientation of the secondary structural elements in the three dimensional structure and the distances between amino acids from different portions of the amino acid sequence. The term “assigmnent” defines a method of analyzing NMR data and identifying which amino acids give rise to signals in the NMR spectrum.

[0229] Screening Methods

[0230] Another aspect of the present invention is the design and identification of agents that inhibit or potentiate an autoinhibition state or active state of an RTK. The rationale design and identification of agents can be accomplished by utilizing the structural coordinates that define a binding pocket of an RTK.

[0231] The structures described herein, and the structures of other polypeptides determined by homology modeling, molecular replacement, and NMR techniques described herein can also be applied to modulator design and identification methods.

[0232] The invention contemplates molecular models, in particular three-dimensional molecular models of RTK proteins, and their use as templates for the design of agents able to mimic or inhibit ligand activation or autophosphorylation or phoshorylation of the proteins (e.g. modulators). A modulator may inhibit or potentiate an autoinhibited state or alternatively an active state.

[0233] In certain embodiments, the present invention provides a method of screening for a ligand that associates with a binding pocket and/or modulates the function of an Eph receptor by using a crystal or a model according to the present invention. The method may involve investigating whether a test compound is capable of associating with or binding a binding pocket, and/or inhibiting or enhancing interactions of atomic contacts in a binding pocket.

[0234] In accordance with an aspect of the present invention, a method is provided for screening for a ligand capable of binding to a binding pocket, wherein the method comprises using a crystal or model according to the invention.

[0235] In another aspect, the invention relates to a method of screening for a ligand capable of binding to a binding pocket, wherein the binding pocket is defined by the structural coordinates given herein, the method comprising contacting the binding pocket with a test compound and determining if the test compound binds to the binding pocket. The binding pocket may be a binding pocket of an autoinhibited state or an active state. In the case of an autoinhibited state binding pocket the screening method may potentially identify an inhibitor that may disrupt catalytic activity of an RTK, for example, by maintaining the RTK in an autoinhibited state. A disruption of catalytic activity may be useful in the treatment of conditions involving increased RTK activity e.g. cancer.

[0236] In one embodiment, the present invention provides a method of screening for a test compound capable of interacting with one or more key amino acid residues of a binding pocket of an RTK. For example, a test compound that interacts with one or more of Tyr/Phe604, Tyr/Phe 610, Tyr 667, Tyr 744, and Tyr 750 of EphB2 receptor may prevent phosphorylation of one or more of the tyrosines and thereby promote the autoinhibited state of the receptor.

[0237] Another aspect of the invention provides a process comprising the steps of:

[0238] (a) performing a method of screening for a ligand described above;

[0239] (b) identifying one or more ligands capable of binding to a binding pocket; and

[0240] (c) preparing a quantity of said one or more ligands.

[0241] A further aspect of the invention provides a process comprising the steps of;

[0242] (a) performing a method of screening for a ligand as described above;

[0243] (b) identifying one or more ligands capable of binding to a binding pocket; and

[0244] (c) preparing a pharmaceutical composition comprising said one or more ligands.

[0245] Once a test compound capable of interacting with one or more key amino acid residues in a binding pocket of an RTK has been identified, further steps may be carried out either to select and/or modify compounds and/or to modify existing compounds, to modulate the interaction with the key amino acid residues in the binding pocket.

[0246] Yet another aspect of the invention provides a process comprising the steps of;

[0247] (a) performing the method of screening for a ligand as described above;

[0248] (b) identifying one or more ligands capable of binding to a binding pocket;

[0249] (c) modifying said one or more ligands capable of binding to a binding pocket;

[0250] (d) performing said method of screening for a ligand as described above; and

[0251] (e) optionally preparing a pharmaceutical composition comprising said one or more ligands.

[0252] In another aspect of the invention, a method of screening for a test compound is provided comprising screening for test compounds that affect (inhibit or potentiate) a juxtamembrane-juxtamembrane interaction (e.g. interactions 25 to 49 in Table 2) or juxtamembrane-kinase interactions (e.g. interactions 1 to 24 in Table 2) described herein.

[0253] As used herein, the term “test compound” means any compound which is potentially capable of associating with a binding pocket, inhibiting or enhancing interactions of atomic contacts in a binding pocket, and/or inhibiting or potentiating an autoinhibited state or active state of an RTK. If, after testing, it is determined that the test compound does bind to the binding pocket, inhibits or enhances interactions of atomic contacts in a binding pocket, and/or inhibits or potentiates an autoinhibited or active state of an RTK, it is known as a “ligand”.

[0254] The test compound may be designed or obtained from a library of compounds which may comprise peptides, as well as other compounds, such as small organic molecules and particularly new lead compounds. By way of example, the test compound may be a natural substance, a biological macromolecule, or an extract made from biological materials such as bacteria, fungi, or animal particularly mammalian) cells or tissues, an organic or an inorganic molecule, a synthetic test compound, a semi-synthetic test compound, a carbohydrate, a monosaccharide, an oligosaccharide or polysaccharide, a glycolipid, a glycopeptide, a saponin, a heterocyclic compound, a structural or functional mimetic, a peptide, a peptidomimetic, a derivatised test compound, a peptide cleaved from a whole protein, or a peptide synthesised synthetically (such as, by way of example, either using a peptide synthesizer or by recombinant techniques or combinations thereof), a recombinant test compound, a natural or a non-natural test compound, a fusion protein or equivalent thereof and mutants, derivatives or combinations thereof.

[0255] The increasing availability of biomacromolecule structures of potential pharmacophoric molecules that have been solved crystallographically has prompted the development of a variety of direct computational methods for molecular design, in which the steric and electronic properties of substrate binding sites are use to guide the design of potential ligands (Cohen et al. (1990) J. Med. Cam. 33: 883-894; Kuntz et al. (1982) J. Mol. Biol 161: 269-288; DesJarlais (1988) J. Med. Cam. 31: 722-729; Bartlett et al. (1989) (Spec. Publ., Roy. Soc. Chem.) 78: 182-196; Goodford et al. (1985) J. Med. Cam. 28: 849-857; DesJarlais et al. J. Med. Cam. 29: 2149-2153). Directed methods generally fall into two categories: (1) design by analogy in which 3-D structures of known molecules (such as from a crystallographic database) are docked to the receptor structure and scored for goodness-of-fit; and (2) de novo design, in which the ligand model is constructed piece-wise in the receptor. The latter approach, in particular, can facilitate the development of novel molecules, uniquely designed to bind to the subject receptor.

[0256] The test compound may be screened as part of a library or a data base of molecules. Modulators of inactivated/activated states of an RTK or binding pocket thereof may be identified by docking a computer representation of compounds from one or more data base of molecules. Data bases which may be used include ACD (Molecular Designs Limited), NCI (National Cancer Institute), CCDC (Cambridge Crystallographic Data Center), CAST (Chemical Abstract Service), Derwent (Derwent Information Limited), Maybridge (Maybridge Chemical Company Ltd), Aldrich (Aldrich Chemical Company), DOCK University of California in San Francisco), and the Directory of Natural Products (Chapman & Hall). Computer programs such as CONCORD (Tripos Associates) or DB-Converter (Molecular Simulations Limited) can be used to convert a data set represented in two dimensions to one represented in three dimensions.

[0257] Test compounds may tested for their capacity to fit spatially into a binding pocket. As used herein, the term “fits spatially” means that the three-dimensional structure of the test compound is accommodated geometrically in a cavity of a binding pocket. The test compound can then be considered to be a ligand.

[0258] A favourable geometric fit occurs when the surface area of the test compound is in close proximity with the surface area of the cavity of a binding pocket without forming unfavorable interactions. A favourable complementary interaction occurs where the test compound interacts by hydrophobic, aromatic, ionic, dipolar, or hydrogen donating and accepting forces. Unfavourable interactions may be steric hindrance between atoms in the test compound and atoms in the binding pocket.

[0259] If a model of the present invention is a computer model, the test compounds may be positioned in a binding pocket through computational docking. If, on the other hand, the model of the present invention is a structural model, the test compounds may be positioned in the binding pocket by, for example, manual docking.

[0260] As used herein the term “docking” refers to a process of placing a compound in close proximity with a binding pocket, or a process of finding low energy conformations of a test compound/binding pocket complex.

[0261] In an illustrative embodiment, the design of potential RTK, in particular EphB2 ligands begins from the general perspective of shape complimentarity for an active site and substrate specificity subsites of the receptor, and a search algorithm is employed which is capable of scanning a database of small molecules of known three-dimensional structure for candidates which fit geometrically into the target protein site. It is not expected that the molecules found in the shape search will necessarily be leads themselves, since no evaluation of chemical interaction need necessarily be made during the initial search. Rather, it is anticipated that such candidates might act as the framework for further design, providing molecular skeletons to which appropriate atomic replacements can be made. Of course, the chemical complimentarily of these molecules can be evaluated, but it is expected that atom types will be changed to maximize the electrostatic, hydrogen bonding, and hydrophobic interactions with the receptor. Most algorithms of this type provide a method for finding a wide assortment of chemical structures that are complementary to the shape of a binding site of the subject receptor. Each of a set of small molecules from a particular data-base, such as the Cambridge Crystallographic Data Bank (CCDB) (Allen et al. (1973) J. Chem. Doc. 13: 119), is individually docked to the binding pocket or site of an RTK, in particular an EphB2 receptor, in a number of geometrically permissible orientations with use of a docking algorithm. In a preferred embodiment, a set of computer algorithms called DOCK, can be used to characterize the shape of invaginations and grooves that form active sites and recognition surfaces of a subject receptor (Kuntz et al. (1982) J. Mol. Biol 161: 269-288). The program can also search a database of small molecules for templates whose shapes are complementary to particular binding pockets or sites of a receptor (DesJarlais et al. (1988) J Med Chem 31: 722-729). These templates normally require modification to achieve good chemical and electrostatic interactions (DesJarlais et al. (1989) ACS Symp Ser 413: 60-69). However, the program has been shown to position accurately known cofactors for ligands based on shape constraints alone.

[0262] The orientations are evaluated for goodness-of-fit and the best are kept for further examination using molecular mechanics programs, such as AMBER or CHARMM. Such algorithms have previously proven successful in finding a variety of molecules that are complementary in shape to a given binding site of a receptor, and have been shown to have several attractive features. First, such algorithms can retrieve a remarkable diversity of molecular architectures. Second, the best structures have, in previous applications to other proteins, demonstrated impressive shape complementarity over an extended surface area. Third, the overall approach appears to be quite robust with respect to small uncertainties in positioning of the candidate atoms.

[0263] Goodford (1985, J Med Chem 28:849-857) and Boobbyer et al. (1989, J Med Chem 32:1083-1094) have produced a computer program (GRID) which seeks to determine regions of high affinity for different chemical groups (termed probes) on the molecular surface of the binding site. GRID hence provides a tool for suggesting modifications to known ligands that might enhance binding. It may be anticipated that some of the sites discerned by GRID as regions of high affinity correspond to “pharmacophoric patterns” determined inferentially from a series of known ligands. As used herein, a pharmacophoric pattern is a geometric arrangement of features of the anticipated ligand that is believed to be important for binding. Attempts have been made to use pharmacophoric patterns as a search screen for novel ligands (Jakes et al. (1987) J Mol Graph 5:41-48; Brint et al. (1987) J Mol Graph 5:49-56; Jakes et al. (1986) J Mol Graph 4:12-20); however, the constraint of steric and “chemical” fit in the putative (and possibly unknown) receptor binding pocket or site is ignored. Goodsell and Olson (1990, Proteins: Struct Funct Genet 8:195-202) have used the Metropolis (simulated annealing) algorithm to dock a single known ligand into a target protein. They allow torsional flexibility in the ligand and use GRID interaction energy maps as rapid lookup tables for computing approximate interaction energies. Given the large number of degrees of freedom available to the ligand, the Metropolis algorithm is time-consuming and is unsuited to searching a candidate database of a few thousand small molecules.

[0264] Yet a further embodiment of the present invention utilizes a computer algorithm such as CLIX which searches such databases as CCDB for small molecules which can be oriented in a receptor binding pocket or site in a way that is both sterically acceptable and has a high likelihood of achieving favorable chemical interactions between the candidate molecule and the surrounding amino acid residues. The method is based on characterizing a binding pocket in terms of an ensemble of favorable binding positions for different chemical groups and then searching for orientations of the candidate molecules that cause maximum spatial coincidence of individual candidate chemical groups with members of the ensemble. The current availability of computer power dictates that a computer-based search for novel ligands follows a breadth-first strategy. A breadth-first strategy aims to reduce progressively the size of the potential candidate search space by the application of increasingly stringent criteria, as opposed to a depth-first strategy wherein a maximally detailed analysis of one candidate is performed before proceeding to the next. CLIX conforms to this strategy in that its analysis of binding is rudimentary it seeks to satisfy the necessary conditions of steric fit and of having individual groups in “correct” places for bonding, without imposing the sufficient condition that favorable bonding interactions actually occur. A ranked “shortlist” of molecules, in their favored orientations, is produced which can then be examined on a molecule-by-molecule basis, using computer graphics and more sophisticated molecular modeling techniques. CLIX is also capable of suggesting changes to the substituent chemical groups of the candidate molecules that might enhance binding.

[0265] The algorithmic details of CLIX is described in Lawerence et al. (1992) Proteins 12:31-41, and the CLIX algorithm can be summarized as follows. The GRID program is used to determine discrete favorable interaction positions (termed target sites) in the binding pocket or site of the protein for a wide variety of representative chemical groups. For each candidate ligand in the CCDB an exhaustive attempt is made to make coincident, in a spatial sense in the binding site of the protein, a pair of the candidate's substituent chemical groups with a pair of corresponding favorable interaction sites proposed by GRID. All possible combinations of pairs of ligand groups with pairs of GRID sites are considered during this procedure. Upon locating such coincidence, the program rotates the candidate ligand about the two pairs of groups and checks for steric hindrance and coincidence of other candidate atomic groups with appropriate target sites. Particular candidate/orientation combinations that are good geometric fits in the binding site and show sufficient coincidence of atomic groups with GRID sites are retained.

[0266] Consistent with the breadth-first strategy, this approach involves simplifying assumptions. Rigid protein and small molecule geometry is maintained throughout. As a first approximation rigid geometry is acceptable as the energy minimized coordinates of an RTK, in particular an EphB2 deduced structure, as described herein, describe an energy minimum for the molecule, albeit a local one. If the surface residues of the site of interest are not involved in crystal contacts then the crystal configuration of those residues is used merely as a starting point for energy minimization, and potential solution structures for those residues determined. The deduced structure described herein should reasonably mimic the mean solution configuration.

[0267] A further assumption implicit in CLIX is that the potential ligand, when introduced into the binding pocket or site of a receptor, does not induce change in the protein's stereochemistry or partial charge distribution and so alter the basis on which the GRID interaction energy maps were computed. It must also be stressed that the interaction sites predicted by GRID are used in a positional and type sense only, i.e., when a candidate atomic group is placed at a site predicted as favorable by GRID, no check is made to ensure that the bond geometry, the state of protonation, or the partial charge distribution favors a strong interaction between the protein and that group. Such detailed analysis should form part of more advanced modeling of candidates identified in the CLIX shortlist Yet another embodiment of a computer-assisted molecular design method for identifying ligands of a binding pocket of an RTK comprises the de novo synthesis of potential ligands by algorithmic connection of small molecular fragments that will exhibit the desired structural and electrostatic complementarity with an active site or binding pocket of the receptor. The methodology employs a large template set of small molecules with are iteratively pieced together in a model of an RTK active site or binding pocket. Each stage of ligand growth is evaluated according to a molecular mechanics-based energy function, which considers van der Waals and coulombic interactions, internal strain energy of the lengthening ligand, and desolvation of both ligand and receptor. The search space can be managed by use of a data tree which is kept under control by pruning according to the binding criteria In an illustrative embodiment, the search space is limited to consider only amino acids and amino acid analogs as the molecular building blocks. Such a methodology generally employs a large template set of amino acid conformations, though need not be restricted to just the 20 natural amino acids, as it can easily be extended to include other related fragments of interest to the medicinal chemist, e.g. amino acid analogs. The putative ligands that result from this construction method are peptides and peptide-like compounds rather than the small organic molecules that are typically the goal of drug design research. The appeal of the peptide building approach is not that peptides are preferable to organics as potential pharmaceutical agents, but rather that: (1) they can be generated relatively rapidly de novo; (2) their energetics can be studied by well-parameterized force field methods; (3) they are much easier to synthesize than are most organics; and (4) they can be used in a variety of ways, for peptidomimetic ligand design, protein-protein binding studies, and even as shape templates in the more commonly used 3D organic database search approach described above.

[0268] Such a de novo peptide design method has been incorporated in a software package called GROW (Moon et al. (1991) Proteins 11:314-328). In a typical design session, standard interactive graphical modeling methods are employed to define the structural environment in which GROW is to operate. For instance, environment could be an active site binding pocket of an RTK, in particular an EphB2, or it could be a set of features on the protein's surface to which the user wishes to bind a peptide-like molecule. The GROW program then operates to generate a set of potential ligand molecules. Interactive modeling methods then come into play again, for examination of the resulting molecules, and for selection of one or more of them for further refinement.

[0269] To illustrate, GROW operates on an atomic coordinate file generated by the user in the interactive modeling session, such as the coordinates provided in Table 3, or the coordinates of a binding pocket or active site as described in Table 2 and 3 plus a small fragment (e.g., an acetyl group) positioned in the active site to provide a starting point for peptide growth. These are referred to as “site” atoms and “seed” atoms, respectively. A second file provided by the user contains a number of control parameters to guide the peptide growth (Moon et al. (1991) Proteins 11:314-328).

[0270] The operation of the GROW algorithm is conceptually fairly simple. GROW proceeds in an iterative fashion, to systematically attach to the seed fragment each amino acid template in a large preconstructed library of amino acid conformations. When a template has been attached, it is scored for goodness-of-fit to the receptor site or binding pocket, and then the next template in the library is attached to the seed. After all the templates have been tested, only the highest scoring ones are retained for the next level of growth. This procedure is repeated for the second growth level; each library template is attached in turn to each of the bonded seed/amino acid molecules that were retained from the first step, and is then scored. Again, only the best of the bonded seed/dipeptide molecules that result are retained for the third level of growth. The growth of peptides can proceed in the N-to-C direction only, the reverse direction only, or in alternating directions, depending on the initial control specifications supplied by the user. Successive growth levels therefore generate peptides that are lengthened by one residue. The procedure terminates when the user-defined peptide length has been reached, at which point the user can select from the constructed peptides those to be studied further. The resulting data provided by the GROW procedure includes not only residue sequences and scores, but also atomic coordinates of the peptides, related directly to the coordinate system of the receptor site atoms.

[0271] In yet another embodiment, potential pharmacophoric compounds can be determined using a method based on an energy minimization-quenched molecular dynamics algorithm for determining energetically favorable positions of functional groups in the binding pockets of the subject receptor. The method can aid in the design of molecules that incorporate such functional groups by modification of known ligands or de novo construction.

[0272] For example, the multiple copy simultaneous search method (MCSS) described by Miranker et al. (1991) Proteins 11: 29-34 may be employed. To determine and characterize a local minima of a functional group in the forcefield of the protein, multiple copies of selected functional groups are first distributed in a binding pocket of interest on the RTK protein. Energy minimization of these copies by molecular mechanics or quenched dynamics yields the distinct local minima. The neighborhood of these minima can then be explored by a grid search or by constrained minimization. In one embodiment, the MCSS method uses the classical time dependent Hartee (TDH) approximation to simultaneously minimize or quench many identical groups in the forcefield of the protein.

[0273] Implementation of the MCSS algorithm requires a choice of functional groups and a molecular mechanics model for each of them. Groups must be simple enough to be easily characterized and manipulated (3-6 atoms, few or no dihedral degrees of freedom), yet complex enough to approximate the steric and electrostatic interactions that the functional group would have in binding to the pocket or site of interest in the RTK protein. A preferred set is, for example, one in which most organic molecules can be described as a collection of such groups (Patai's Guide to the Chemistry of Functional Groups, ed. S. Patai (New York: John Wiley, and Sons, (1989)). This includes fragments such as acetonitrile, methanol, acetate, methyl ammonium, dimethyl ether, methane, and acetaldehyde.

[0274] Determination of the local energy minima in the binding pocket or site requires that many starting positions be sampled. This can be achieved by distributing, for example, 1,000-5,000 groups at random inside a sphere centered on the binding site; only the space not occupied by the protein needs to be considered. If the interaction energy of a particular group at a certain location with the protein is more positive than a given cut-off (e.g. 5.0 kcal/mole) the group is discarded from that site. Given the set of starting positions, all the fragments are minimized simultaneously by use of the TDH approximation (Elber et al. (1990) J Am Chem Soc 112: 9161-9175). In this method, the forces on each fragment consist of its internal forces and those due to the protein. The essential element of this method is that the interactions between the fragments are omitted and the forces on the protein are normalized to those due to a single fragment. In this way simultaneous minimization or dynamics of any number of functional groups in the field of a single protein can be performed.

[0275] Minimization is performed successively on subsets of, for example 100, of the randomly placed groups. After a certain number of step intervals, such as 1,000 intervals, the results can be examined to eliminate groups converging to the same minimum. This process is repeated until minimization is complete (e.g. RMS gradient of 0.01 kcal/mole/C). Thus the resulting energy minimized set of molecules comprises what amounts to a set of disconnected fragments in three dimensions representing potential pharmacophores.

[0276] The next step then is to connect the pharmacophoric pieces with spacers assembled from small chemical entities (atoms, chains, or ring moieties). In a preferred embodiment, each of the disconnected can be linked in space to generate a single molecule using such computer programs as, for example, NEWLEAD (Tschinke et al. (1993) J Med Chem 36: 3863, 3870). The procedure adopted by NEWLEAD executes the following sequence of commands (1) connect two isolated moieties, (2) retain the intermediate solutions for further processing, (3) repeat the above steps for each of the intermediate solutions until no disconnected units are found, and (4) output the final solutions, each of which is a single molecule. Such a program can use for example, three types of spacers: library spacers, single-atom spacers, and fuse-ring spacers. The library spacers are optimized structures of small molecules such as ethylene, benzene and methylamide. The output produced by programs such as NEWLEAD consist of a set of molecules containing the original fragments now connected by spacers. The atoms belonging to the input fragments maintain their original orientations in space. The molecules are chemically plausible because of the simple makeup of the spacers and functional groups, and energetically acceptable because of the rejection of solutions with van-der Waals radii violations.

[0277] A screening method of the present invention may comprise the following steps:

[0278] (i) generating a computer model of a binding pocket using a crystal according to the invention;

[0279] (ii) docking a computer representation of a test compound with the computer model;

[0280] (iii) analysing the fit of the compound in the binding pocket.

[0281] In an aspect of the invention, a method is provided comprising the following steps:

[0282] (a) docking a computer representation of a structure of a test compound into a computer representation of a binding pocket of an RTK defined in accordance with the invention using a computer program, or by interactively moving the representation of the test compound into the representation of the binding pocket;

[0283] (b) characterizing the geometry and the complementary interactions formed between the atoms of the binding pocket and the compound; optionally

[0284] (c) searching libraries for molecular fragments which can fit into the empty space between the compound and the binding pocket and can be linked to the compound; and

[0285] (d) linking the fragments found in (c) to the compound and evaluating the new modified compound.

[0286] In an embodiment of the invention, a method is provided which comprises the following steps:

[0287] (a) docking a computer representation of a test compound from a computer data base with a computer representation of a selected binding pocket on an RTK defined in accordance with the invention to define a complex;

[0288] (b) determining a conformation of the complex with a favorable fit and favourable complementary interactions; and

[0289] (c) identifying test compounds that best fit the selected binding pocket as potential modulators of the RTK.

[0290] In another embodiment of the invention, a method is provided which comprises docking a computer representation of a selected binding pocket of an RTK defined by the atomic interactions, atomic contacts, or structural coordinates in accordance with the invention to define a complex. In particular a method is provided comprising:

[0291] (a) docking a computer representation of a test compound from a computer database with a computer representation of a selected binding pocket of an RTK defined by the atomic interactions, atomic contacts, or structural coordinates described herein;

[0292] (b) determining a conformation of the complex with a favorable fit and favourable complementary interactions; and

[0293] (c) identifying test compounds that best fit the selected binding pocket as potential modulators of the RTK.

[0294] A model used in a screening method may comprise a binding pocket either alone or in association with one or more ligands and/or cofactors. For example, the model may comprise the binding pocket in association with a nucleotide (or analogue thereof), a substrate (or analogue thereof), and/or modulator.

[0295] If the model comprises an unassociated binding pocket, then the selected site under investigation may be the binding pocket itself. The test compound may, for example, mimic a known ligand (e.g. nucleotide or substrate) for an RTK in order to interact with the binding pocket The selected site may alternatively be another site on the RTK.

[0296] If the model comprises an associated binding pocket, for example a binding pocket in association with a ligand, the selected site may be the binding pocket or a site made up of the binding pocket and the complexed ligand, or a site on the ligand itself. The test compound may be investigated for its capacity to modulate the interaction with the associated molecule.

[0297] The screening methods described herein may be applied to a plurality of test compounds, to identify those that best fit the selected site. The screening methods may be used to identify a modulator that changes an autoinhibited state of an RTK to an active state, or an active state to an autoinhibited state.

[0298] A test compound (or plurality of test compounds) may be selected on the basis of their similarity to a known ligand for an RTK, in particular an Eph receptor. For example, the screening method may comprise the following steps:

[0299] (i) generating a computer model of a binding pocket in complex with a ligand;

[0300] (ii) searching for a test compound with a similar three dimensional structure and/or similar chemical groups as the ligand; and

[0301] (iii) evaluating the fit of the test compound in the binding pocket

[0302] Searching may be carried out using a database of computer representations of potential compounds, using methods known in the art.

[0303] The present invention also provides a method for designing ligands for RTKs. It is well known in the art to use a screening method as described above to identify a test compound with promising fit, but then to use this test compound as a starting point to design a ligand with improved fit to the model. Such techniques are known as “structure-based ligand design” (See Kuntz et al., 1994, Acc. Chem. Res. 27:117; Guida, 1994, Current Opinion in Struc. Biol. 4: 777; and Colman, 1994, Current Opinion in Struc. Biol. 4: 868, for reviews of structure-based drug design and identification; and Kuntz et al 1982, J. Mol. Biol. 162:269; Kuntz et al., 1994, Acc. Chem. Res. 27: 117; Meng et al., 1992, J. Compt. Chem. 13: 505; Bohm, 1994, J. Comp. Aided Molec. Design 8: 623 for methods of structure-based modulator design).

[0304] Examples of computer programs that may be used for structure-based ligand design are CAVEAT (Bartlett et al., 1989, in “Chemical and Biological Problems in Molecular Recognition”, Roberts, S. M. Ley, S. V.; Campbell, N. M. eds; Royal Society of Chemistry: Cambridge, pp 182-196); FLOG (Miller et al., 1994, J. Comp. Aided Molec. Design 8:153); PRO Modulator (Clark et al., 1995 J. Comp. Aided Molec. Design 9:13); MCSS (Miranker and Karplus, 1991, Proteins: Structure, Fuction, and Genetics 8:195); and, GRID (Goodford, 1985, J. Med. Chem. 28:849).

[0305] The method may comprise the following steps:

[0306] (i) docking a model of a test compound with a model of a binding pocket;

[0307] (ii) identifying one or more groups on the test compound which may be modified to improve their fit in the binding pocket;

[0308] (iii) replacing one or more identified groups to produce a modified test compound model; and

[0309] (iv) docking the modified test compound model with the model of the binding pocket.

[0310] Evaluation of fit may comprise the following steps:

[0311] (a) mapping chemical features of a test compound such as by hydrogen bond donors or acceptors, hydrophobic/lipophilic sites, positively ionizable sites, or negatively ionizable sites; and

[0312] (b) adding geometric constraints to selected mapped features.

[0313] The fit of the modified test compound may then be evaluated using the same criteria.

[0314] The chemical modification of a group may either enhance or reduce hydrogen bonding interaction, charge interaction, hydrophobic interaction, Van Der Waals interaction or dipole interaction between the test compound and the key amino acid residue(s) of the binding pocket. Preferably the group modifications involve the addition removal or replacement of substituents onto the test compound such that the substituents are positioned to collide or to bind preferentially with one or more amino acid residues that correspond to the key amino acid residues of the binding pocket.

[0315] If a modified test compound model has an improved fit, then it may bind to a binding pocket and be considered to be a “ligand”. Rational modification of groups may be made with the aid of libraries of molecular fragments which may be screened for their capacity to fit into the available space and to interact with the appropriate atoms. Databases of computer representations of libraries of chemical groups are available commercially, for this purpose.

[0316] The test compound may also be modified “in situ” (i.e. once docked into the potential binding pocket), enabling immediate evaluation of the effect of replacing selected groups. The computer representation of the test compound may be modified by deleting a chemical group or groups, or by adding a chemical group or groups. After each modification to a compound, the atoms of the modified compound and potential binding pocket can be shifted in conformation and the distance between the modulator and the binding pocket atoms may be scored on the basis of geometric fit and favourable complementary interactions between the molecules. This technique is described in detail in Molecular Simulations User Manual, 1995 in LUDI.

[0317] Examples of ligand building and/or searching computer programs include programs in the Molecular Simulations Package (Catalyst), ISIS/HOST, ISIS/BASE, and ISIS/DRAW (Molecular Designs Limited), and UNITY (Tripos Associates).

[0318] The “starting point” for rational ligand design may be a known ligand for the enzyme. For example, in order to identify potential modulators of an RTK, in particular an Eph receptor, a logical approach would be to start with a known ligand (for example a nucleotide or known kinase inhibitors) to produce a molecule which mimics the binding of the ligand. Such a molecule may, for example, act as a competitive inhibitor for the true ligand, or may bind so strongly that the interaction (and inhibition) is effectively irreversible.

[0319] Such a method may comprise the following steps:

[0320] (i) generating a computer model of a binding pocket in complex with a ligand;

[0321] (ii) replacing one or more groups on the ligand model to produce a modified ligand; and

[0322] (iii) evaluating the fit of the modified ligand in the binding pocket.

[0323] The replacement groups could be selected and replaced using a compound construction program which replaces computer representations of chemical groups with groups from a computer database, where the representations of the compounds are defined by structural coordinates.

[0324] In an embodiment, a screening method is provided for identifying a ligand of an RTK, in particular an Eph receptor, comprising the step of using the structural coordinates of a nucleotide or component thereof, defined in relation to its spatial association with a binding pocket of the invention, to generate a compound that is capable of associating with the binding pocket In an embodiment of the invention, a screening method is provided for identifying a ligand of an RTK, in particular an Eph receptor, comprising the step of using the structural coordinates of adenosine adenine, or ATP listed in Table 3 to generate a compound for associating with a binding pocket of RTK, in particular an Eph receptor as described herein. The following steps are employed in a particular method of the invention: (a) generating a computer representation of adenosine adenine, or ATP, defined by its structural coordinates listed in Table 3; (b) searching for molecules in a data base that are structurally or chemically similar to the defined adenosine adenine, or ATP, using a searching computer program, or replacing portions of the adenosine adenine, or ATP with similar chemical structures from a database using a compound building computer program.

[0325] A screening method is provided for identifying a ligand of an RTK, in particular an Eph receptor, comprising the step of using the structural coordinates of a binding pocket comprising a juxtamembrane region or part thereof listed in Table 3 to generate a compound for associating with a kinase domain of an RTK, in particular an Eph receptor. The following steps are employed in a particular method of the invention: (a) generating a computer representation of a binding pocket comprising a juxtamembrane region or part thereof defined by its structural coordinates listed in Table 3; and (b) searching for molecules in a data base that are structurally or chemically similar to the defined binding pocket using a searching computer program, or replacing portions of the binding pocket with structures from a database using a compound building computer program.

[0326] The screening methods of the present invention may be used to identify compounds or entities that associate with a molecule that associates with an RTK, in particular an Eph receptor (for example, a nucleotide).

[0327] Compounds and entities (e.g. ligands) of RTKs, in particular Eph receptors, identified using the above-described methods may be prepared using methods described in standard reference sources utilized by those skilled in the art. For example, organic compounds may be prepared by organic synthetic methods described in references such as March, 1994, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, New York, McGraw Hill.

[0328] Test compounds and ligands which are identified using a crystal or model of the present invention can be screened in assays such as those well known in the art. Screening may be for example in vitro, in cell culture, and/or in vivo. Biological screening assays preferably centre on activity-based response models, binding assays (which measure how well a compound binds to a binding pocket of a receptor), and bacterial, yeast, and animal cell lines (which measure the biological effect of a compound in a cell). The assays may be automated for high throughput screening in which large numbers of compounds can be tested to identify compounds with the desired activity. The biological assay may also be an assay for the binding activity of a compound that selectively binds to the binding pocket compared to other receptors.

[0329] Ligands/Compounds Identified by Screening Methods

[0330] The present invention provides a ligand or compound identified by a screening method of the present invention. A ligand or compound may have been designed rationally by using a model according to the present invention. A ligand or compound identified using the screening methods of the invention may specifically associate with a target compound, or part thereof (e.g. a binding pocket). In the present invention the target compound may be the RTK (e.g. Eph receptor) or part thereof, or a molecule that is capable of associating with the RTK or part thereof (for example a nucleotide). In an embodiment, the ligand is capable of binding to phosphoregulatory sites of a binding pocket, in particular phosphoregulatory sites of a juxtamembrane region or kinase domain. In another embodiment, the ligand is capable of binding to the activation segment of a kinase domain of an Eph receptor.

[0331] A ligand or compound identified using a screening method of the invention may act as a “modulator”, i.e. a compound which affects the activity of an RTK in particular an Eph receptor. A modulator may reduce, enhance or alter the biological function of an RTK, in particular an Eph receptor. For example a modulator may modulate the capacity of the RTK to autophosphorylate. An alteration in biological function may be characterised by a change in specificity. For example, a modulator may cause the RTK to accept a different nucleotide, to phosphorylate a different amino acid residue, or to work with a different metal cofactor. A modulator may dispose an RTK to favor the autoinhibited state or active state. In order to exert its function, the modulator commonly binds to a binding pocket.

[0332] A “modulator” which is capable of reducing the biological function of the enzyme may also be known as an inhibitor. Preferably an inhibitor reduces or blocks the capacity of the enzyme to autophosphorylate. An inhibitor may promote the autoinhibition state of an RTK. The inhibitor may mimic the binding of a nucleotide or substrate, for example, it may be a nucleotide or substrate analogue. A nucleotide analogue may be designed by considering the interactions between the nucleotide and the RTK (for example, by using information derivable from the crystal of the invention) and specifically altering one or more groups (as described above).

[0333] The present invention also provides a method for modulating the activity of an RTK, in particular an Eph receptor, using a modulator according to the present invention. The invention also provides a method for inhibiting autophosphorylation of an RTK, preferably an Eph receptor, by potentiating the autoinhibition state of an RTK, or inhibiting the active state of the RTK. Inhibition of phosphorylation of an RTK may decrease signaling by the RTK and inhibit cellular processes that may be involved in disease. It would be possible to monitor receptor activity following such treatments by a number of methods known in the art.

[0334] A modulator may be an agonist, partial agonist, partial inverse agonist or antagonist of an RTK.

[0335] As used herein, the term “agonist” means any ligand, which is capable of binding to a binding pocket and which is capable of increasing a proportion of the receptor that is in an active form, resulting in an increased biological response. The term includes partial agonists and inverse agonists.

[0336] As used herein, the term “partial agonist” means an agonist that is unable to evoke the maximal response of a biological system, even at a concentration sufficient to saturate the specific receptors.

[0337] As used herein, the term “partial inverse agonist” is an inverse agonist that evokes a submaximal response to a biological system, even at a concentration sufficient to saturate the specific receptors. At high concentrations, it will diminish the actions of a full inverse agonist.

[0338] As used herein, the term “antagonist” means any agent that reduces the action of another agent, such as an agonist. The antagonist may act at the same site as the agonist (competitive antagonism). The antagonistic action may result from a combination of the substance being antagonised (chemical antagonism) or the production of an opposite effect through a different receptor (functional antagonism or physiological antagonism) or as a consequence of competition for the binding site of an intermediate that links receptor activation to the effect observed (indirect antagonism).

[0339] As used herein, the term “competitive antagonism” refers to the competition between an agonist and an antagonist for a binding pocket of a receptor that occurs when the binding of agonist and antagonist becomes mutually exclusive. This may be because the agonist and antagonist compete for the same binding sites or pockets, or combine with adjacent but overlapping sites. A third possibility is that different sites are involved but that they influence the receptor macromolecules in such a way that agonist and antagonist molecules cannot be bound at the same time. If the agonist and antagonist form only short lived combinations with a binding pocket of a receptor so that equilibrium between agonist, antagonist and receptor is reached during the presence of the agonist, the antagonism will be surmountable over a wide range of concentrations. In contrast, some antagonists, when in close enough proximity to their binding site, may form a stable covalent bond with it and the antagonism becomes insurmountable when no spare receptors remain.

[0340] As mentioned above, an identified ligand or compound may act as a ligand model (for example, a template) for the development of other compounds. A modulator may be a mimetic of a ligand.

[0341] Like the test compound (see above) a modulator may be one or a variety of different sorts of molecule. (See examples herein.) A modulator may be an endogenous physiological compound, or it may be a natural or synthetic compound. The modulators of the present invention may be natural or synthetic. The term “modulator” also refers to a chemically modified ligand or compound.

[0342] The technique suitable for preparing a modulator will depend on its chemical nature. For example, peptides can be synthesized by solid phase techniques (Roberge J Y et al (1995) Science 269: 202-204) and automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perlin Elmer) in accordance with the instructions provided by the manufacturer. Once cleaved from the resin, the peptide may be purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures and Molecular Principles, W H Freeman and Co, New York N.Y.). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra).

[0343] If a modulator is a nucleotide, or a polypeptide expressable therefrom, it may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers M H et al (1980) Nuc Acids Res Symp Ser 215-23, Horn T et al (1980) Nuc Acids Res Symp Ser 225-232), or it may be prepared using recombinant techniques well known in the art.

[0344] Organic compounds may be prepared by organic synthetic methods described in references such as March, 1994, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, New York, McGraw Hill.

[0345] The invention also relates to classes of modulators of RTKs based on the structure and shape of a nucleotide, or component thereof, or a substrate or component thereof; defined in relation to the nucleotide's or substrate's spatial association with a crystal structure of the invention or part thereof.

[0346] A class of modulators may comprise a compound containing a structure of adenine, adenosine, ribose, pyrophosphate, or ATP, and having one or more, preferably all, of the structural coordinates of adenine, adenosine, ribose, pyrophosphate, or ATP of Table 4. Functional groups in the adenine, adenosine, ribose, pyrophosphate, or ATP modulators may be substituted with, for example, alkyl, alkoxy, hydroxyl, aryl, cycloalkyl, alkenyl, alkynyl, thiol, thioalkyl, thioaryl, amino, or halo, or they may be modified using techniques known in the art.

[0347] Another class of modulators defined by the invention are compounds comprising an adenine triphosphate group having the structural coordinates of adenine triphosphate in the active site binding pocket of an Eph receptor.

[0348] The invention contemplates all optical isomers and racemic forms of the modulators of the invention.

[0349] Pharmaceutical Composition

[0350] The present invention also provides for the use of a modulator according to the invention, in the manufacture of a medicament to treat and/or prevent a disease in a mammalian patient. There is also provided a pharmaceutical composition comprising such a modulator and a method of treating and/or preventing a disease comprising the step of administering such a modulator or pharmaceutical composition to a subject, preferably a mammalian patient.

[0351] The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise a pharmaceutically acceptable carrier, diluent, excipient, adjuvant or combination thereof.

[0352] Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R1 Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as—or in addition to—the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).

[0353] Preservatives, stabilizers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

[0354] The routes for administration (delivery) include, but are not limited to, one or more of: oral (e.g. as a tablet, capsule, or as an ingestable solution), topical, mucosal (e.g. as a nasal spray or aerosol for inhalation), nasal, parenteral (e.g. by an injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal, buccal, vaginal, epidural, sublingual.

[0355] Where the pharmaceutical composition is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.

[0356] Where appropriate, the pharmaceutical compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, gel, hydrogel, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose or chalk, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

[0357] If the agent of the present invention is administered parenterally, then examples of such administration include one or more of intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrastemally, intracranially, intramuscularly or subcutaneously administering the agent; and/or by using infusion techniques.

[0358] For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

[0359] The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

[0360] Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elxirs, the agent may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

[0361] As indicated, a therapeutic agent (e.g. modulator) of the present invention can be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134 μm) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA™), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the agent and a suitable powder base such as lactose or starch.

[0362] Therapeutic administration of polypeptide modulators may also be accomplished using gene therapy. A nucleic acid including a promoter operatively linked to a heterologous polypeptide may be used to produce high-level expression of the polypeptide in cells transfected with the nucleic acid. DNA or isolated nucleic acids may be introduced into cells of a subject by conventional nucleic acid delivery systems. Suitable delivery systems include liposomes, naked DNA, and receptor-mediated delivery systems, and viral vectors such as retroviruses, herpes viruses, and adenoviruses.

[0363] Applications

[0364] The invention provides a method for inhibiting kinase activity of an RTK comprising maintaining the RTK or a binding pocket thereof involved in regulating the kinase domain in an autoinhibited state, or potentiating an autoinhibited state for the RTK or binding pocket thereof involved in regulating the kinase domain. An autoinhibited state may be maintained or potentiated by inhibiting phosphorylation of phosphoregulatory sites of the juxtamembrane segment and/or kinase domain (e.g. activation segment). Inhibition may be accomplished using modulators, or altering the structure of a binding pocket of the RTK comprising the phosphoregulatory sites, to prevent phosphorylation of the sites.

[0365] The invention contemplates a method for altering the stability of an autoinhibited state of an RTK comprising phosphorylating phosphoregulatory sites of a juxtamembrane region of the RTK.

[0366] In an aspect the invention relates to a method for changing an RTK from an autoinhibited state to an active state comprising phosphorylating phosphoregulatory sites of a juxtamembrane region of the RTK.

[0367] In another aspect the invention provides a method for activating kinase activity of an RTK comprising phosphorylating phosphoregulatory sites of a juxtamembrane region and kinase domain (e.g. activation segment) of the RTK.

[0368] The invention further provides a method of treating a mammal, the method comprising administering to a mammal a modulator or pharmaceutical composition of the present invention.

[0369] In particular, the invention contemplates a method of treating or preventing a condition or disease associated with an RTK in a cellular organism, comprising:

[0370] (c) administering a modulator of the invention in an acceptable pharmaceutical preparation; and

[0371] (d) activating or inhibiting the RTK to treat or prevent the disease.

[0372] In an aspect the invention provides a method for treating or preventing a condition or disease involving increased RTK activity comprising maintaining the RTK or a binding pocket thereof involved in regulating the kinase domain of the RTK in an autoinhibited state. An autoinhibited state may be maintained as described herein. In an embodiment the condition or disease is cancer.

[0373] The invention provides for the use of a modulator identified by the methods of the invention in the preparation of a medicament to treat or prevent a disease in a cellular organism. Use of modulators of the invention to manufacture a medicament is also provided.

[0374] Typically, a physician will determine the actual dosage of a modulator or pharmaceutical composition of the invention that will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient and severity of the condition. There can, of course, be individual instances where higher or lower dosage ranges are merited.

[0375] The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. By way of example, the pharmaceutical composition of the present invention may be administered in accordance with a regimen of 1 to 10 times per day, such as once or twice per day.

[0376] For oral and parenteral administration to human patients, the daily dosage level of the agent may be in single or divided doses.

[0377] The modulators and compositions of the invention may be useful in the prevention and treatment of conditions involving aberrant RTKs.

[0378] Conditions which may be prevented or treated in accordance with the invention include but are not limited to lymphoproliferative conditions, malignant and pre-malignant conditions, arthritis, inflammation, and autoimmune disorders. Malignant and pre-malignant conditions may include solid tumors, B cell lymphomas, chronic lymphocytic leukemia, chronic myelogenous leukemia, prostate hypertrophy, Hirschsprung disease, glioblastoma, breast and ovarian cancer, adenocarcinoma of the salivary gland, premyelocytic leukemia, prostate cancer, multiple endocrine neoplasia type IIA and IIB, medullary thyroid carcinoma, papillary carcinoma, papillary renal carcinoma, hepatocellular carcinoma, gastrointestinal stromal tumors, sporadic mastocytosis, acute myeloid leukemia, large cell lymphoma or Alk lymphoma, chronic myeloid leukemia, hematological/solid tumors, papillary thyroid carcinoma, stem cell leukemia/lymphoma syndrome, acute myelogenous leukemia, osteosarcoma, multiple myeloma, preneoplastic liver foci, and resistance to chemotherapy. Diseases associated with increased cell survival, or the inhibition of apoptosis, include cancers (e.g. follicular lymphomas, carcinomas with p53 mutations, hormone-dependent tumors such as breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as lupus erythematosus and immune-related glomerulonephritis rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses, and adenoviruses); inflammation, graft vs. host disease, acute graft rejection and chronic graft rejection.

[0379] Eph receptors and ephrins mediate contact-dependent repulsive guidance of migrating cells and axons in culture and in vivo. Many Eph family members are prominently expressed in the developing nervous system, and epbrin stimulation of growing primary axons in vitro results in axonal retraction or repulsion, characterized by a collapse of actin-rich growth cone structures at the leading edge of the cell. Mice bearing homozygous null mutations in EphA8 or in both EphB2 and EphB3 exhibit abnormal migration of axon tracts in the brain. Ephrin-induced retraction of exploratory actin filopodia has also been described in vivo in migrating Eph receptor-expressing neural crest cells.

[0380] The Eph receptors and ephrins have also been implicated in cell sorting and boundary formation. Eph-receptor signaling is able to modulate both cell-cell and cell-substrate attachment. Bidirectional Eph receptor-ephrin signaling is important for the formation of boundaries between rhombomeres of the hind brain. These cellular responses to Eph receptor stimulation indicate that they may regulate signaling events which control cytoskeletal architecture and cell adhesion functions.

[0381] Therefore, modulators of Eph receptors may be used to modulate axonogenesis, nerve cell interactions and regeneration, to treat conditions such as neurodegenerative diseases and conditions involving trauma and injury to the nervous system, for example Alzheimer's disease, Parkinson's disease, Huntington's disease, demylinating diseases, such as multiple sclerosis, amyotrophic lateral sclerosis, bacterial and viral infections of the nervous system, deficiency diseases, such as Wernicke's disease and nutritional polyneuropathy, progressive supranuclear palsy, Shy Drager's syndrome, multistem degeneration and olivo ponto cerebellar atrophy, peripheral nerve damage, trauma and ischemia resulting from stroke.

[0382] Therapeutic efficacy and toxicity of compositions and modulators of the invention may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED50 (the dose therapeutically effective in 50% of the population) or LD50 (the dose lethal to 50% of the population) statistics. The therapeutic index is the dose ratio of therapeutic to toxic effects and it can be expressed as the ED50/LD50 ratio. Pharmaceutical compositions that exhibit large therapeutic indices are preferred.

[0383] The invention will now be illustrated by the following non-limiting example:

EXAMPLE

[0384] The following methods were used in the investigation described in the example:

[0385] Methods

[0386] Protein Expression and Purification

[0387] Mutagenesis of the juxtamembrane tyrosines (Y604/61° F.) of murine EphB2 was performed using a PCR-based approach. The amplified cDNA sequence, corresponding to the receptor's juxtamembrane region and kinase domain (residues 595-906), was cloned into pGEX-4T-1 (Pharmacia). The glutathione-S transferase (GST)-EphB2 construct was transformed into Escherichia coli B834 cells and the cells grown in minimal media supplemented with selenomethionine, with overnight induction at 15° C., and 0.15 mM IPTG (isopropyl-β-D-thiogalactopyranoside, BioShop). Cells were lysed by homogenization and sonication in 25 mM HEPES (pH 7.5), 50 mM NaCl, 20% glycerol, 2 mM DTT, 2 mM phenyl-methyl sulphonyl fluoride. Purification of the selenomethionyl derivative of EphB2 was performed as previously described (Binns et al., 2000), with the exception that the buffer used for gel filtration (buffer C) was 10 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM DTT.

[0388] Crystallization, Data Collection, and Structure Determination

[0389] Hanging drops containing 1 μl of 12.5 mg/ml protein in buffer C were mixed with equal volumes of reservoir buffer containing 0.1 M HEPES (pH 7.0), 0.2 M magnesium chloride, 10% (w/v) PEG 4000, 10% (v/v) isopropanol, and 15% (v/v) ethylene glycol. Rod-like crystals were obtained overnight at 28° C. after streak seeding with smaller crystals obtained initially. The crystals belong to primitive space group P21, (a=47.86 Å, b=98.09 Å, c=68.18 Å, α=γ=90°, β=104.97°), with two molecules of EphB2 in the asymmetric unit. Crystals were flash frozen by immersion in liquid nitrogen. A MAD experiment was performed on a frozen crystal at APS beamline BM 14-D (λ1=0.9790 Å, λ2=0.9788 Å, λ3=0.9770 Å) using a Quantum 4 ADSC CCD detector. Data processing and reduction was carried out with the HKL program suite (Otwinowski and Minor, 1997). The programs SHARP (La Fortelle and Bricogne, 1997) and SnB (Miller et al., 1994) were used in combination to locate and subsequently refine positions for 22 of the possible 30 Se sites. Following density modification with Solomon (Abrahams and Leslie, 1996), a partial model was generated using 0 (Jones et al., 1991) and refined using CNS (Brunger et al., 1998) (R-factors>40%). Consequently, crystals of EphB2 in complex with 2 μM AMP-PNP were grown as described above (space group —P1, a=47.05 Å, b=57.62 Å, c=67.74 Å, α=112.95°, β=103.170, γ=91.58°), with two molecules per asymmetric unit Diffraction data was collected to 1.9 Å at APS beamline BM 14-C (λ=1.00 Å) using a Quantum 4 ADSC CCD detector and processed with the HKL program suite. Molecular replacement solutions were determined with AMoRe (Navaza, 1994; CCP4, 1994), using one monomer of the P21-derived model as a search molecule. The two AMoRe solutions, which correspond to the two EphB2 molecules in the asymmetric unit, refined readily in CNS. With minimal modification to the starting model, the model has been refined to a working R value of 24.1% and a free R value of 27.7%. As defined in PROCHECK (Laskowski et al., 1993), 90.8% of protein residues are in the most favored regions of the Ramachandran plot, with none in the disallowed regions. Pertinent statistics for data collection and refinement are shown in Table 1.

[0390] Mutagenesis

[0391] The cDNA sequence of the juxtamembrane region and kinase domain of murine EphA4 (amino acids 591-896), corresponding to residues 599-906 of murine EphB2, was cloned into pGEX-4T-2 (Pharmacia). The murine EphB2 numbering scheme was employed, and the corresponding EphA4 residue numbers are listed in parentheses. Using a PCR-based approach, Tyr 604 (Tyr596) and Tyr 610 (Tyr602) were mutated to phenylalanine. The following site-directed mutants were then generated using this doubly mutated construct: (1) ΔJXall; deletion of 599-621 (591-613), (2) ΔJX1; deletion of 599-606 (591-598), (3) ΔJX1+2; deletion of 599-610 (591-602), (4) Pro607Gly (Pro599Gly), (5) Phe608Asp (Phe600Asp), (6) Phe620Asp (Phe612Asp), (7) Ser680Trp (Ser672Trp), (8) ΔJX1+2 plus Phe620Asp, and (9) Tyr604/610 Glu (Tyr596/602Glu). The GST-EphA4 constructs were transformed into E. coli BL21 codon plus cells and grown in LB supplemented with ampicillin, with overnight induction at 15° C., 0.15 mM IPTG. Purification was performed as described for EphB2. The mutations Tyr604Phe, Tyr610Phe, Pro607Gly, Phe620Asp, Ser680Trp, Gln684Trp, deletion of 599-606 (ΔJX1), deletion of 599-610 (ΔJX1+2), and deletion of 600-621 (ΔJX1all) in murine EphB2 were generated by site-directed mutagenesis using overlapping oligonucleotide primers containing the above indicated point mutations or deletions. All mutations were confirmed by DNA sequencing.

[0392] Western Blotting

[0393] GST-EphA4 proteins expressed in E. coli (BL21 codon plus), and EphB2 proteins transiently expressed in COS-1 cells, were harvested as previously described (Binns et al, 2000; Holland et al, 1997). Proteins were resolved using 12% denaturing polyacrylamide gel electrophoresis (PAGE), transferred onto a polyvinylidene difluoride membrane (Millipore), blotted with anti-pTyr (Upstate Biotechnology), anti-GST (Santa Cruz Laboratories), or anti-EphB2 antibodies (Holland et al., 1997), and visualized using enhanced chemiluminescence (ECL Plus; Amersham).

[0394] In Vitro Kinase Reactions

[0395] In vitro kinase reactions using GST fusion EphA4 proteins bound to glutathione sepharose or immunoprecipitated EphB2 proteins transiently expressed in COS-1 cells were performed with 5 μg and 2 μg of acid-denatured enolase, respectively, and 5 μCi of [γ32P]ATP at room temperature as previously described (Binns et al., 2000).

[0396] Spectrophotometric Coupling Assay

[0397] Kinetic analysis of the bacterial expressed EphA4 proteins was performed using a coupled in vitro spectrophotometric kinase assay where production of ADP is coupled to the oxidation of NADH through pyruvate kinase and lactic dehydrogenase (Barker et al., 1995; Binns et al, 2000). The 100-μl reaction volume contained 1 U lactic dehydrogenase, 1 U pyruvate kinase, 1 mM phosphoenolpyruvate, 0.2 mM NADH, and 0.5 mM ATP (in 20 mM MgCl2, 0.1 mM DTT, 60 mM HEPES [pH 7.5], 20 μg/mL bovine serum albumin). Wild type and mutant EphA4 activity was measured by monitoring absorbance at 340 nm (Varian UV-Visible spectrophotometer) for 90 minutes at a fixed enzyme concentration (0.5 μM) and 1 mM S-1 synthetic peptide (GEEIYGEFD; amide at carboxy terminus) concentrations. For accuracy, protein concentrations were determined by UV spectrometry at 280 nm using molar extinction coefficients. (Andersson, 1998; Collaborative Computational Project, 1994).

[0398] Results and Discussions

[0399] Structure Determination

[0400] Since the expression of active EphB2 polypeptides in E. coli is toxic, efforts were focused on the catalytically repressed Tyr 604/610 Phe double mutant. For the purposes of discussion, these sites are referred to as Tyr/Phe 604 and Tyr/Phe 610. A cytoplasmic fragment (residues 595 to 906) of the murine EphB2 RTK consisting of the latter half of the juxtamembrane region and the entire catalytic domain was expressed as a GST fusion in E. coli and purified to homogeneity (see Methods). The predicted boundaries of the juxtamembrane region are residues 573-620, while those of the kinase and SAM domains are residues 621-892 and residues 919-994, respectively. Protein crystals of two different space groups were grown and the EphB2 structure was determined using a combination of seleno-methionine multiwavelength anomalous dispersion (SeMet MAD) and molecular replacement (MR) methods (see Methods). The EphB2 crystal structure reported here corresponds to the juxtamembrane-catalytic domain fragment in complex with AMP-PNP (β,γ-imidoadenosine-5′-triphosphate). Overall, the EphB2 structure is well ordered except for the first seven and last six amino acid residues, kinase domain residues 651 to 653 connecting β-strands 2 and 3 of the N-terminal catalytic lobe, and residues 774 to 796 corresponding to the kinase activation segment within the C-terminal lobe. Only the adenine ring of AMP-PNP is ordered in experimental and model based electron density maps, and hence the sugar and phosphate groups have not been modeled. Data collection and refinement statistics are listed in Table 1 and a representative alignment of the EphB2 receptor and other protein kinase family members is provided in FIG. 1.

[0401] Overall Description of the Autoinhibited Structure

[0402] The structure of the catalytic domain of EphB2 conforms to that generally observed for protein kinases, consisting of two lobes, a smaller N-terminal lobe and larger C-terminal lobe (FIGS. 2a,b). Protein kinases are capable of a range of conformations owing to an inherent inter-lobe flexibility that allows for both open and closed conformations. However, the catalytically competent conformation is generally a closed structure in which the two catalytic lobes clamp together to form an interfacial nucleotide binding site and catalytic cleft. Surprisingly, the autoinhibited EphB2 catalytic domain adopts a closed conformation that resembles an ‘active’ state.

[0403] The N-terminal lobe of protein kinases consists minimally of a twisted 5-strand β-sheet (denoted β1 to β5 as first described for the cAMP dependent protein kinase (cAPK) and a single helix αC (Knighton et al., 1991). The N-terminal lobe functions to assist in the binding and coordination of ATP for the productive transfer of the y-phosphate to a substrate oriented by the C-terminal lobe. In this regard, β-strands 1 and 2 and the glycine rich connecting segment (g-loop) form a flexible flap that interacts with the adenine base, ribose sugar and the non-hydrolyzable phosphate groups of ATP. Furthermore, an invariant salt bridge between a lysine side chain (sub-domain 2 in the protein kinase nomenclature of Hanks et al., 1988) in β-strand 3 and a glutamic acid side chain (sub-domain 3) in helix αC coordinates the β-phosphate of ATP. In the EphB2 crystal structure, all N-terminal lobe elements implicated in nucleotide binding are well ordered and adopt a prototypical protein kinase arrangement. However, distortions in helix αC and the g-loop arising from interactions with the juxtamembrane segment are evident.

[0404] The C-terminal lobe of protein kinases consists minimally of two β-strands (β7 and β8) and a series of α-helices (αD to αI). Strands β7 and β8 locate to the cleft region between the N- and C-terminal lobes where they contribute side chains that participate in catalysis and the binding of magnesium for the coordination of ATP phosphate groups. In the EphB2 crystal structure, all lower lobe residues implicated in catalysis and ATP coordination appear optimally oriented (FIG. 3c). The activation segment, which is also located in the large catalytic lobe, is disordered as in several other protein kinase structures in which the activation segment is not phosphorylated (reviewed by Johnson et al., 1996). The remaining C-terminal lobe elements, including α-helices αD to αI, are well ordered and adopt the prototypical protein kinase configuration. Terminating the catalytic domain structure is a short helix αJ.

[0405] The EphB2 juxtamembrane region preceding the catalytic domain is highly ordered and adopts an identical conformation in the four unique environments sampled in the two different crystal forms studied. From the amino-terminus, the conformation consists of an extended strand segment Ex1, a single turn 3/10 helix αA′, and a four-turn helix αB′. These elements associate intimately with helix αC of the N-terminal catalytic lobe and also make limited interactions with the C-terminal lobe. As a consequence of the association of the juxtamembrane segment with the N-terminal kinase lobe, significant curvature is imposed on helix αC. This distortion couples directly to local distortions in other N-terminal lobe elements, most critically the g-loop and the invariant lysine-glutamate salt bridge. Together the N-terminal lobe distortions appear to impinge on catalytic function by adversely affecting the coordination of the sugar and phosphate groups of the bound nucleotide.

[0406] With limited contacts to the lower lobe of the catalytic domain, the juxtamembrane segment also sterically impedes the activation segment from adopting the productive conformation that typifies the active state of protein-serine/threonine and tyrosine kinases. Together, the effects on nucleotide coordination and the activation segment form the basis for autoinhibition of EphB2 by the juxtamembrane segment.

[0407] Depending on the splice variant of EphB2, there are 29-45 juxtamembrane residues between the start of strand Ex1 (Lys 602) and the plasma membrane (Connor and Pasquale, 1995). This relatively lengthy sequence makes it impossible to predict whether the autoinhibited structure observed here would be oriented in a specific fashion with respect to the inner surface of the membrane.

[0408] Detailed Analysis of Juxtamembrane Structure

[0409] The juxtamembrane strand segment Ex1, corresponding to amino acid residues Lys 602 to lie 605, extends along the cleft region between the N- and C-terminal lobes (FIGS. 2c,d). The phosphoregulatory residue Tyr/Phe 604 orients into a solvent-exposed hydrophobic pocket composed of the side chains of Met 748 and Tyr 750 of the C-terminal kinase lobe, Ile 681 and Phe 685 from helix αC and Pro 607 from the juxtamembrane helix αA′. This site has been termed ‘switch region 1’ since Tyr/Phe 604 appears well placed to influence the association of the juxtamembrane region with the catalytic domain. Further stabilizing the interaction of strand Ex1 with the lower catalytic lobe are hydrogen bonds between the amide group of Tyr/Phe 604 and the carbonyl group of Met 748 and between the side chain of Gln 684 and the backbone amide and carbonyl groups of lie 605.

[0410] Helix αA′ is composed of a single rigid turn initiated by an Asp606Pro607 sequence and terminated by Thr 609. This helix appears stabilized by the conformational rigidity of Pro 607 and the capping interactions involving the side chains of Asp 606 and Thr 609 with the free backbone amino group and carbonyl groups of Phe 608 and Asp 606. A short linker and then a three-turn helix αB′, initiated by Asp 612Pro613 and extending to Phe 620, follow helix αA′. Helix αB′ is also initiated by an Asp Pro sequence (residues 612 and 613) and Asp 612 makes similar capping interactions with the backbone amino and side chain of Asn 614. Helices αA′ and αB′ form an interface with the N-terminal lobe of the kinase that centers on helix αC. Hydrophobic side chains projecting from αA′ and αB′ include Pro 607, Phe 608, Pro 613, Val 617, Phe620 and Ala 621. These residues associate intimately with Arg 673, Leu 676, and Ile 681 from helix αC and Leu 693 and Val 696 from α-strand 4. In addition, a hydrogen bond interaction (2.9 Å) is observed between Asn 614 and Arg 672 (FIG. 2c), and the small side chains at positions 616 (Ala), 677 (Ser) and 680 (Ser) facilitate the close packing of helices αA′, αB′ and αC.

[0411] Opposite to, but contiguous with, the site of association with helix αC, strand Ex1 and helices αA′ and αB′ form an interface composed primarily of hydrophobic interactions. The side chain of the phosphoregulatory residue Tyr/Phe 610 projects onto the surface of this site and appears well positioned to exert an influence on the local juxtamembrane structure. This interface, termed ‘switch region 2’, is composed of the side chains of Ile 605 from strand Ex1 and the side chains of Ala 616 and Phe 620 from helix αB′.

[0412] Effect of the Juxtamembrane Engagement on the N-Terminal Lobe Structure

[0413] Comparison of the EphB2 crystal structure with that of the ‘active’ triply phosphorylated insulin receptor tyrosine kinase (active IRK (Hubbard, 1997)) indicates the mechanism by which the juxtamembrane region of EphB2 inhibits the catalytic domain. Superposition of C-terminal kinase lobe elements places the majority of N-terminal lobe elements into close correspondence (FIGS. 3a-d). A distinguishing feature of the EphB2 structure is a 14° kink midway along helix αC centered at Glu 678. This kink, which coincides with the site of association with the juxtamembrane elements Ex1, αA′ and αB′, displaces the forward facing N-terminus of helix αC 6.8 Å upward and outward from the equivalent position observed in IRK (FIGS. 3a,c). Stabilizing this kink internally are side chain/main chain interactions involving Ser 677 and Ser 680.

[0414] The kink in helix αC places its forward projecting terminus in close proximity to β-strands 3, 4, and 5, forming a tighter interface than that observed in active IRK (FIG. 3b). Residues participating in this interface include Arg 672, Phe 675, and Leu 676 from helix αC, Tyr 667 from the β3/αC linker and Leu 663, Val 696, Thr 698, Val 703, and Ile 705 from the β-strands. Interestingly, tyrosine 667, which is centrally positioned within this interface and is highly conserved amongst the Eph receptor family members, has been identified as an in vivo site of phosphorylation (Kalo and Pasquale, 1999), suggesting a possible phosphoregulatory role.

[0415] The close association of helix αC with β-strands 3, 4 and 5 is achieved with a local alteration to the twist of the forward projecting termini of β-strands 1, 2 and 3 that leaves the bulk of the N-terminal sheet structure unperturbed. The g-loop side chain Phe 640 plays a role in coupling the β-strand movements to that of helix αC through a direct interaction with Phe 675. The altered twist of the β1, β2 and β3-strand termini displace main chain atoms at the end of the g-loop (Glu 639 and Phe 640) by approximately 3.3 Å. In addition, together with the kink in helix αC, the altered twist of the β-strands displaces the invariant glutamate and lysine side chains by 2.4 and 2.1 Å, respectively, relative to their positions in active IRK (FIG. 3c). As a consequence, the ability of the catalytic domain to coordinate the sugar and phosphate groups of bound nucleotide is compromised (FIGS. 3a-c). Since the domain closure and the bulk of the N-terminal β-sheet structure is not perturbed, the adenine binding pocket is well formed and indeed the adenine base of bound AMP-PNP is ordered and orients in a manner similar to that in the crystal structure of active IRK.

[0416] Steric Influence of the Juxtamembrane Region on the Activation Segment

[0417] While the majority of interactions between the juxtamembrane segment and the catalytic domain are directed towards the N-terminal lobe, strand Ex1 forms a limited set of interactions with the C-terminal lobe that may serve a regulatory role. Superposition of EphB2 with active IRK illustrates how the side chain of the phosphoregulatory residue Tyr/Phe 604 impedes the activation segment from adopting a productive conformation (FIG. 3d). In autoinhibited EphB2, the side chain of Tyr 750 adopts an alternate conformation from that of the corresponding residue Phe 1128 in active IRK. This avoids a steric clash with the side chain of Tyr/Phe604. The alternate conformation of Tyr 750, in turn, impedes the activation segment from adopting a path observed in active IRK due to a steric clash with Ser 776 (Thr 1154 in IRK). Interestingly, the side chain conformation of Tyr 750 in EphB2, Tyr 382 in Src and Hck, and Phe 1128 in IRK all correlate with their activation segments adopting non-productive conformations. This may be indicative of a more general function in protein kinases for position 750 in regulating the conformation of the activation segment.

[0418] The Phosphoregulatory Switch

[0419] The ability to oscillate between catalytically active and repressed states in a regulated manner is the key to the function of protein kinases as versatile molecular switches. In EphB2, EphA4, and most likely Eph RTKs in general, the switch to an active state is coordinated by phosphorylation at highly conserved sites within both the juxtamembrane region and the catalytic domain. The mechanism by which phosphorylation at sites within the activation segment stimulate protein kinases is relatively well understood (reviewed by Johnson et al., 1996) and by inference, phosphorylation of EphB2 at Tyr 788 likely promotes the ordering of the activation segment to a catalytically competent conformation.

[0420] In contrast, phosphorylation at Tyr/Phe 604 and 610 may serve to destabilize the juxtamembrane structure and cause it to dissociate from the catalytic domain. This would allow for a return of the N-terminal lobe to an undistorted active conformation.

[0421] The EphB2 crystal structure helps to explain how phosphorylation at each of the two phosphoregulatory sites could destabilize the juxtamembrane structure and cause its release from the catalytic domain. The environment around each of the two switch regions is hydrophobic, but solvent exposed, and thus could accommodate either tyrosine or phenylalanine at positions 604 and 610 with little or no reorganization of the juxtamembrane structure. However, substitution with phosphotyrosine appears less tolerable due to steric and electrostatic clashes involving the bulky anionic phosphate group. In ‘switch region 1’, the phosphorylation of Tyr/Phe 604 would place a phosphate group within van der Waals contact of Asp 606, Pro 607 and Ile 681. Furthermore, the side chain of Asp 606 dominates the electrostatic environment around Tyr/Phe 604 such that the introduction of a phosphate group would generate repulsive electrostatic forces (FIG. 4). The electrostatic environment around ‘switch region 2’ is also dominated by negatively charged amino acids, namely Asp 606, Glu 611, Asp 612, Glu 615, and Glu 619. Thus, phosphorylation of Tyr 610 would also generate repulsive electrostatic forces, which are likely essential for the expulsion of this residue from its binding pocket since a phosphate group could be accommodated sterically.

[0422] Three other highly conserved tyrosine residues have been identified as in vivo phosphorylation sites in EphB2 and EphB5, namely tyrosines 667, 744 and 750 (FIG. 3c). Although their roles in regulating Eph receptor kinase activity have not been probed by mutagenesis, all three sites appear well positioned to influence the stability of the autoinhibited structure and hence Eph receptor activity (FIG. 3). For example, phosphorylation of Tyr 667 could promote a catalytically competent state by destabilizing the tight association of helix αC with β-strands 3, 4 and 5 observed in the autoinhibited state. In addition, phosphorylation of Tyr 744 and Tyr 750, which line the cleft region through which the juxtamembrane strand Ex1 navigates, could amplify the effect of phosphorylation at Tyr 604.

[0423] Function of the EphA4 Juxtamembrane Segment Probed by Mutagenesis

[0424] Previously, a cytoplasmic fragment of the EphA4 receptor tyrosine kinase, consisting of the juxtamembrane segment, the catalytic domain and the SAM domain, has been shown to require autophosphorylation for maximal activation (Binns et al., 2000). The importance of autophosphorylation was revealed by a lag period at the start of in vitro kinase reactions employing the dephosphorylated form of the EphA4 enzyme. This lag period was greatly reduced by pre-incubation of the EphA4 fragment with ATP or by deletion of the entire juxtamembrane segment. In contrast, mutation to phenylalanine of either Tyr 604 or Tyr 610 reduced the specific activity of the enzyme, while mutation of both sites in tandem drastically impaired catalytic function (<10% relative to WT). These results are consistent with the mechanism of autoinhibition suggested by the EphB2 crystal structure.

[0425] In order to test the crystallographic findings and to probe the regulation of Eph receptor catalytic activity in more detail, additional site-directed mutations were generated in the full-length murine EphB2 receptor expressed in COS-1 cells and in a murine EphA4 receptor fragment expressed in bacteria, corresponding in content to the EphB2 construct used for the structure determination. For the sake of discussion, the murine EphB2 numbering scheme has been employed for all mutants and the corresponding EphA4 residue numbers are listed in parentheses. Each mutation was generated in the catalytically repressed Tyr 604/610 Phe double mutant background and was tested for its ability to restore catalytic function. The mutations include a small N-terminal deletion of residues 595 to 606 (ΔJX1) encompassing strand Ex1 and the first phosphoregulatory site, an intermediate N-terminal deletion of residues 599 to 610 (ΔJX1+2) that encompasses strand Ex1, the first phosphoregulatory site, helix αA′ and the second phosphoregulatory site, and a full juxtamembrane segment deletion of residues 599 to 621 (ΔJXall). In addition, six separate point mutations were generated in both the juxtamembrane region and the kinase domain (Pro607Gly, Phe608Asp, Phe620Asp, Tyr604/610Glu, Ser680Trp, Gln684Trp) that were predicted to destabilize the interaction of the kinase domain with the juxtamembrane segment. Lastly, the ΔJX1+2 mutation was combined with the Phe620Asp mutation (ΔJX1+2 plus Phe620Asp) and the Ser680Trp mutation was combined with the Gln684Trp mutation (Ser680Trp/Gln684Trp). The Tyr604/610Phe double mutant and the wild type proteins were analyzed concomitantly as reference points for the fully repressed (0%) and active (100%) states, respectively. The activities of the EphA4 proteins expressed in bacteria were tested for their ability to induce protein tyrosine phosphorylation in vivo (FIG. 5a), and to autophosphorylate and to phosphorylate enolase in vitro (FIG. 5b). EphA4 proteins were also tested for their ability to phosphorylate a peptide substrate using a continuous spectophotometric assay (FIG. 5c). Lastly, full-length EphB2 proteins expressed in COS-1 cells were tested for their ability to autophosphorylate in vivo and to autophosphorylate and phosphorylate enolase in vitro (FIG. 5d).

[0426] The two partial N-terminal juxtamembrane deletions when introduced into the EphA4 construct significantly increased kinase activity in all four assays, restoring catalytic function as measured by the spectrophotometric assay to 136% and 216% of wild-type activity in the case of ΔJX1 and the ΔJX1+2 deletions, respectively. A similar effect was observed for the ΔJX1+2 deletion introduced into full-length EphB2.

[0427] Mutation of Phe 608 in EphA4, which locates to helix αA′, gave very weak restoration of catalytic function. This result is consistent with the variability of position 608 amongst the Eph receptor family members (42% identity). In contrast, mutation in both EphA4 and EphB2 constructs of the highly conserved Pro 607 (95% identity), which initiates helix αA′, to Gly greatly enhanced catalytic function in all four assays, quantitated at 122% of wild-type activity by the spectrophotometric assay. This result is consistent with a role for Pro607, suggested by the crystal structure, in stabilizing helix αA′ by imposing conformational rigidity, or in promoting the association of juxtamembrane and N-terminal kinase lobe elements through hydrophobic interactions. Similarly, mutation of the highly conserved Phe 620 (95% identity) at the terminus of helix aB′ to Asp also restored catalytic function in the four assays tested. Phe 620 is notable because it contributes to the hydrophobic pocket into which the phosphoregulatory residue Tyr/Phe 610 binds; its substitution with Asp is predicted to disrupt the hydrophobic interaction with Tyr/Phe 610, and to clash electrostatically with the surrounding negatively charged groups in a manner mimicking phosphorylation of Tyr/Phe 610.

[0428] The introduction of point mutations into the kinase domain at the interface with the juxtamembrane region also restored catalytic function. Mutation of Ser680 (82% identity) to Trp in both EphA4 and EphB2 constructs gave modest restoration with the phosphorylation of peptide substrate being restored to 41% of wild-type activity. Mutation of the absolutely conserved Gln684 (100% identity) to Trp in EphB2 resulted in a greater increase in kinase activity, as did the double mutation Ser 680Trp/Gln684Trp. Both mutations map to helix αC and are predicted to sterically perturb the association of the juxtamembrane region with the N-terminal catalytic lobe.

[0429] Robust restoration of activity was also observed for the EphA4 and EphB2 mutants ΔJXall, Tyr604/610Glu, and ΔJX1+2 plus Phe620Asp, although the relative restoration as measured by the various assays differed to a small degree. The restoration of activity by the ΔJX1 mutant confirms that the juxtamembrane segment is not absolutely required for kinase function, the restoration by the Tyr604/610 Glu mutation suggests that the addition of negative charges at positions 604 and 610 is an important component of juxatmembrane destabilization and the relief of autoinhibition. Lastly, the finding that none of the EphB2 mutants are as active as the wild-type enzyme may indicate that these mutants have perturbed some aspect of the oligomerization event that is needed for maximal activation of the full-length receptor.

[0430] Overall, the mutagenesis results support a model for the regulation of receptor catalytic function by the juxtamembrane segment, shown in FIG. 6. Strand Ex1 and helix αA′ of the juxtamembrane segment contribute to the inhibitory effect on the catalytic domain, and the release of these elements from their association with the catalytic domain is a requirement for catalytic activation. Physiologically, this would be accomplished by phosphorylation at the Tyr 604 and 610 regulatory sites and potentially at additional sites. The strong conservation of residues involved in the inhibitory interaction suggests that this regulatory mechanism is conserved for all Eph receptor family members.

[0431] Comparison of Autoinhibitory Mechanisms of EphB2 and TGFβR1 Receptor Kinase

[0432] Analysis of the TGFβR1 serine/threonine kinase has revealed a role for the juxtamembrane Gly/Ser/Thr-rich motif (“GS segment”) in regulating catalytic activity. As with Eph receptor tyrosine kinases, TGFβR1 kinases require phosphorylation at sites within the juxtamembrane segment for subsequent phosphorylation of target Smad proteins (Macias-Silva et al, 1996). The regulatory mechanism revealed by the X-ray crystal structure of a cytoplasmic fragment of TGFβR1 in complex with FKBP12 (Huse et al, 1999) shows some parallels to EphB2. In both structures, the intramolecular engagement of the juxtamembrane segment induces conformational distortions in the catalytic domain that impinge on kinase function. In addition, the induced distortions impact on the relative positioning and/or conformation of helix αC. Beyond these similarities, however, the inhibitory mechanisms, including the mode of juxtamembrane association with the catalytic domain and the resulting basis for inhibition, diverge. Perhaps the most significant difference relates to the potential involvement of FKBP12 in stabilizing the inhibited structure of TGFβR1, whereas EphB2 achieves an autoinhibited state independently. Nonetheless, the data for EphB2 indicate that receptor tyrosine kinases and receptor serine/threonine kinases have in some cases converged on a related regulatory mechanism in which the juxtamembrane region inhibits the kinase domain in the inactive state, and is potentially liberated to interact with downstream targets upon autophosphorylation.

[0433] Discussion

[0434] Why does EphB2 employ a rather complex mechanism of autoregulation, involving the non-catalytic juxtamembrane region? One possible benefit may be to block any potential signaling activity intrinsic to the juxtamembrane sequence. In particular, phosphorylation of tyrosines 604 and 610 in EphB2 creates docking sites for SH2 domain proteins. Sequestering these tyrosines decreases their chance of becoming adventitiously phosphorylated and thereby inappropriately transmitting a signal through the recruitment of downstream targets. The coordination of kinase activation with the release of binding sites for targets is reminiscent of Src family cytoplasmic tyrosine kinase, in which the SH2 and SH3 domains engage internal ligands in a fashion that both inhibits the activity of the kinase domain and hinders interactions of the SH2 and SH3 domains with other binding partners (Sicheri et al., 1997; Xu et al., 1997).

[0435] The involvement of the juxtamembrane sequence in autoregulation of EphB2 activity may also set a phosphorylation threshold that must be exceeded to induce receptor activation. Full stimulation of Eph receptors apparently requires autophosphorylation at multiple sites within both the activation segment and juxtamembrane region. The use of at least two distinct phosphoregulatory steps may preclude inappropriate Eph receptor activation resulting from basal levels of kinase activity. Since Eph receptors have powerful biological activities during embryogenesis and postnatally, their aberrant activation would be expected to have severe phenotypic consequences, which could be avoided by requiring multi-site phosphorylation of the receptor.

[0436] Are the Eph receptors unique among RTKs in employing cytoplasmic elements outside the catalytic domain to regulate kinase activity? A variety of data obtained for the platelet-derived growth factor β receptor (PDGFR), the closely related colony stimulating factor-1 receptor (c-Fms), stem cell factor receptor (Kit), and the Flt3 receptor raise the possibility that this may in fact be a more widespread phenomenon. Biochemical analysis and mutagenesis of the PDGFR-β has suggested that autophosphorylation of juxtamembrane tyrosines 579 and 581 is required for stimulation of receptor kinase activity by PDGF, potentially by allowing subsequent phosphorylation of tyrosine 857 in the activation segment (Baxter et al., 1998). Conversion of these juxtamembrane tyrosines to phenylalanine inhibits receptor activation, while their phosphorylation creates a binding site for the Src SH2 domain, resulting in Src recruitment to the receptor. Thus, autophosphorylation within the juxtamembrane region of the PDGFR-β may couple receptor activation to the exposure of SH2 domain-binding sites, as appears to be the case for Eph receptors. Consistent with the notion that the juxtamembrane region of the PDGFR-β exerts an inhibitory influence on kinase activity, substitution of a valine residue, just N-terminal to the regulatory tyrosines, results in constitutive receptor activation in vitro and in vivo (Irusta and DiMaio, 1998). In addition to the PDGFR-β, the juxtamembrane regions of c-Fms (Myles et al., 1994), Kit, and Flt3 receptors have been implicated in regulation of tyrosine kinase activity. Oncogenic variants of Kit identified in human and murine mast cell leukemias carry either amino acid substitutions or deletions in the juxtamembrane region, which result in constitutive activation of the kinase domain (Tsujimura et al., 1996)(see FIG. 1). Remarkably a majority of human gastrointestinal stromal tumors (GIST) have activating Kit mutations that introduce substitutions or deletions into a short segment of the juxtamembrane region, and are strongly implicated in the etiology of these tumors (Hirota et al, 1998; Nakahara et al; 1998; Anderson, 1998). Furthermore, approximately 20% of acute myeloid leukemias have internal tandem duplications of Flt3 that create in-frame insertions of variable length in the juxtamembrane region, leading to ligand-independent kinase activity and oncogenic acitvation (Nakao et al, 1996; Yokota et al, 1997; Hayakawa et al, 2000). Thus, Kit and Flt3 juxtamembrane regions may repress kinase activity, and juxtamembrane mutations that relieve this inhibition can result in human cancers.

[0437] A similar situation may pertain for the insulin receptor, which upon activation becomes autophosphorylated within the juxtamembrane region and consequently binds targets such as IRS-1 and ShcA, which possess PTB domains. Kinetic analysis of wild type and mutant insulin receptors has suggested that the insulin receptor juxtamembrane region acts as an intrasteric inhibitor to block the kinase domain active site, in a fashion that is relieved by autophosphorylation of juxtamembrane tyrosines (Cann et al., 2000).

[0438] Many RTKs have C-terminal tails that upon activation become phosphorylated at SH2/PTB domain-binding sites. Structural analysis of the Tie2/Tek receptor cytoplasmic region has indicated that in the inactive state the tail interacts with the kinase domain in a way that partially occludes the C-terminal tyrosines and the peptide binding site (Shewchuk et al., 2000). This raises the possibility that autophosphorylation of the Tie2 tail causes a conformational change that exposes both C-terminal phosphotyrosine sites as well as the substrate binding site of the kinase domain.

[0439] Thus the juxtamembrane and C-terminal segments of RTKs may play a pivotal role in regulating the kinase domain, and in coordinating enzymatic activation with the exposure of motifs that bind cytoplasmic targets.

[0440] In addition to revealing an unexpected level of complexity in the regulation of RTKs, these observations have interesting implications for the design of RTK inhibitors. The structure of the Ab1 kinase bound to the inhibitor STI-571 suggests that this compound binds selectively to the inactive form of the kinase (Schindler et al., 2000). The unusual structure of autoinhibited EphB2 suggests the possibility of isolating inhibitors that bind specifically to the inactive conformation of the kinase. Indeed, if this mode of intrasteric regulation is a more common feature of RTKs, this might be a general strategy for the identification of selective RTK inhibitors.

[0441] The structure of EphB2 reveals an entirely novel mechanism for RTK autoregulation.

[0442] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biology or related fields are intended to be within the scope of the following claims.

1TABLE 1Data collection, structure determination and refinement statisticsSeMet MAD AnalysisNativeλ1 = 0.9790 Åλ2 = 0.9788 Åλ3 = 0.9770 Å(AMP-PNP)SpacegroupP21P21P21P1Resolution (Å)2.32.32.31.9Reflections total/unique185034/52252188170/52750186452/52855 80035/43865Completeness (%)*97.5 (91.2)97.6 (91.7)97.6 (90.8)90.3 (55.9)Rsym (%)*† 7.4 (26.2) 7.5 (27.9) 8.5 (38.7) 3.3 (14.6)<I/σ>*15.6 (4.0) 16.1 (3.9) 14.9 (3.1) 20.9 (4.1) Phasing Power‡ 0/2.59 2.71/4.11 2.08/3.40—(ISO/ANO)Refinement (AMP-PNP complex)Resolution Range (Å)30-1.9Average B value (Å2)24.9ReflectionsRmsd for B values (Å2)1.42all data42903Rmsd for bonds (Å)0.007|F|>2σ39931Rmsd for angles (°)1.09Rfactor/Rfree(%)**Number of non-hydrogen protein atoms4311all data24.1/27.7Number of non-hydrogen nucleotide atoms20|F|>2σ23.2/26.9Number of water molecules263*Numbers given in parentheses refer to data for the highest resolution shell. † Rsym = 100 × Σ|I-<I>|/Σ<I>, where I is the observed intensity and <I> is the average intensity from multiple observations of symmetry-related reflections. ‡ Phasing power for isomorphous and anomalous acentric reflections, where Ppower = <[|Fh(calc)/phase-integrated lack of closure]>. **Free R-value was calculated with 8.7% of the data.

[0443]

2

TABLE 2

Intermolecular contacts of the Juxtamembrane Region

and Kinase Domain of an Eph Receptor

No.

Kinase

of

Domain/

Atom-

Juxtamembrane
Distance

ic
Juxtamembrane
Region
Between
Atomic

Inter-
Region
Atomic
Atomic
Interaction

action
Atomic Contact
Contact
Contacts
Property

1
Phe/Tyr 604 CB
Met 748 CE
4.58
hydrophobic

2
Phe/Tyr 604 N
Met 748 O
2.83
H-bond

3
Phe/Tyr 604 CD2
Tyr 750 CD1
3.78
Hydrophobic

4
Phe/Tyr 604 CE2
Tyr 750 CE1
4.12
Hydrophobic

5
Phe/Tyr 604 CD1
Phe 685 CE2
4.06
Hydrophobic

6
Ile 605 N
Gln 684 OE1
2.83
H-bond

7
Ile 605 O
Gln 684 NE2
3.00
H-bond

8
Phe/Tyr 604 CE1
Gln 684 CD
4.11
van der Waal

9
Pro 607 CD
Ile 681 CG1
3.85
Hydrophobic

10
Pro 607 CB
Ser 680 OG
3.16
van der Waal

11
Phe 608 CZ
Asp 674 OD1
3.27
van der Waal

12
Phe 608 CZ
Ser 677 CB
4.35
van der Waal

13
Phe 608 CE2
Arg 673 CG
3.79
Hydrophobic

14
Pro 613 CB
Arg 673 CD
3.60
Hydrophobic

15
Asn 614 OD1
Arg 672 NH1
2.87
H-bond

16
Val 617 CG2
Leu 676 CD1
4.69
Hydrophobic

17
Val 617 CG2
Ser 680 CB
4.15
Hydrophobic

18
Val 617 CG2
Leu 676 CB
4.10
Hydrophobic

19
Ala 621 CB
Leu 693 CD2
3.98
Hydrophobic

20
Phe 620 CD1
Gln 684 CG
3.60
Hydrophobic

21
Phe 620 CE1
Gln 684 CD
3.77
Hydrophobic

22
Phe 620 CB
Gln 683 O
4.15
van der Waal

23
Phe 620 O
Gln 683 O
3.41
H-bond

24
Ala 616 CA
Ser 680 CB
3.8
Hydrophobic

25
Tyr/Phe 604 CE2
Asp 606 CB
4.26
Hydrophobic

26
Pro 607 CD
Asp 606 OD1
3.28
van der Waal

27
Asp 606 O
Thr 609 OG1
2.72
Hydrogen bond

28
Asp 606 O
Thr 609 N
2.90
Hydrogen bond

29
Asp 606 CB
Thr 609 CG2
3.56
Hydrophobic

30
Phe 604 CZ
Pro 607 CD
3.90
Hydrophobic

31
Pro 607 O
Phe 610 N
3.08
Hydrogen bond

32
Phe 608 CD2
Pro 613 CG
4.43
Hydrophobic

33
Phe 610 CE1
Ile 605 CG2
3.48
Hydrophobic

34
Phe 610 CZ
Phe 620 CE1
3.91
Hydrophobic

35
Phe 610 CE1
Ala 616 CB
3.91
Hydrophobic

36
Phe 610 CD2
Glu 615 CG
3.74
Hydrophobic

37
Asp 612 O
Glu 615 N
2.73
Hydrogen bond

38
Phe 608 N
Asp 606 OD1
2.83
Hydrogen

Bond

39
Asp 612 OD1
Asn 614 ND2
3.09
Hydrogen bond

40
Asp 612 OD1
Asn 614 N
3.11
Hydrogen bond

41
Asn 614 O
Arg 618 N
3.16
Hydrogen bond

42
Pro 613 O
Val 617 N
3.59
Weak hydrogen

bond, van der

Waal

43
Glu 615 O
Glu 619 N
3.06
Hydrogen

Bond

44
Glu 615 OE1
Glu 619 OE1
2.64
Hydrogen

Bond

45
Phe 620 N
Ala 616 O
2.96
Hydorgen

Bond

46
Glu 619 OE2
Phe 620 CZ
4.03
Van der waals

47
Ala 621 N
Val 617 O
2.91
Hydrogen

Bond

48
Ala 621 O
Val 617 O
3.25
Hydrogen

Bond

49
Ala 621 CB
Val 617 CG1
4.16
Hydrophobic

[0444]

3

TABLE 3

REMARK coordinates from minimization and B-factor refinement

□

REMARK refinement resolution: 30 - 1.9 A

REMARK starting r= 0.2330 free_r= 0.2672

REMARK final r= 0.2316 free_r= 0.2691

REMARK rmsd bonds= 0.007125 rmsd angles= 1.07641

REMARK B rmsd for bonded mainchain atoms= 1.398 target= 1.5

REMARK B rmsd for bonded sidechain atoms= 1.963 target= 2

REMARK B rmsd for angle mainchain atoms= 2.128 target= 2

REMARK B rmsd for angle sidechain atoms= 2.727 target= 2.5

REMARK target= mlf final wa= 1.79025

REMARK final rweight= 0.1000 (with wa= 1.79025)

REMARK md-method= torsion annealing schedule= slowcool

REMARK starting temperature= 3000 total md steps= 30 * 6

REMARK cycles= 2 coordinate steps= 20 B-factor steps= 10

REMARK sg= P1 a= 47.052 b= 57.616 c= 67.742 alpha= 112.949 beta= 103.173 gamma=

91.577

REMARK topology file 1 : CNS_TOPPAR:protein.top

REMARK topology file 2 : CNS_TOPPAR:dna-rna.top

REMARK topology file 3 : CNS_TOPPAR:water.top

REMARK topology file 4 : CNS_TOPPAR:ion.top

REMARK topology file 5 : adenine.top

REMARK parameter file 1 : CNS_TOPPAR:proteinrep.param

REMARK parameter file 2 : CNS_TOPPAR:dna-rna_rep.param

REMARK parameter file 3 : CNS_TOPPAR:water_rep.param

REMARK parameter file 4 : CNS_TOPPAR:ion.param

REMARK parameter file 5 : adenine.par

REMARK molecular structure file: gen_ab.mtf

REMARK input coordinates: ref7b.pdb

REMARK reflection file= ../cycle1/amp.cv

REMARK ncs= none

REMARK B-correction resolution: 6.0 - 1.9

REMARK B-factor correction applied to coordinate arrayB:
0.210

REMARK bulk solvent: density level= 0.37437 e/A{circumflex over ( )}3, B-factor= 62.0599 A{circumflex over ( )}2

REMARK reflections with |Fobs| /sigma_F < 2 rejected

REMARK reflections with |Fobs| > 10000 * rms(Fobs) rejected

REMARK theoretical total number of refl. in resol. range:
49847 (100.0%)

REMARK number of unobserved reflections (no entry or |F|=0):
6944 ( 13.9%)

REMARK number of reflections rejected:
2972 ( 6.0%)

REMARK total number of reflections used:
39931 ( 80.1%)

REMARK number of reflections in working set:
35881 ( 72.0%)

REMARK number of reflections in test set:
4050 ( 8.1%)

CRYST1 47.052 57.616 67.742 112.95 103.17 91.58 P 1

REMARK FILENAME=′ref7c.pdb′

REMARK DATE:18-Jan-01 11:50:14 created by user: groot

REMARK VERSION:1.0

ATOM
1
CB
LYS
A
602
−9.305
−0.312
−16.924
1.00
36.55
A

ATOM
2
CG
LYS
A
602
−9.592
−1.380
−17.964
1.00
40.76
A

ATOM
3
CD
LYS
A
602
−9.801
−2.735
−17.332
1.00
43.15
A

ATOM
4
CE
LYS
A
602
−10.202
−3.766
−18.379
1.00
46.04
A

ATOM
5
NZ
LYS
A
602
−10.292
−5.135
−17.793
1.00
47.27
A

ATOM
6
C
LYS
A
602
−9.501
2.125
−16.413
1.00
30.61
A

ATOM
7
O
LYS
A
602
−8.689
3.021
−16.178
1.00
31.27
A

ATOM
8
N
LYS
A
602
−7.962
1.290
−18.245
1.00
34.40
A

ATOM
9
CA
LYS
A
602
−9.247
1.097
−17.512
1.00
33.41
A

ATOM
10
N
ILE
A
603
−10.653
1.995
−15.761
1.00
26.03
A

ATOM
11
CA
ILE
A
603
−11.041
2.890
−14.680
1.00
21.35
A

ATOM
12
CB
ILE
A
603
−12.110
3.916
−15.127
1.00
23.23
A

ATOM
13
CG2
ILE
A
603
−13.424
3.183
−15.474
1.00
23.72
A

ATOM
14
CG1
ILE
A
603
−12.383
4.899
−13.988
1.00
22.54
A

ATOM
15
CD1
ILE
A
603
−13.398
5.974
−14.316
1.00
27.41
A

ATOM
16
C
ILE
A
603
−11.648
2.050
−13.553
1.00
17.90
A

ATOM
17
O
ILE
A
603
−12.280
1.022
−13.815
1.00
16.74
A

ATOM
18
N
PHE
A
604
−11.460
2.501
−12.313
1.00
12.95
A

ATOM
19
CA
PHE
A
604
−11.981
1.815
−11.122
1.00
14.58
A

ATOM
20
CB
PHE
A
604
−11.309
2.347
−9.848
1.00
13.58
A

ATOM
21
CG
PHE
A
604
−11.978
1.890
−8.569
1.00
10.12
A

ATOM
22
CD1
PHE
A
604
−11.890
0.565
−8.165
1.00
12.14
A

ATOM
23
CD2
PHE
A
604
−12.683
2.785
−7.770
1.00
12.13
A

ATOM
24
CE1
PHE
A
604
−12.493
0.132
−6.972
1.00
12.82
A

ATOM
25
CE2
PHE
A
604
−13.293
2.368
−6.574
1.00
13.41
A

ATOM
26
CZ
PEE
A
604
−13.194
1.036
−6.176
1.00
11.79
A

ATOM
27
C
PHE
A
604
−13.488
2.027
−10.968
1.00
14.34
A

ATOM
28
O
PEE
A
604
−13.972
3.155
−11.068
1.00
14.17
A

ATOM
29
N
ILE
A
605
−14.205
0.946
−10.671
1.00
14.05
A

ATOM
30
CA
ILE
A
605
−15.658
0.985
−10.471
1.00
15.51
A

ATOM
31
CB
ILE
A
605
−16.376
−0.024
−11.404
1.00
14.91
A

ATOM
32
CG2
ILE
A
605
−17.892
0.062
−11.203
1.00
17.40
A

ATOM
33
CG1
ILE
A
605
−16.034
0.269
−12.868
1.00
16.70
A

ATOM
34
CD1
ILE
A
605
−16.412
1.664
−13.326
1.00
20.67
A

ATOM
35
C
ILE
A
605
−15.976
0.616
−9.010
1.00
15.81
A

ATOM
36
O
ILE
A
605
−15.679
−0.491
−8.569
1.00
17.18
A

ATOM
37
N
ASP
A
606
−16.547
1.548
−8.253
1.00
16.32
A

ATOM
38
CA
ASP
A
606
−16.902
1.291
−6.855
1.00
17.50
A

ATOM
39
CB
ASP
A
606
−17.542
2.550
−6.253
1.00
18.47
A

ATOM
40
CG
ASP
A
606
−17.884
2.403
−4.775
1.00
19.43
A

ATOM
41
OD1
ASP
A
606
−17.942
1.262
−4.272
1.00
20.86
A

ATOM
42
OD2
ASP
A
606
−18.114
3.440
−4.115
1.00
20.82
A

ATOM
43
C
ASP
A
606
−17.899
0.128
−6.844
1.00
18.22
A

ATOM
44
O
ASP
A
606
−19.001
0.249
−7.371
1.00
17.14
A

ATOM
45
N
PRO
A
607
−17.517
−1.014
−6.247
1.00
17.74
A

ATOM
46
CD
PRO
A
607
−16.268
−1.278
−5.509
1.00
17.77
A

ATOM
47
CA
PRO
A
607
−18.427
−2.164
−6.209
1.00
18.01
A

ATOM
48
CB
PRO
A
607
−17.621
−3.247
−5.470
1.00
17.22
A

ATOM
49
CG
PRO
A
607
−16.645
−2.465
−4.633
1.00
18.76
A

ATOM
50
C
PRO
A
607
−19.753
−1.836
−5.536
1.00
16.89
A

ATOM
51
O
PRO
A
607
−20.780
−2.404
−5.878
1.00
17.69
A

ATOM
52
N
PEE
A
608
−19.744
−0.897
−4.602
1.00
17.34
A

ATOM
53
CA
PEE
A
608
−20.989
−0.557
−3.946
1.00
19.12
A

ATOM
54
CB
PHE
A
608
−20.738
0.133
−2.613
1.00
18.91
A

ATOM
55
CG
PEE
A
608
−20.319
−0.799
−1.511
1.00
18.72
A

ATOM
56
CD1
PHE
A
608
−20.047
−0.291
−0.251
1.00
18.84
A

ATOM
57
CD2
PEE
A
608
−20.171
−2.166
−1.729
1.00
18.90
A

ATOM
58
CE1
PHE
A
608
−19.632
−1.114
0.776
1.00
20.59
A

ATOM
59
CE2
PHE
A
608
−19.750
−3.011
−0.693
1.00
22.14
A

ATOM
60
CZ
PHE
A
608
−19.482
−2.478
0.559
1.00
19.82
A

ATOM
61
C
PEE
A
608
−21.928
0.292
−4.795
1.00
18.67
A

ATOM
62
O
PHE
A
608
−22.993
0.678
−4.319
1.00
18.02
A

ATOM
63
N
TER
A
609
−21.546
0.609
−6.031
1.00
18.97
A

ATOM
64
CA
THR
A
609
−22.463
1.373
−6.868
1.00
19.12
A

ATOM
65
CB
THR
A
609
−21.748
2.284
−7.911
1.00
17.33
A

ATOM
66
OG1
THR
A
609
−20.955
1.487
−8.799
1.00
17.70
A

ATOM
67
CG2
THR
A
609
−20.886
3.309
−7.216
1.00
18.57
A

ATOM
68
C
THR
A
609
−23.313
0.342
−7.606
1.00
19.52
A

ATOM
69
O
THR
A
609
−24.302
0.683
−8.247
1.00
19.06
A

ATOM
70
N
PHE
A
610
−22.925
−0.928
−7.524
1.00
18.09
A

ATOM
71
CA
PHE
A
610
−23.709
−1.967
−8.181
1.00
19.10
A

ATOM
72
CB
PHE
A
610
−22.955
−3.299
−8.223
1.00
20.69
A

ATOM
73
CG
PHE
A
610
−21.861
−3.357
−9.240
1.00
21.23
A

ATOM
74
CD1
PHE
A
610
−20.707
−2.610
−9.082
1.00
22.59
A

ATOM
75
CD2
PHE
A
610
−21.973
−4.184
−10.350
1.00
23.50
A

ATOM
76
CE1
PHE
A
610
−19.678
−2.681
−10.007
1.00
21.63
A

ATOM
77
CE2
PHE
A
610
−20.942
−4.261
−11.285
1.00
23.79
A

ATOM
78
CZ
PHE
A
610
−19.791
−3.504
−11.107
1.00
20.78
A

ATOM
79
C
PHE
A
610
−25.000
−2.167
−7.386
1.00
20.52
A

ATOM
80
O
PHE
A
610
−24.986
−2.148
−6.150
1.00
20.43
A

ATOM
81
N
GLU
A
611
−26.111
−2.343
−8.095
1.00
20.58
A

ATOM
82
CA
GLU
A
611
−27.404
−2.571
−7.459
1.00
21.53
A

ATOM
83
CB
GLU
A
611
−28.485
−2.853
−8.523
1.00
22.75
A

ATOM
84
CG
GLU
A
611
−28.714
−1.718
−9.518
0.00
23.28
A

ATOM
85
CD
GLU
A
611
−29.783
−2.041
−10.554
0.00
23.79
A

ATOM
86
OE1
GLU
A
611
−30.061
−1.175
−11.409
0.00
24.06
A

ATOM
87
OE2
GLU
A
611
−30.345
−3.158
−10.516
0.00
24.06
A

ATOM
88
C
GLU
A
611
−27.257
−3.790
−6.546
1.00
21.14
A

ATOM
89
O
GLU
A
611
−27.861
−3.857
−5.479
1.00
20.93
A

ATOM
90
N
ASP
A
612
−26.445
−4.746
−6.992
1.00
21.15
A

ATOM
91
CA
ASP
A
612
−26.160
−5.966
−6.239
1.00
21.15
A

ATOM
92
CB
ASP
A
612
−26.738
−7.203
−6.946
1.00
22.41
A

ATOM
93
CG
ASP
A
612
−26.407
−8.504
−6.220
1.00
26.34
A

ATOM
94
OD1
ASP
A
612
−25.869
−8.451
−5.091
1.00
26.08
A

ATOM
95
OD2
ASP
A
612
−26.693
−9.588
−6.776
1.00
28.73
A

ATOM
96
C
ASP
A
612
−24.641
−6.106
−6.114
1.00
20.74
A

ATOM
97
O
ASP
A
612
−23.967
−6.530
−7.051
1.00
18.43
A

ATOM
98
N
PRO
A
613
−24.085
−5.745
−4.948
1.00
22.26
A

ATOM
99
CD
PRO
A
613
−24.796
−5.172
−3.790
1.00
21.09
A

ATOM
100
CA
PRO
A
613
−22.642
−5.825
−4.692
1.00
23.09
A

ATOM
101
CB
PRO
A
613
−22.551
−5.579
−3.188
1.00
23.78
A

ATOM
102
CG
PRO
A
613
−23.662
−4.598
−2.957
1.00
24.95
A

ATOM
103
C
PRO
A
613
−22.001
−7.149
−5.112
1.00
24.21
A

ATOM
104
O
PRO
A
613
−20.830
−7.182
−5.486
1.00
24.14
A

ATOM
105
N
ASN
A
614
−22.764
−8.238
−5.060
1.00
24.32
A

ATOM
106
CA
ASN
A
614
−22.232
−9.544
−5.445
1.00
24.86
A

ATOM
107
CB
ASN
A
614
−23.242
−10.652
−5.162
1.00
28.16
A

ATOM
108
CG
ASN
A
614
−23.520
−10.813
−3.699
1.00
30.99
A

ATOM
109
OD1
ASN
A
614
−22.600
−10.994
−2.903
1.00
33.48
A

ATOM
110
ND2
ASN
A
614
−24.795
−10.750
−3.325
1.00
34.69
A

ATOM
111
C
ASN
A
614
−21.866
−9.598
−6.912
1.00
25.02
A

ATOM
112
O
ASN
A
614
−21.035
−10.412
−7.329
1.00
23.77
A

ATOM
113
N
GLU
A
615
−22.498
−8.742
−7.706
1.00
22.98
A

ATOM
114
CA
GLU
A
615
−22.213
−8.728
−9.129
1.00
23.04
A

ATOM
115
CB
GLU
A
615
−23.144
−7.762
−9.863
1.00
24.89
A

ATOM
116
CG
GLU
A
615
−22.838
−7.681
−11.345
1.00
29.63
A

ATOM
117
CD
GLU
A
615
−22.902
−9.036
−12.032
1.00
33.50
A

ATOM
118
OE1
GLU
A
615
−22.270
−9.188
−13.103
1.00
35.83
A

ATOM
119
OE2
GLU
A
615
−23.589
−9.949
−11.511
1.00
35.71
A

ATOM
120
C
GLU
A
615
−20.766
−8.312
−9.348
1.00
19.65
A

ATOM
121
O
GLU
A
615
−20.079
−8.854
−10.211
1.00
19.95
A

ATOM
122
N
ALA
A
616
−20.306
−7.340
−8.569
1.00
19.06
A

ATOM
123
CA
ALA
A
616
−18.932
−6.884
−8.702
1.00
16.63
A

ATOM
124
CB
ALA
A
616
−18.676
−5.728
−7.763
1.00
16.24
A

ATOM
125
C
ALA
A
616
−17.982
−8.037
−8.401
1.00
15.74
A

ATOM
126
O
ALA
A
616
−16.929
−8.180
−9.031
1.00
15.81
A

ATOM
127
N
VAL
A
617
−18.353
−8.880
−7.447
1.00
15.22
A

ATOM
128
CA
VAL
A
617
−17.493
−10.001
−7.106
1.00
14.30
A

ATOM
129
CB
VAL
A
617
−18.003
−10.750
−5.865
1.00
14.79
A

ATOM
130
CG1
VAL
A
617
−17.028
−11.869
−5.501
1.00
16.38
A

ATOM
131
CG2
VAL
A
617
−18.123
−9.781
−4.703
1.00
11.27
A

ATOM
132
C
VAL
A
617
−17.337
−10.979
−8.256
1.00
14.61
A

ATOM
133
O
VAL
A
617
−16.215
−11.372
−8.608
1.00
14.99
A

ATOM
134
N
ARG
A
618
−18.445
−11.370
−8.868
1.00
16.24
A

ATOM
135
CA
ARG
A
618
−18.353
−12.322
−9.964
1.00
18.27
A

ATOM
136
CB
ARG
A
618
−19.752
−12.808
−10.375
1.00
20.76
A

ATOM
137
CG
ARG
A
618
−20.691
−11.740
−10.838
0.00
21.07
A

ATOM
138
CD
ARG
A
618
−22.044
−12.351
−11.112
0.00
22.19
A

ATOM
139
NE
ARG
A
618
−22.650
−12.891
−9.899
0.00
22.97
A

ATOM
140
CZ
ARG
A
618
−23.853
−13.451
−9.857
0.00
23.42
A

ATOM
141
NH1
ARG
A
618
−24.575
−13.545
−10.965
0.00
23.69
A

ATOM
142
NH2
ARG
A
618
−24.342
−13.903
−8.711
0.00
23.69
A

ATOM
143
C
ARG
A
618
−17.626
−11.746
−11.168
1.00
18.93
A

ATOM
144
O
ARG
A
618
−16.988
−12.479
−11.928
1.00
21.33
A

ATOM
145
N
GLU
A
619
−17.707
−10.430
−11.334
1.00
18.93
A

ATOM
146
CA
GLU
A
619
−17.059
−9.777
−12.463
1.00
20.86
A

ATOM
147
CB
GLU
A
619
−17.728
−8.439
−12.745
1.00
23.33
A

ATOM
148
CG
GLU
A
619
−19.148
−8.576
−13.213
1.00
30.69
A

ATOM
149
CD
GLU
A
619
−19.640
−7.325
−13.876
1.00
34.05
A

ATOM
150
OE1
GLU
A
619
−20.842
−7.271
−14.214
1.00
37.39
A

ATOM
151
OE2
GLU
A
619
−18.821
−6.396
−14.065
1.00
36.23
A

ATOM
152
C
GLU
A
619
−15.564
−9.548
−12.338
1.00
20.77
A

ATOM
153
O
GLU
A
619
−14.829
−9.748
−13.300
1.00
21.23
A

ATOM
154
N
PHE
A
620
−15.113
−9.128
−11.161
1.00
19.38
A

ATOM
155
CA
PHE
A
620
−13.697
−8.841
−10.977
1.00
20.53
A

ATOM
156
CB
PHE
A
620
−13.544
−7.472
−10.326
1.00
19.14
A

ATOM
157
CG
PHE
A
620
−14.366
−6.393
−10.987
1.00
18.99
A

ATOM
158
CD1
PHE
A
620
−15.303
−5.672
−10.258
1.00
18.77
A

ATOM
159
CD2
PHE
A
620
−14.193
−6.091
−12.339
1.00
22.01
A

ATOM
160
CE1
PHE
A
620
−16.061
−4.661
−10.859
1.00
20.15
A

ATOM
161
CE2
PHE
A
620
−14.947
−5.082
−12.951
1.00
20.05
A

ATOM
162
CZ
PHE
A
620
−15.879
−4.369
−12.205
1.00
18.18
A

ATOM
163
C
PHE
A
620
−12.892
−9.871
−10.190
1.00
22.13
A

ATOM
164
O
PHE
A
620
−11.677
−9.719
−10.037
1.00
21.27
A

ATOM
165
N
ALA
A
621
−13.562
−10.915
−9.704
1.00
22.03
A

ATOM
166
CA
ALA
A
621
−12.906
−11.959
−8.922
1.00
23.00
A

ATOM
167
CB
ALA
A
621
−13.365
−11.873
−7.469
1.00
22.11
A

ATOM
168
C
ALA
A
621
−13.159
−13.368
−9.456
1.00
24.84
A

ATOM
169
O
ALA
A
621
−14.300
−13.748
−9.731
1.00
24.07
A

ATOM
170
N
LYS
A
622
−12.088
−14.146
−9.587
1.00
24.59
A

ATOM
171
CA
LYS
A
622
−12.195
−15.517
−10.074
1.00
26.44
A

ATOM
172
CB
LYS
A
622
−10.842
−16.000
−10.600
1.00
29.90
A

ATOM
173
CG
LYS
A
622
−10.862
−17.445
−11.086
1.00
34.03
A

ATOM
174
CD
LYS
A
622
−9.455
−18.030
−11.189
1.00
36.96
A

ATOM
175
CE
LYS
A
622
−8.623
−17.304
−12.231
1.00
39.89
A

ATOM
176
NZ
LYS
A
622
−7.211
−17.795
−12.281
1.00
41.83
A

ATOM
177
C
LYS
A
622
−12.647
−16.453
−8.956
1.00
26.32
A

ATOM
178
O
LYS
A
622
−12.038
−16.482
−7.885
1.00
25.37
A

ATOM
179
N
GLU
A
623
−13.713
−17.211
−9.202
1.00
25.42
A

ATOM
180
CA
GLU
A
623
−14.222
−18.161
−8.214
1.00
25.52
A

ATOM
181
CB
GLU
A
623
−15.657
−18.582
−8.566
1.00
26.37
A

ATOM
182
CG
GLU
A
623
−16.289
−19.613
−7.627
1.00
26.38
A

ATOM
183
CD
GLU
A
623
−16.521
−19.094
−6.212
1.00
28.72
A

ATOM
184
OE1
GLU
A
623
−16.905
−17.909
−6.053
1.00
28.21
A

ATOM
185
OE2
GLU
A
623
−16.328
−19.876
−5.253
1.00
29.13
A

ATOM
186
C
GLU
A
623
−13.302
−19.371
−8.231
1.00
24.77
A

ATOM
187
O
GLU
A
623
−13.131
−20.015
−9.261
1.00
25.82
A

ATOM
188
N
ILE
A
624
−12.686
−19.663
−7.094
1.00
26.18
A

ATOM
189
CA
ILE
A
624
−11.777
−20.798
−6.993
1.00
25.31
A

ATOM
190
CB
ILE
A
624
−10.466
−20.400
−6.263
1.00
25.34
A

ATOM
191
CG2
ILE
A
624
−9.588
−21.641
−6.048
1.00
23.96
A

ATOM
192
CG1
ILE
A
624
−9.730
−19.327
−7.070
1.00
24.39
A

ATOM
193
CD1
ILE
A
624
−8.450
−18.815
−6.426
1.00
25.55
A

ATOM
194
C
ILE
A
624
−12.427
−21.950
−6.236
1.00
25.18
A

ATOM
195
O
ILE
A
624
−13.012
−21.763
−5.170
1.00
25.10
A

ATOM
196
N
ASP
A
625
−12.324
−23.144
−6.801
1.00
26.96
A

ATOM
197
CA
ASP
A
625
−12.890
−24.323
−6.167
1.00
27.95
A

ATOM
198
CB
ASP
A
625
−12.781
−25.528
−7.089
1.00
30.55
A

ATOM
199
CG
ASP
A
625
−13.634
−26.679
−6.625
1.00
34.97
A

ATOM
200
OD1
ASP
A
625
−14.850
−26.648
−6.907
1.00
37.34
A

ATOM
201
OD2
ASP
A
625
−13.095
−27.597
−5.963
1.00
34.92
A

ATOM
202
C
ASP
A
625
−12.088
−24.580
−4.902
1.00
27.84
A

ATOM
203
O
ASP
A
625
−10.857
−24.583
−4.937
1.00
26.54
A

ATOM
204
N
ILE
A
626
−12.781
−24.807
−3.791
1.00
27.43
A

ATOM
205
CA
ILE
A
626
−12.111
−25.042
−2.518
1.00
28.27
A

ATOM
206
CB
ILE
A
626
−13.149
−25.293
−1.398
1.00
29.15
A

ATOM
207
CG2
ILE
A
626
−13.897
−26.591
−1.660
1.00
28.78
A

ATOM
208
CG1
ILE
A
626
−12.455
−25.330
−0.040
1.00
30.32
A

ATOM
209
CD1
ILE
A
626
−13.412
−25.148
1.122
1.00
35.27
A

ATOM
210
C
ILE
A
626
−11.099
−26.195
−2.563
1.00
27.88
A

ATOM
211
O
ILE
A
626
−10.116
−26.198
−1.825
1.00
27.54
A

ATOM
212
N
SER
A
627
−11.319
−27.164
−3.442
1.00
29.17
A

ATOM
213
CA
SER
A
627
−10.393
−28.289
−3.538
1.00
29.60
A

ATOM
214
CB
SER
A
627
−10.942
−29.350
−4.483
1.00
29.57
A

ATOM
215
OG
SER
A
627
−10.885
−28.887
−5.818
1.00
31.25
A

ATOM
216
C
SER
A
627
−9.009
−27.858
−4.028
1.00
29.72
A

ATOM
217
O
SER
A
627
−8.072
−28.657
−4.025
1.00
29.55
A

ATOM
218
N
CYS
A
628
−8.888
−26.606
−4.465
1.00
29.03
A

ATOM
219
CA
CYS
A
628
−7.616
−26.075
−4.951
1.00
29.50
A

ATOM
220
CB
CYS
A
628
−7.840
−25.103
−6.128
1.00
28.94
A

ATOM
221
SG
CYS
A
628
−8.593
−25.809
−7.641
1.00
30.55
A

ATOM
222
C
CYS
A
628
−6.871
−25.335
−3.836
1.00
29.48
A

ATOM
223
O
CYS
A
628
−5.665
−25.115
−3.928
1.00
28.60
A

ATOM
224
N
VAL
A
629
−7.592
−24.955
−2.785
1.00
30.83
A

ATOM
225
CA
VAL
A
629
−7.004
−24.218
−1.671
1.00
31.02
A

ATOM
226
CB
VAL
A
629
−7.999
−23.159
−1.128
1.00
32.23
A

ATOM
227
CG1
VAL
A
629
−7.317
−22.303
−0.052
1.00
32.29
A

ATOM
228
CG2
VAL
A
629
−8.509
−22.280
−2.264
1.00
31.46
A

ATOM
229
C
VAL
A
629
−6.578
−25.107
−0.498
1.00
32.09
A

ATOM
230
O
VAL
A
629
−7.324
−25.985
−0.063
1.00
32.06
A

ATOM
231
N
LYS
A
630
−5.377
−24.869
0.017
1.00
31.20
A

ATOM
232
CA
LYS
A
630
−4.889
−25.641
1.148
1.00
32.57
A

ATOM
233
CB
LYS
A
630
−3.847
−26.667
0.686
1.00
32.81
A

ATOM
234
CG
LYS
A
630
−4.348
−27.600
−0.410
0.00
33.76
A

ATOM
235
CD
LYS
A
630
−3.253
−28.548
−0.871
0.00
34.37
A

ATOM
236
CE
LYS
A
630
−3.749
−29.493
−1.955
0.00
34.80
A

ATOM
237
NZ
LYS
A
630
−4.215
−28.765
−3.167
0.00
35.13
A

ATOM
238
C
LYS
A
630
−4.286
−24.708
2.190
1.00
31.65
A

ATOM
239
O
LYS
A
630
−3.204
−24.162
2.000
1.00
31.96
A

ATOM
240
N
ILE
A
631
−5.009
−24.513
3.285
1.00
32.38
A

ATOM
241
CA
ILE
A
631
−4.542
−23.655
4.359
1.00
33.62
A

ATOM
242
CB
ILE
A
631
−5.700
−23.282
5.314
1.00
33.44
A

ATOM
243
CG2
ILE
A
631
−5.155
−22.539
6.532
1.00
33.62
A

ATOM
244
CG1
ILE
A
631
−6.740
−22.438
4.565
1.00
33.30
A

ATOM
245
CD1
ILE
A
631
−7.916
−21.992
5.416
1.00
31.54
A

ATOM
246
C
ILE
A
631
−3.464
−24.396
5.142
1.00
34.94
A

ATOM
247
O
ILE
A
631
−3.709
−25.490
5.646
1.00
34.77
A

ATOM
248
N
GLU
A
632
−2.278
−23.797
5.237
1.00
35.73
A

ATOM
249
CA
GLU
A
632
−1.154
−24.396
5.958
1.00
37.69
A

ATOM
250
CB
GLU
A
632
0.167
−24.114
5.236
1.00
38.83
A

ATOM
251
CG
GLU
A
632
0.312
−24.785
3.892
0.00
39.80
A

ATOM
252
CD
GLU
A
632
0.374
−26.290
4.006
0.00
40.38
A

ATOM
253
OE1
GLU
A
632
1.254
−26.793
4.735
0.00
40.70
A

ATOM
254
OE2
GLU
A
632
−0.455
−26.970
3.367
0.00
40.70
A

ATOM
255
C
GLU
A
632
−1.047
−23.892
7.394
1.00
38.57
A

ATOM
256
O
GLU
A
632
−1.118
−24.681
8.342
1.00
38.93
A

ATOM
257
N
GLN
A
633
−0.868
−22.583
7.556
1.00
38.28
A

ATOM
258
CA
GLN
A
633
−0.744
−21.995
8.889
1.00
39.08
A

ATOM
259
CB
GLN
A
633
0.739
−21.825
9.250
1.00
40.39
A

ATOM
260
CG
GLN
A
633
1.001
−21.4821
0.712
0.00
41.11
A

ATOM
261
CD
GLN
A
633
2.481
−21.3671
1.028
0.00
41.66
A

ATOM
262
OE1
GLN
A
633
3.235
−22.3311
0.891
0.00
41.94
A

ATOM
263
NE2
GLN
A
633
2.904
−20.1831
1.455
0.00
41.94
A

ATOM
264
C
GLN
A
633
−1.455
−20.650
8.994
1.00
39.45
A

ATOM
265
O
GLN
A
633
−1.725
−20.000
7.982
1.00
36.93
A

ATOM
266
N
VAL
A
634
−1.762
−20.238
10.221
1.00
39.17
A

ATOM
267
CA
VAL
A
634
−2.425
−18.960
10.445
1.00
41.26
A

ATOM
268
CB
VAL
A
634
−3.371
−19.013
11.653
1.00
41.53
A

ATOM
269
CG1
VAL
A
634
−4.109
−17.694
11.778
1.00
42.05
A

ATOM
270
CG2
VAL
A
634
−4.344
−20.164
11.501
1.00
40.57
A

ATOM
271
C
VAL
A
634
−1.368
−17.900
10.719
1.00
42.58
A

ATOM
272
O
VAL
A
634
−0.569
−18.044
11.649
1.00
43.99
A

ATOM
273
N
ILE
A
635
−1.365
−16.842
9.910
1.00
43.60
A

ATOM
274
CA
ILE
A
635
−0.398
−15.749
10.045
1.00
44.51
A

ATOM
275
CB
ILE
A
635
−0.214
−14.995
8.699
1.00
45.18
A

ATOM
276
CG2
ILE
A
635
0.860
−13.925
8.841
0.00
45.30
A

ATOM
277
CG1
ILE
A
635
0.154
−15.979
7.586
0.00
45.40
A

ATOM
278
CD1
ILE
A
635
1.450
−16.730
7.817
0.00
45.70
A

ATOM
279
C
ILE
A
635
−0.846
−14.744
11.103
1.00
45.18
A

ATOM
280
O
ILE
A
635
−0.281
−14.678
12.197
1.00
45.07
A

ATOM
281
N
GLY
A
636
−1.871
−13.966
10.768
1.00
45.30
A

ATOM
282
CA
GLY
A
636
−2.385
−12.968
11.689
1.00
45.00
A

ATOM
283
C
GLY
A
636
−3.900
−12.992
11.798
1.00
44.80
A

ATOM
284
O
GLY
A
636
−4.550
−13.951
11.375
1.00
44.35
A

ATOM
285
N
ALA
A
637
−4.462
−11.934
12.373
1.00
44.33
A

ATOM
286
CA
ALA
A
637
−5.906
−11.818
12.545
1.00
43.81
A

ATOM
287
CB
ALA
A
637
−6.238
−11.493
13.996
1.00
43.88
A

ATOM
288
C
ALA
A
637
−6.450
−10.729
11.634
1.00
43.58
A

ATOM
289
O
ALA
A
637
−5.828
−9.677
11.465
1.00
42.65
A

ATOM
290
N
GLY
A
638
−7.612
−10.988
11.044
1.00
43.24
A

ATOM
291
CA
GLY
A
638
−8.218
−10.012
10.157
1.00
41.84
A

ATOM
292
C
GLY
A
638
−9.481
−9.419
10.741
1.00
41.01
A

ATOM
293
O
GLY
A
638
−9.978
−9.880
11.773
1.00
41.22
A

ATOM
294
N
GLU
A
639
−10.006
−8.397
10.075
1.00
40.04
A

ATOM
295
CA
GLU
A
639
−11.222
−7.733
10.525
1.00
37.62
A

ATOM
296
CB
GLU
A
639
−11.469
−6.470
9.695
1.00
39.78
A

ATOM
297
CG
GLU
A
639
−12.702
−5.688
10.127
1.00
44.07
A

ATOM
298
CD
GLU
A
639
−13.102
−4.611
9.134
1.00
46.08
A

ATOM
299
OE1
GLU
A
639
−14.145
−3.961
9.358
1.00
48.25
A

ATOM
300
OE2
GLU
A
639
−12.381
−4.416
8.128
1.00
47.68
A

ATOM
301
C
GLU
A
639
−12.448
−8.645
10.431
1.00
35.13
A

ATOM
302
O
GLU
A
639
−13.392
−8.509
11.219
1.00
34.00
A

ATOM
303
N
PHE
A
640
−12.430
−9.574
9.477
1.00
31.83
A

ATOM
304
CA
PHE
A
640
−13.560
−10.482
9.278
1.00
30.48
A

ATOM
305
CB
PHE
A
640
−14.083
−10.366
7.832
1.00
29.76
A

ATOM
306
CG
PHE
A
640
−14.482
−8.966
7.433
1.00
28.46
A

ATOM
307
CD1
PHE
A
640
−13.531
−8.058
6.974
1.00
28.70
A

ATOM
308
CD2
PHE
A
640
−15.802
−8.545
7.548
1.00
28.68
A

ATOM
309
CE1
PHE
A
640
−13.889
−6.745
6.636
1.00
27.42
A

ATOM
310
CE2
PHE
A
640
−16.172
−7.237
7.215
1.00
26.96
A

ATOM
311
CZ
PHE
A
640
−15.211
−6.337
6.759
1.00
27.64
A

ATOM
312
C
PHE
A
640
−13.242
−11.952
9.591
1.00
29.44
A

ATOM
313
O
PHE
A
640
−14.118
−12.817
9.499
1.00
27.82
A

ATOM
314
N
GLY
A
641
−11.998
−12.230
9.966
1.00
28.09
A

ATOM
315
CA
GLY
A
641
−11.611
−13.597
10.266
1.00
28.09
A

ATOM
316
C
GLY
A
641
−10.105
−13.766
10.389
1.00
28.66
A

ATOM
317
O
GLY
A
641
−9.402
−12.833
10.777
1.00
27.98
A

ATOM
318
N
GLU
A
642
−9.609
−14.955
10.052
1.00
27.87
A

ATOM
319
CA
GLU
A
642
−8.185
−15.249
10.140
1.00
28.48
A

ATOM
320
CB
GLU
A
642
−7.969
−16.715
10.559
1.00
31.49
A

ATOM
321
CG
GLU
A
642
−8.655
−17.116
11.879
1.00
35.67
A

ATOM
322
CD
GLU
A
642
−8.289
−18.526
12.345
1.00
38.58
A

ATOM
323
OE1
GLU
A
642
−8.407
−19.482
11.544
1.00
39.58
A

ATOM
324
OE2
GLU
A
642
−7.884
−18.676
13.521
1.00
39.80
A

ATOM
325
C
GLU
A
642
−7.419
−14.989
8.844
1.00
27.74
A

ATOM
326
O
GLU
A
642
−7.980
−15.043
7.744
1.00
26.91
A

ATOM
327
N
VAL
A
643
−6.130
−14.693
8.989
1.00
27.11
A

ATOM
328
CA
VAL
A
643
−5.252
−14.461
7.853
1.00
26.42
A

ATOM
329
CB
VAL
A
643
−4.478
−13.134
7.996
1.00
25.95
A

ATOM
330
CG1
VAL
A
643
−3.732
−12.838
6.728
1.00
25.98
A

ATOM
331
CG2
VAL
A
643
−5.427
−12.014
8.334
1.00
25.96
A

ATOM
332
C
VAL
A
643
−4.268
−15.620
7.891
1.00
26.37
A

ATOM
333
O
VAL
A
643
−3.519
−15.763
8.858
1.00
24.39
A

ATOM
334
N
CYS
A
644
−4.267
−16.433
6.836
1.00
25.63
A

ATOM
335
CA
CYS
A
644
−3.409
−17.616
6.763
1.00
25.19
A

ATOM
336
CB
CYS
A
644
−4.275
−18.883
6.718
1.00
25.46
A

ATOM
337
SG
CYS
A
644
−5.746
−18.849
7.750
1.00
28.36
A

ATOM
338
C
CYS
A
644
−2.483
−17.655
5.551
1.00
25.37
A

ATOM
339
O
CYS
A
644
−2.556
−16.811
4.657
1.00
25.07
A

ATOM
340
N
SER
A
645
−1.615
−18.658
5.532
1.00
23.66
A

ATOM
341
CA
SER
A
645
−0.709
−18.873
4.419
1.00
24.21
A

ATOM
342
CB
SER
A
645
0.750
−18.801
4.862
1.00
24.10
A

ATOM
343
OG
SER
A
645
1.107
−19.968
5.574
1.00
27.96
A

ATOM
344
C
SER
A
645
−1.025
−20.280
3.949
1.00
23.74
A

ATOM
345
O
SER
A
645
−1.558
−21.088
4.715
1.00
22.82
A

ATOM
346
N
GLY
A
646
−0.703
−20.575
2.695
1.00
24.19
A

ATOM
347
CA
GLY
A
646
−0.985
−21.896
2.171
1.00
22.79
A

ATOM
348
C
GLY
A
646
−0.559
−22.070
0.730
1.00
22.27
A

ATOM
349
O
GLY
A
646
0.255
−21.304
0.208
1.00
21.87
A

ATOM
350
N
HIS
A
647
−1.097
−23.100
0.091
1.00
22.58
A

ATOM
351
CA
HIS
A
647
−0.776
−23.367
−1.296
1.00
24.03
A

ATOM
352
CB
HIS
A
647
−0.024
−24.698
−1.445
1.00
25.12
A

ATOM
353
CG
HIS
A
647
1.316
−24.725
−0.770
1.00
27.93
A

ATOM
354
CD2
HIS
A
647
2.572
−24.751
−1.278
1.00
28.54
A

ATOM
355
ND1
HIS
A
647
1.460
−24.753
0.602
1.00
29.81
A

ATOM
356
CE1
HIS
A
647
2.745
−24.800
0.909
1.00
28.73
A

ATOM
357
NE2
HIS
A
647
3.441
−24.799
−0.214
1.00
30.01
A

ATOM
358
C
HIS
A
647
−2.054
−23.392
−2.125
1.00
22.29
A

ATOM
359
O
HIS
A
647
−3.134
−23.758
−1.643
1.00
22.95
A

ATOM
360
N
LEU
A
648
−1.913
−22.977
−3.376
1.00
22.24
A

ATOM
361
CA
LEU
A
648
−3.010
−22.935
−4.323
1.00
22.62
A

ATOM
362
CB
LEU
A
648
−3.302
−21.488
−4.743
1.00
22.09
A

ATOM
363
CG
LEU
A
648
−4.285
−21.306
−5.898
1.00
21.11
A

ATOM
364
CD1
LEU
A
648
−5.639
−21.893
−5.505
1.00
22.47
A

ATOM
365
CD2
LEU
A
648
−4.418
−19.834
−6.250
1.00
21.63
A

ATOM
366
C
LEU
A
648
−2.565
−23.737
−5.532
1.00
24.96
A

ATOM
367
O
LEU
A
648
−1.525
−23.447
−6.131
1.00
25.51
A

ATOM
368
N
LYS
A
649
−3.343
−24.755
−5.879
1.00
27.46
A

ATOM
369
CA
LYS
A
649
−3.029
−25.596
−7.024
1.00
31.13
A

ATOM
370
CB
LYS
A
649
−2.997
−27.078
−6.610
1.00
31.15
A

ATOM
371
CG
LYS
A
649
−2.185
−27.994
−7.529
0.00
31.95
A

ATOM
372
CD
LYS
A
649
−2.818
−28.177
−8.903
0.00
32.48
A

ATOM
373
CE
LYS
A
649
−1.940
−29.047
−9.801
0.00
32.88
A

ATOM
374
NZ
LYS
A
649
−1.650
−30.379
−9.199
0.00
33.21
A

ATOM
375
C
LYS
A
649
−4.115
−25.373
−8.066
1.00
33.65
A

ATOM
376
O
LYS
A
649
−5.272
−25.744
−7.858
1.00
35.71
A

ATOM
377
N
LEU
A
650
−3.740
−24.753
−9.178
1.00
35.03
A

ATOM
378
CA
LEU
A
650
−4.681
−24.498
−10.265
1.00
36.97
A

ATOM
379
CB
LEU
A
650
−4.654
−23.021
−10.659
1.00
36.46
A

ATOM
380
CG
LEU
A
650
−5.111
−22.027
−9.592
1.00
37.31
A

ATOM
381
CD1
LEU
A
650
−4.809
−20.616
−10.058
1.00
37.80
A

ATOM
382
CD2
LEU
A
650
−6.596
−22.211
−9.307
1.00
35.30
A

ATOM
383
C
LEU
A
650
−4.308
−25.361
−11.465
1.00
36.94
A

ATOM
384
O
LEU
A
650
−3.274
−26.027
−11.458
1.00
35.61
A

ATOM
385
N
ARG
A
654
1.042
−25.384
−11.878
1.00
38.79
A

ATOM
386
CA
ARG
A
654
1.946
−25.673
−10.770
1.00
38.06
A

ATOM
387
CB
ARG
A
654
3.360
−25.184
−11.092
1.00
40.05
A

ATOM
388
CG
ARG
A
654
4.377
−25.516
−10.010
0.00
40.81
A

ATOM
389
CD
ARG
A
654
5.712
−24.843
−10.271
0.00
41.95
A

ATOM
390
NE
ARG
A
654
6.674
−25.111
−9.205
0.00
42.83
A

ATOM
391
CZ
ARG
A
654
7.878
−24.553
−9.127
0.00
43.31
A

ATOM
392
NH1
ARG
A
654
8.273
−23.692
−10.055
0.00
43.60
A

ATOM
393
NH2
ARG
A
654
8.687
−24.854
−8.121
0.00
43.60
A

ATOM
394
C
ARG
A
654
1.477
−25.019
−9.473
1.00
36.96
A

ATOM
395
O
ARG
A
654
0.953
−23.905
−9.479
1.00
37.48
A

ATOM
396
N
GLU
A
655
1.677
−25.718
−8.360
1.00
34.47
A

ATOM
397
CA
GLU
A
655
1.279
−25.210
−7.055
1.00
32.62
A

ATOM
398
CB
GLU
A
655
1.489
−26.287
−5.995
1.00
34.16
A

ATOM
399
CG
GLU
A
655
0.948
−25.913
−4.628
1.00
37.34
A

ATOM
400
CD
GLU
A
655
1.042
−27.056
−3.638
1.00
39.34
A

ATOM
401
OE1
GLU
A
655
2.177
−27.464
−3.306
1.00
40.30
A

ATOM
402
OE2
GLU
A
655
−0.017
−27.547
−3.200
1.00
40.82
A

ATOM
403
C
GLU
A
655
2.075
−23.963
−6.672
1.00
30.39
A

ATOM
404
O
GLU
A
655
3.263
−23.864
−6.970
1.00
30.59
A

ATOM
405
N
ILE
A
656
1.417
−23.006
−6.024
1.00
26.58
A

ATOM
406
CA
ILE
A
656
2.096
−21.787
−5.599
1.00
24.21
A

ATOM
407
CB
ILE
A
656
1.779
−20.575
−6.522
1.00
22.73
A

ATOM
408
CG2
ILE
A
656
2.273
−20.843
−7.949
1.00
21.90
A

ATOM
409
CG1
ILE
A
656
0.271
−20.283
−6.494
1.00
22.82
A

ATOM
410
CD1
ILE
A
656
−0.124
−18.937
−7.096
1.00
20.25
A

ATOM
411
C
ILE
A
656
1.680
−21.399
−4.185
1.00
22.57
A

ATOM
412
O
ILE
A
656
0.593
−21.758
−3.731
1.00
21.28
A

ATOM
413
N
PHE
A
657
2.552
−20.669
−3.493
1.00
22.56
A

ATOM
414
CA
PHE
A
657
2.251
−20.190
−2.147
1.00
21.07
A

ATOM
415
CB
PHE
A
657
3.516
−19.648
−1.462
1.00
27.21
A

ATOM
416
CG
PHE
A
657
4.424
−20.719
−0.909
1.00
32.40
A

ATOM
417
CD1
PHE
A
657
5.499
−21.203
−1.652
1.00
36.01
A

ATOM
418
CD2
PHE
A
657
4.188
−21.258
0.355
1.00
35.51
A

ATOM
419
CE1
PHE
A
657
6.327
−22.209
−1.144
1.00
36.52
A

ATOM
420
CE2
PHE
A
657
5.009
−22.265
0.872
1.00
37.58
A

ATOM
421
CZ
PHE
A
657
6.080
−22.740
0.120
1.00
37.93
A

ATOM
422
C
PHE
A
657
1.240
−19.054
−2.292
1.00
20.47
A

ATOM
423
O
PHE
A
657
1.295
−18.298
−3.261
1.00
19.30
A

ATOM
424
N
VAL
A
658
0.314
−18.941
−1.347
1.00
17.78
A

ATOM
425
CA
VAL
A
658
−0.676
−17.873
−1.392
1.00
16.97
A

ATOM
426
CB
VAL
A
658
−2.004
−18.311
−2.112
1.00
15.03
A

ATOM
427
CG1
VAL
A
658
−1.779
−18.443
−3.615
1.00
13.58
A

ATOM
428
CG2
VAL
A
658
−2.504
−19.636
−1.546
1.00
17.13
A

ATOM
429
C
VAL
A
658
−1.034
−17.421
0.012
1.00
17.80
A

ATOM
430
O
VAL
A
658
−0.782
−18.140
0.990
1.00
16.73
A

ATOM
431
N
ALA
A
659
−1.596
−16.218
0.102
1.00
14.11
A

ATOM
432
CA
ALA
A
659
−2.060
−15.671
1.367
1.00
15.67
A

ATOM
433
CB
ALA
A
659
−1.810
−14.162
1.433
1.00
14.67
A

ATOM
434
C
ALA
A
659
−3.552
−15.965
1.283
1.00
16.07
A

ATOM
435
O
ALA
A
659
−4.150
−15.830
0.207
1.00
16.48
A

ATOM
436
N
ILE
A
660
−4.145
−16.377
2.401
1.00
17.28
A

ATOM
437
CA
ILE
A
660
−5.557
−16.756
2.453
1.00
20.16
A

ATOM
438
CB
ILE
A
660
−5.691
−18.307
2.608
1.00
20.13
A

ATOM
439
CG2
ILE
A
660
−7.149
−18.725
2.565
1.00
22.79
A

ATOM
440
CG1
ILE
A
660
−4.938
−19.019
1.483
1.00
21.87
A

ATOM
441
CD1
ILE
A
660
−4.887
−20.538
1.654
1.00
22.50
A

ATOM
442
C
ILE
A
660
−6.309
−16.101
3.609
1.00
22.05
A

ATOM
443
O
ILE
A
660
−5.937
−16.254
4.774
1.00
22.45
A

ATOM
444
N
LYS
A
661
−7.367
−15.372
3.274
1.00
22.33
A

ATOM
445
CA
LYS
A
661
−8.201
−14.707
4.267
1.00
23.44
A

ATOM
446
CB
LYS
A
661
−8.507
−13.270
3.850
1.00
26.63
A

ATOM
447
CG
LYS
A
661
−7.426
−12.272
4.175
1.00
30.21
A

ATOM
448
CD
LYS
A
661
−7.924
−10.863
3.894
1.00
32.40
A

ATOM
449
CE
LYS
A
661
−6.980
−9.824
4.450
1.00
34.22
A

ATOM
450
NZ
LYS
A
661
−7.561
−8.466
4.315
1.00
34.03
A

ATOM
451
C
LYS
A
661
−9.509
−15.479
4.376
1.00
23.79
A

ATOM
452
O
LYS
A
661
−10.109
−15.837
3.366
1.00
22.44
A

ATOM
453
N
THR
A
662
−9.940
−15.751
5.599
1.00
22.77
A

ATOM
454
CA
THR
A
662
−11.174
−16.485
5.800
1.00
23.53
A

ATOM
455
CB
THR
A
662
−10.938
−17.758
6.637
1.00
25.15
A

ATOM
456
OG1
THR
A
662
−10.286
−17.399
7.860
1.00
26.34
A

ATOM
457
CG2
THR
A
662
−10.065
−18.748
5.872
1.00
25.34
A

ATOM
458
C
THR
A
662
−12.183
−15.608
6.518
1.00
24.19
A

ATOM
459
O
THR
A
662
−11.814
−14.694
7.256
1.00
25.54
A

ATOM
460
N
LEU
A
663
−13.459
−15.887
6.282
1.00
24.00
A

ATOM
461
CA
LEU
A
663
−14.542
−15.146
6.904
1.00
26.09
A

ATOM
462
CB
LEU
A
663
−15.667
−14.907
5.887
1.00
23.14
A

ATOM
463
CG
LEU
A
663
−16.886
−14.132
6.383
1.00
24.98
A

ATOM
464
CD1
LEU
A
663
−16.448
−12.756
6.855
1.00
22.54
A

ATOM
465
CD2
LEU
A
663
−17.931
−14.012
5.263
1.00
23.69
A

ATOM
466
C
LEU
A
663
−15.067
−15.977
8.076
1.00
27.30
A

ATOM
467
O
LEU
A
663
−15.493
−17.115
7.888
1.00
28.82
A

ATOM
468
N
LYS
A
664
−15.026
−15.401
9.275
1.00
28.84
A

ATOM
469
CA
LYS
A
664
−15.490
−16.061
10.490
1.00
30.02
A

ATOM
470
CB
LYS
A
664
−15.417
−15.086
11.674
1.00
31.14
A

ATOM
471
CG
LYS
A
664
−16.325
−13.865
11.549
0.00
31.57
A

ATOM
472
CD
LYS
A
664
−16.193
−12.935
12.752
0.00
32.18
A

ATOM
473
CE
LYS
A
664
−17.158
−11.760
12.657
0.00
32.50
A

ATOM
474
NZ
LYS
A
664
−16.945
−10.972
11.413
0.00
32.80
A

ATOM
475
C
LYS
A
664
−16.923
−16.572
10.334
1.00
30.86
A

ATOM
476
O
LYS
A
664
−17.802
−15.841
9.880
1.00
31.20
A

ATOM
477
N
SER
A
665
−17.152
−17.829
10.711
1.00
31.16
A

ATOM
478
CA
SER
A
665
−18.479
−18.439
10.628
1.00
30.42
A

ATOM
479
CB
SER
A
665
−18.431
−19.861
11.178
1.00
32.90
A

ATOM
480
OG
SER
A
665
−18.013
−19.840
12.532
1.00
35.02
A

ATOM
481
C
SER
A
665
−19.487
−17.629
11.439
1.00
30.07
A

ATOM
482
O
SER
A
665
−19.128
−17.026
12.455
1.00
29.38
A

ATOM
483
N
GLY
A
666
−20.743
−17.622
10.993
1.00
28.66
A

ATOM
484
CA
GLY
A
666
−21.781
−16.876
11.689
1.00
27.97
A

ATOM
485
C
GLY
A
666
−21.789
−15.423
11.250
1.00
27.26
A

ATOM
486
O
GLY
A
666
−22.273
−14.535
11.958
1.00
25.74
A

ATOM
487
N
TYR
A
667
−21.239
−15.186
10.065
1.00
27.08
A

ATOM
488
CA
TYR
A
667
−21.156
−13.847
9.494
1.00
25.72
A

ATOM
489
CB
TYR
A
667
−20.169
−13.854
8.325
1.00
25.23
A

ATOM
490
CG
TYR
A
667
−20.563
−14.793
7.207
1.00
24.76
A

ATOM
491
CD1
TYR
A
667
−21.567
−14.447
6.301
1.00
23.91
A

ATOM
492
CR1
TYR
A
667
−21.951
−15.314
5.284
1.00
25.60
A

ATOM
493
CD2
TYR
A
667
−19.947
−16.040
7.067
1.00
23.97
A

ATOM
494
CE2
TYR
A
667
−20.323
−16.917
6.051
1.00
25.46
A

ATOM
495
CZ
TYR
A
667
−21.325
−16.546
5.163
1.00
26.43
A

ATOM
496
OH
TYR
A
667
−21.691
−17.392
4.143
1.00
28.78
A

ATOM
497
C
TYR
A
667
−22.515
−13.345
9.004
1.00
25.35
A

ATOM
498
O
TYR
A
667
−23.358
−14.125
8.569
1.00
24.49
A

ATOM
499
N
THR
A
668
−22.720
−12.037
9.081
1.00
24.59
A

ATOM
500
CA
THR
A
668
−23.963
−11.444
8.612
1.00
24.16
A

ATOM
501
CB
THR
A
668
−24.265
−10.131
9.344
1.00
25.06
A

ATOM
502
OG1
THR
A
668
−23.182
−9.209
9.132
1.00
28.51
A

ATOM
503
CG2
THR
A
668
−24.432
−10.381
10.837
1.00
24.37
A

ATOM
504
C
THR
A
668
−23.801
−11.147
7.119
1.00
24.62
A

ATOM
505
O
THR
A
668
−22.689
−11.224
6.585
1.00
21.64
A

ATOM
506
N
GLU
A
669
−24.908
−10.809
6.459
1.00
22.42
A

ATOM
507
CA
GLU
A
669
−24.901
−10.493
5.031
1.00
23.71
A

ATOM
508
CB
GLU
A
669
−26.315
−10.102
4.578
1.00
24.53
A

ATOM
509
CG
GLU
A
669
−26.450
−9.796
3.099
0.00
25.34
A

ATOM
510
CD
GLU
A
669
−26.084
−10.978
2.226
0.00
25.88
A

ATOM
511
OE1
GLU
A
669
−26.743
−12.033
2.345
0.00
26.22
A

ATOM
512
0E2
GLU
A
669
−25.137
−10.853
1.422
0.00
26.22
A

ATOM
513
C
GLU
A
669
−23.926
−9.351
4.744
1.00
21.59
A

ATOM
514
O
GLU
A
669
−23.152
−9.408
3.797
1.00
23.09
A

ATOM
515
N
LYS
A
670
−23.956
−8.329
5.589
1.00
22.19
A

ATOM
516
CA
LYS
A
670
−23.092
−7.171
5.439
1.00
20.44
A

ATOM
517
CB
LYS
A
670
−23.522
−6.073
6.415
1.00
19.94
A

ATOM
518
CG
LYS
A
670
−22.615
−4.846
6.417
1.00
19.99
A

ATOM
519
CD
LYS
A
670
−23.150
−3.762
7.315
1.00
19.07
A

ATOM
520
CE
LYS
A
670
−22.130
−2.654
7.524
1.00
21.46
A

ATOM
521
NZ
LYS
A
670
−21.542
−2.169
6.255
1.00
21.44
A

ATOM
522
C
LYS
A
670
−21.622
−7.527
5.668
1.00
22.01
A

ATOM
523
O
LYS
A
670
−20.750
−7.100
4.913
1.00
18.77
A

ATOM
524
N
GLN
A
671
−21.339
−8.305
6.707
1.00
20.25
A

ATOM
525
CA
GLN
A
671
−19.957
−8.678
6.969
1.00
21.57
A

ATOM
526
CB
GLN
A
671
−19.857
−9.540
8.226
1.00
19.59
A

ATOM
527
CG
GLN
A
671
−20.174
−8.772
9.489
1.00
23.04
A

ATOM
528
CD
GLN
A
671
−20.090
−9.646
10.715
1.00
23.12
A

ATOM
529
OE1
GLN
A
671
−20.591
−10.765
10.716
1.00
25.03
A

ATOM
530
NE2
GLN
A
671
−19.454
−9.141
11.766
1.00
26.08
A

ATOM
531
C
GLN
A
671
−19.360
−9.412
5.774
1.00
19.09
A

ATOM
532
O
GLN
A
671
−18.203
−9.188
5.419
1.00
19.45
A

ATOM
533
N
ARG
A
672
−20.159
−10.263
5.143
1.00
17.49
A

ATOM
534
CA
ARG
A
672
−19.711
−11.014
3.978
1.00
18.83
A

ATOM
535
CB
ARG
A
672
−20.775
−12.044
3.584
1.00
18.14
A

ATOM
536
CG
ARG
A
672
−20.482
−12.799
2.295
1.00
19.76
A

ATOM
537
CD
ARG
A
672
−21.620
−13.754
1.961
1.00
21.40
A

ATOM
538
NE
ARG
A
672
−21.459
−14.337
0.633
1.00
23.81
A

ATOM
539
CZ
ARG
A
672
−21.574
−13.656
−0.506
1.00
23.75
A

ATOM
540
NH1
ARG
A
672
−21.863
−12.361
−0.486
1.00
24.13
A

ATOM
541
NH2
ARG
A
672
−21.377
−14.267
−1.665
1.00
23.85
A

ATOM
542
C
ARG
A
672
−19.461
−10.053
2.813
1.00
19.29
A

ATOM
543
O
AEG
A
672
−18.476
−10.172
2.080
1.00
17.76
A

ATOM
544
N
ARG
A
673
−20.376
−9.102
2.655
1.00
19.04
A

ATOM
545
CA
ARG
A
673
−20.280
−8.113
1.592
1.00
18.53
A

ATOM
546
CB
ARG
A
673
−21.499
−7.192
1.599
1.00
16.97
A

ATOM
547
CG
ARG
A
673
−21.472
−6.171
0.481
1.00
16.95
A

ATOM
548
CD
ARG
A
673
−22.763
−5.428
0.403
1.00
18.42
A

ATOM
549
NE
ARG
A
673
−22.963
−4.625
1.595
1.00
23.56
A

ATOM
550
CZ
ARG
A
673
−24.042
−4.692
2.366
1.00
20.84
A

ATOM
551
NE1
ARG
A
673
−25.022
−5.533
2.066
1.00
22.79
A

ATOM
552
NH2
ARG
A
673
−24.140
−3.912
3.434
1.00
21.92
A

ATOM
553
C
ARG
A
673
−19.028
−7.269
1.740
1.00
17.35
A

ATOM
554
O
ARG
A
673
−18.245
−7.137
0.802
1.00
18.98
A

ATOM
555
N
ASP
A
674
−18.852
−6.678
2.915
1.00
15.56
A

ATOM
556
CA
ASP
A
674
−17.690
−5.846
3.151
1.00
17.33
A

ATOM
557
CB
ASP
A
674
−17.810
−5.182
4.527
1.00
18.55
A

ATOM
558
CG
ASP
A
674
−19.012
−4.227
4.609
1.00
22.12
A

ATOM
559
OD1
ASP
A
674
−19.522
−3.825
3.542
1.00
19.41
A

ATOM
560
OD2
ASP
A
674
−19.441
−3.870
5.722
1.00
23.46
A

ATOM
561
C
ASP
A
674
−16.376
−6.635
2.991
1.00
16.55
A

ATOM
562
O
ASP
A
674
−15.400
−6.145
2.412
1.00
14.92
A

ATOM
563
N
PHE
A
675
−16.362
−7.864
3.476
1.00
14.43
A

ATOM
564
CA
PHE
A
675
−15.190
−8.711
3.345
1.00
15.13
A

ATOM
565
CB
PHE
A
675
−15.483
−10.070
3.969
1.00
14.23
A

ATOM
566
CG
PHE
A
675
−14.376
−11.054
3.821
1.00
14.93
A

ATOM
567
CD1
PHE
A
675
−13.181
−10.873
4.501
1.00
17.64
A

ATOM
568
CD2
PHE
A
675
−14.543
−12.188
3.040
1.00
16.15
A

ATOM
569
CE1
PHE
A
675
−12.164
−11.815
4.414
1.00
17.02
A

ATOM
570
CE2
PHE
A
675
−13.536
−13.135
2.944
1.00
19.09
A

ATOM
571
CZ
PHE
A
675
−12.342
−12.949
3.636
1.00
18.72
A

ATOM
572
C
PHE
A
675
−14.812
−8.912
1.873
1.00
14.71
A

ATOM
573
O
PHE
A
675
−13.672
−8.655
1.464
1.00
13.96
A

ATOM
574
N
LEU
A
676
−15.780
−9.363
1.080
1.00
13.97
A

ATOM
575
CA
LEU
A
676
−15.550
−9.638
−0.331
1.00
12.30
A

ATOM
576
CB
LEU
A
676
−16.721
−10.465
−0.913
1.00
13.83
A

ATOM
577
CG
LEU
A
676
−16.885
−11.904
−0.364
1.00
13.35
A

ATOM
578
CD1
LEU
A
676
−18.132
−12.553
−0.923
1.00
14.68
A

ATOM
579
CD2
LEU
A
676
−15.665
−12.732
−0.726
1.00
14.56
A

ATOM
580
C
LEU
A
676
−15.318
−8.387
−1.172
1.00
12.89
A

ATOM
581
O
LEU
A
676
−14.816
−8.488
−2.292
1.00
14.97
A

ATOM
582
N
SER
A
677
−15.663
−7.212
−0.651
1.00
11.83
A

ATOM
583
CA
SER
A
677
−15.448
−5.999
−1.438
1.00
14.06
A

ATOM
584
CB
SER
A
677
−16.016
−4.758
−0.733
1.00
12.26
A

ATOM
585
OG
SER
A
677
−15.253
−4.398
0.404
1.00
15.09
A

ATOM
586
C
SER
A
677
−13.952
−5.848
−1.665
1.00
14.36
A

ATOM
587
O
SER
A
677
−13.524
−5.330
−2.685
1.00
14.36
A

ATOM
588
N
GLU
A
678
−13.144
−6.304
−0.715
1.00
16.37
A

ATOM
589
CA
GLU
A
678
−11.705
−6.200
−0.916
1.00
15.57
A

ATOM
590
CB
GLU
A
678
−10.946
−6.832
0.246
1.00
19.69
A

ATOM
591
CG
GLU
A
678
−9.443
−6.939
−0.008
1.00
24.61
A

ATOM
592
CD
GLU
A
678
−8.694
−7.521
1.166
1.00
27.65
A

ATOM
593
OE1
GLU
A
678
−9.219
−8.448
1.807
1.00
29.80
A

ATOM
594
OE2
GLU
A
678
−7.571
−7.052
1.440
1.00
32.17
A

ATOM
595
CG
GLU
A
678
−11.328
−6.885
−2.240
1.00
14.92
A

ATOM
596
O
GLU
A
678
−10.498
−6.386
−2.996
1.00
16.05
A

ATOM
597
N
ALA
A
679
−11.958
−8.015
−2.532
1.00
12.66
A

ATOM
598
CA
ALA
A
679
−11.659
−8.737
−3.761
1.00
13.34
A

ATOM
599
CB
ALA
A
679
−12.111
−10.186
−3.650
1.00
9.98
A

ATOM
600
C
ALA
A
679
−12.296
−8.084
−4.981
1.00
13.80
A

ATOM
601
O
ALA
A
679
−11.699
−8.078
−6.059
1.00
12.31
A

ATOM
602
N
SER
A
680
−13.503
−7.544
−4.829
1.00
14.31
A

ATOM
603
CA
SER
A
680
−14.156
−6.915
−5.975
1.00
14.70
A

ATOM
604
CB
SER
A
680
−15.637
−6.606
−5.666
1.00
13.71
A

ATOM
605
OG
SER
A
680
−15.802
−5.683
−4.610
1.00
19.60
A

ATOM
606
C
SER
A
680
−13.369
−5.665
−6.365
1.00
15.81
A

ATOM
607
O
SER
A
680
−13.449
−5.192
−7.496
1.00
17.05
A

ATOM
608
N
ILE
A
681
−12.566
−5.160
−5.431
1.00
15.14
A

ATOM
609
CA
ILE
A
681
−11.739
−3.998
−5.695
1.00
14.43
A

ATOM
610
CB
ILE
A
681
−11.583
−3.167
−4.412
1.00
15.35
A

ATOM
611
CG2
ILE
A
681
−10.483
−2.107
−4.589
1.00
13.45
A

ATOM
612
CG1
ILE
A
681
−12.955
−2.582
−4.050
1.00
12.39
A

ATOM
613
CD1
ILE
A
681
−12.965
−1.725
−2.820
1.00
13.10
A

ATOM
614
C
ILE
A
681
−10.382
−4.441
−6.241
1.00
15.72
A

ATOM
615
O
ILE
A
681
−10.014
−4.091
−7.371
1.00
15.31
A

ATOM
616
N
MET
A
682
−9.658
−5.247
−5.465
1.00
14.71
A

ATOM
617
CA
MET
A
682
−8.349
−5.746
−5.871
1.00
14.74
A

ATOM
618
CE
MET
A
682
−7.862
−6.775
−4.835
1.00
15.19
A

ATOM
619
CG
MET
A
682
−6.417
−7.225
−5.012
1.00
17.99
A

ATOM
620
SD
MET
A
682
−5.958
−8.505
−3.763
1.00
18.16
A

ATOM
621
CE
MET
A
682
−6.407
−7.626
−2.305
1.00
8.10
A

ATOM
622
C
MET
A
682
−8.384
−6.381
−7.267
1.00
13.42
A

ATOM
623
O
MET
A
682
−7.472
−6.179
−8.076
1.00
12.39
A

ATOM
624
N
GLY
A
683
−9.463
−7.108
−7.563
1.00
11.77
A

ATOM
625
CA
GLY
A
683
−9.598
−7.780
−8.856
1.00
10.35
A

ATOM
626
C
GLY
A
683
−9.632
−6.903
−10.105
1.00
11.65
A

ATOM
627
O
GLY
A
683
−9.492
−7.388
−11.229
1.00
10.47
A

ATOM
628
N
GLN
A
684
−9.816
−5.607
−9.911
1.00
11.57
A

ATOM
629
CA
GLN
A
684
−9.862
−4.670
−11.032
1.00
13.82
A

ATOM
630
CE
GLN
A
684
−10.759
−3.487
−10.680
1.00
14.52
A

ATOM
631
CG
GLN
A
684
−12.201
−3.851
−10.377
1.00
13.27
A

ATOM
632
CD
GLN
A
684
−13.003
−2.641
−9.965
1.00
13.03
A

ATOM
633
OE1
GLN
A
684
−12.961
−1.601
−10.637
1.00
14.53
A

ATOM
634
NE2
GLN
A
684
−13.730
−2.754
−8.857
1.00
9.41
A

ATOM
635
C
GLN
A
684
−8.475
−4.129
−11.345
1.00
14.70
A

ATOM
636
O
GLN
A
684
−8.274
−3.438
−12.347
1.00
13.46
A

ATOM
637
N
PHE
A
685
−7.521
−4.425
−10.469
1.00
14.39
A

ATOM
638
CA
PHE
A
685
−6.156
−3.925
−10.639
1.00
14.62
A

ATOM
639
CE
PHE
A
685
−5.644
−3.368
−9.313
1.00
13.37
A

ATOM
640
CG
PHE
A
685
−6.545
−2.344
−8.696
1.00
13.64
A

ATOM
641
CD1
PHE
A
685
−6.742
−2.327
−7.318
1.00
10.36
A

ATOM
642
CD2
PHE
A
685
−7.187
−1.385
−9.483
1.00
12.29
A

ATOM
643
CE1
PHE
A
685
−7.573
−1.361
−6.724
1.00
12.32
A

ATOM
644
CE2
PHE
A
685
−8.016
−0.419
−8.899
1.00
11.59
A

ATOM
645
CE
PHE
A
685
−8.210
−0.406
−7.520
1.00
13.91
A

ATOM
646
C
PHE
A
685
−5.187
−4.968
−11.146
1.00
14.76
A

ATOM
647
O
PHE
A
685
−5.306
−6.144
−10.836
1.00
15.69
A

ATOM
648
N
ASP
A
686
−4.231
−4.531
−11.952
1.00
15.75
A

ATOM
649
CA
ASP
A
686
−3.230
−5.441
−12.477
1.00
16.52
A

ATOM
650
CB
ASP
A
686
−3.648
−6.006
−13.833
1.00
17.91
A

ATOM
651
CG
ASP
A
686
−2.696
−7.075
−14.319
1.00
20.72
A

ATOM
652
OD1
ASP
A
686
−2.813
−7.517
−15.481
1.00
22.99
A

ATOM
653
OD2
ASP
A
686
−1.820
−7.480
−13.526
1.00
21.44
A

ATOM
654
C
ASP
A
686
−1.929
−4.668
−12.626
1.00
16.22
A

ATOM
655
O
ASP
A
686
−1.645
−4.100
−13.681
1.00
13.75
A

ATOM
656
N
HIS
A
687
−1.143
−4.636
−11.557
1.00
15.08
A

ATOM
657
CA
HIS
A
687
0.114
−3.912
−11.585
1.00
14.22
A

ATOM
658
CB
HIS
A
687
−0.119
−2.460
−11.160
1.00
13.14
A

ATOM
659
CG
HIS
A
687
1.084
−1.585
−11.315
1.00
16.93
A

ATOM
660
CD2
HIS
A
687
1.406
−0.672
−12.264
1.00
15.49
A

ATOM
661
HD1
HIS
A
687
2.141
−1.610
−10.431
1.00
15.21
A

ATOM
662
CE1
HIS
A
687
3.062
−0.748
−10.828
1.00
17.79
A

ATOM
663
NE2
HIS
A
687
2.639
−0.166
−11.937
1.00
16.56
A

ATOM
664
C
HIS
A
687
1.120
−4.598
−10.671
1.00
14.95
A

ATOM
665
O
HIS
A
687
0.766
−5.125
−9.617
1.00
13.04
A

ATOM
666
N
PRO
A
688
2.394
−4.612
−11.076
1.00
15.37
A

ATOM
667
CD
PRO
A
688
2.933
−4.106
−12.355
1.00
13.76
A

ATOM
668
CA
PRO
A
688
3.441
−5.250
−10.275
1.00
14.14
A

ATOM
669
CB
PRO
A
688
4.716
−4.909
−11.048
1.00
16.35
A

ATOM
670
CG
PRO
A
688
4.244
−4.857
−12.483
1.00
16.48
A

ATOM
671
C
PRO
A
688
3.496
−4.785
−8.816
1.00
14.10
A

ATOM
672
O
PRO
A
688
3.868
−5.558
−7.936
1.00
15.51
A

ATOM
673
N
ASN
A
689
3.107
−3.543
−8.545
1.00
12.52
A

ATOM
674
CA
ASN
A
689
3.171
−3.048
−7.173
1.00
12.26
A

ATOM
675
CB
ASN
A
689
3.949
−1.741
−7.144
1.00
11.33
A

ATOM
676
CG
ASN
A
689
5.370
−1.919
−7.629
1.00
13.08
A

ATOM
677
OD1
ASN
A
689
6.237
−2.433
−6.907
1.00
14.93
A

ATOM
678
ND2
ASN
A
689
5.618
−1.524
−8.864
1.00
10.50
A

ATOM
679
C
ASN
A
689
1.840
−2.910
−6.458
1.00
9.30
A

ATOM
680
O
ASN
A
689
1.685
−2.116
−5.543
1.00
9.38
A

ATOM
681
N
VAL
A
690
0.872
−3.696
−6.901
1.00
10.15
A

ATOM
682
CA
VAL
A
690
−0.438
−3.738
−6.275
1.00
10.17
A

ATOM
683
CB
VAL
A
690
−1.523
−3.159
−7.189
1.00
11.34
A

ATOM
684
CG1
VAL
A
690
−2.907
−3.458
−6.593
1.00
5.73
A

ATOM
685
CG2
VAL
A
690
−1.320
−1.643
−7.296
1.00
8.17
A

ATOM
686
C
VAL
A
690
−0.655
−5.232
−6.053
1.00
10.12
A

ATOM
687
O
VAL
A
690
−0.445
−6.038
−6.959
1.00
10.79
A

ATOM
688
N
ILE
A
691
−1.030
−5.601
−4.835
1.00
12.68
A

ATOM
689
CA
ILE
A
691
−1.225
−7.005
−4.482
1.00
12.48
A

ATOM
690
CB
ILE
A
691
−1.833
−7.136
−3.061
1.00
15.22
A

ATOM
691
CG2
ILE
A
691
−2.079
−8.597
−2.729
1.00
17.18
A

ATOM
692
CG1
ILE
A
691
−0.876
−6.555
−2.027
1.00
18.01
A

ATOM
693
CD1
ILE
A
691
0.426
−7.349
−1.935
1.00
24.97
A

ATOM
694
C
ILE
A
691
−2.122
−7.724
−5.478
1.00
14.49
A

ATOM
695
O
ILE
A
691
−3.213
−7.256
−5.798
1.00
14.87
A

ATOM
696
N
HIS
A
692
−1.662
−8.877
−5.948
1.00
14.15
A

ATOM
697
CA
HIS
A
692
−2.409
−9.658
−6.922
1.00
14.37
A

ATOM
698
CB
HIS
A
692
−1.444
−10.529
−7.729
1.00
17.27
A

ATOM
699
CG
HIS
A
692
−2.113
−11.404
−8.745
1.00
19.90
A

ATOM
700
CD2
HIS
A
692
−2.301
−12.743
−8.775
1.00
19.49
A

ATOM
701
ND1
HIS
A
692
−2.671
−10.913
−9.909
1.00
21.64
A

ATOM
702
CE1
HIS
A
692
−3.172
−11.914
−10.610
1.00
20.32
A

ATOM
703
NE2
HIS
A
692
−2.961
−13.035
−9.944
1.00
21.85
A

ATOM
704
C
HIS
A
692
−3.472
−10.542
−6.286
1.00
14.88
K

ATOM
705
O
HIS
A
692
−3.212
−11.229
−5.294
1.00
14.72
A

ATOM
706
N
LEU
A
693
−4.673
−10.513
−6.854
1.00
11.54
A

ATOM
707
CA
LEU
A
693
−5.759
−11.355
−6.369
1.00
11.27
A

ATOM
708
CB
LEU
A
693
−7.113
−10.655
−6.518
1.00
11.71
A

ATOM
709
CG
LEU
A
693
−8.362
−11.527
−6.311
1.00
12.06
A

ATOM
710
CD1
LEU
A
693
−8.584
−11.777
−4.808
1.00
13.97
A

ATOM
711
CD2
LEU
A
693
−9.568
−10.827
−6.903
1.00
12.36
A

ATOM
712
C
LEU
A
693
−5.766
−12.629
−7.202
1.00
12.80
A

ATOM
713
O
LEU
A
693
−5.744
−12.569
−8.432
1.00
12.66
A

ATOM
714
N
GLU
A
694
−5.749
−13.784
−6.541
1.00
9.91
A

ATOM
715
CA
GLU
A
694
−5.803
−15.043
−7.275
1.00
12.11
A

ATOM
716
CB
GLU
A
694
−5.190
−16.197
−6.463
1.00
12.53
A

ATOM
717
CG
GLU
A
694
−3.663
−16.209
−6.417
1.00
15.99
A

ATOM
718
CD
GLU
A
694
−3.024
−16.390
−7.786
1.00
19.23
A

ATOM
719
OE1
GLU
A
694
−3.596
−17.118
−8.633
1.00
22.30
A

ATOM
720
OE2
GLU
A
694
−1.939
−15.817
−8.019
1.00
21.66
A

ATOM
721
C
GLU
A
694
−7.284
−15.311
−7.497
1.00
12.27
A

ATOM
722
O
GLU
A
694
−7.706
−15.664
−8.589
1.00
14.65
A

ATOM
723
N
GLY
A
695
−8.072
−15.126
−6.446
1.00
12.00
A

ATOM
724
CA
GLY
A
695
−9.501
−15.363
−6.554
1.00
13.93
A

ATOM
725
C
GLY
A
695
−10.162
−15.460
−5.191
1.00
14.27
A

ATOM
726
O
GLY
A
695
−9.562
−15.139
−4.165
1.00
15.08
A

ATOM
727
N
VAL
A
696
−11.407
−15.914
−5.185
1.00
16.25
A

ATOM
728
CA
VAL
A
696
−12.160
−16.051
−3.959
1.00
18.13
A

ATOM
729
CB
VAL
A
696
−13.213
−14.920
−3.813
1.00
20.10
A

ATOM
730
CG1
VAL
A
696
−12.523
−13.577
−3.711
1.00
18.53
A

ATOM
731
CG2
VAL
A
696
−14.164
−14.947
−5.011
1.00
18.11
A

ATOM
732
C
VAL
A
696
−12.900
−17.385
−3.944
1.00
20.73
A

ATOM
733
O
VAL
A
696
−13.078
−18.040
−4.984
1.00
20.59
A

ATOM
734
N
VAL
A
697
−13.324
−17.776
−2.752
1.00
20.83
A

ATOM
735
CA
VAL
A
697
−14.086
−18.990
−2.564
1.00
22.28
A

ATOM
736
CB
VAL
A
697
−13.365
−19.983
−1.621
1.00
24.02
A

ATOM
737
CG1
VAL
A
697
−14.184
−21.265
−1.499
1.00
22.46
A

ATOM
738
CG2
VAL
A
697
−11.960
−20.284
−2.156
1.00
22.98
A

ATOM
739
C
VAL
A
697
−15.344
−18.486
−1.880
1.00
22.94
A

ATOM
740
O
VAL
A
697
−15.268
−17.994
−0.758
1.00
22.04
A

ATOM
741
N
THR
A
698
−16.484
−18.568
−2.568
1.00
25.19
A

ATOM
742
CA
THR
A
698
−17.751
−18.113
−2.006
1.00
27.59
A

ATOM
743
CB
THR
A
698
−18.298
−16.854
−2.736
1.00
25.83
A

ATOM
744
OG1
THR
A
698
−18.578
−17.176
−4.099
1.00
23.74
A

ATOM
745
CG2
THR
A
698
−17.287
−15.713
−2.687
1.00
24.82
A

ATOM
746
C
THR
A
698
−18.828
−19.193
−2.076
1.00
31.39
A

ATOM
747
O
THR
A
698
−19.826
−19.119
−1.362
1.00
32.32
A

ATOM
748
N
LYS
A
699
−18.634
−20.186
−2.939
1.00
34.91
A

ATOM
749
CA
LYS
A
699
−19.606
−21.265
−3.085
1.00
38.78
A

ATOM
750
CB
LYS
A
699
−19.632
−21.763
−4.533
1.00
39.53
A

ATOM
751
CG
LYS
A
699
−20.129
−20.728
−5.533
1.00
41.69
A

ATOM
752
CD
LYS
A
699
−20.157
−21.281
−6.953
1.00
43.87
A

ATOM
753
CE
LYS
A
699
−20.685
−20.252
−7.943
1.00
44.93
A

ATOM
754
NZ
LYS
A
699
−20.775
−20.824
−9.328
1.00
47.77
A

ATOM
755
C
LYS
A
699
−19.295
−22.422
−2.145
1.00
40.28
A

ATOM
756
O
LYS
A
699
−19.761
−23.544
−2.342
1.00
42.43
A

ATOM
757
N
SER
A
700
−18.505
−22.139
−1.117
1.00
41.32
A

ATOM
758
CA
SER
A
700
−18.129
−23.146
−0.139
1.00
41.46
A

ATOM
759
CE
SER
A
700
−16.927
−23.949
−0.642
1.00
41.67
A

ATOM
760
OG
SER
A
700
−17.243
−24.643
−1.836
1.00
42.85
A

ATOM
761
C
SER
A
700
−17.776
−22.463
1.176
1.00
41.64
A

ATOM
762
O
SER
A
700
−17.550
−21.252
1.215
1.00
41.74
A

ATOM
763
N
THR
A
701
−17.725
−23.250
2.246
1.00
40.19
A

ATOM
764
CA
THR
A
701
−17.400
−22.745
3.575
1.00
40.36
A

ATOM
765
CB
THR
A
701
−18.451
−23.208
4.618
1.00
41.83
A

ATOM
766
OG1
THR
A
701
−19.763
−22.837
4.175
1.00
44.63
A

ATOM
767
CG2
THR
A
701
−18.190
−22.558
5.973
1.00
42.59
A

ATOM
768
C
THR
A
701
−16.024
−23.256
4.011
1.00
38.95
A

ATOM
769
O
THR
A
701
−15.702
−24.434
3.844
1.00
39.42
A

ATOM
770
N
PRO
A
702
−15.185
−22.370
4.565
1.00
37.14
A

ATOM
771
CD
PRO
A
702
−13.936
−22.766
5.239
1.00
37.05
A

ATOM
772
CA
PRO
A
702
−15.465
−20.949
4.792
1.00
34.67
A

ATOM
773
CB
PRO
A
702
−14.551
−20.611
5.955
1.00
35.95
A

ATOM
774
CG
PRO
A
702
−13.338
−21.429
5.630
1.00
36.79
A

ATOM
775
C
PRO
A
702
−15.158
−20.095
3.569
1.00
31.84
A

ATOM
776
O
PRO
A
702
−14.339
−20.469
2.734
1.00
31.01
A

ATOM
777
N
VAL
A
703
−15.831
−18.952
3.472
1.00
30.06
A

ATOM
778
CA
VAL
A
703
−15.620
−18.017
2.372
1.00
26.08
A

ATOM
779
CB
VAL
A
703
−16.592
−16.830
2.475
1.00
27.78
A

ATOM
780
CG1
VAL
A
703
−16.399
−15.886
1.292
1.00
26.07
A

ATOM
781
CG2
VAL
A
703
−18.028
−17.343
2.525
1.00
27.11
A

ATOM
782
C
VAL
A
703
−14.183
−17.510
2.493
1.00
23.65
A

ATOM
783
O
VAL
A
703
−13.727
−17.199
3.591
1.00
22.73
A

ATOM
784
N
MET
A
704
−13.480
−17.422
1.367
1.00
21.87
A

ATOM
785
CA
MET
A
704
−12.091
−16.985
1.371
1.00
19.85
A

ATOM
786
CE
MET
A
704
−11.154
−18.194
1.287
1.00
20.58
A

ATOM
787
CG
MET
A
704
−11.394
−19.298
2.324
1.00
22.83
A

ATOM
788
SD
MET
A
704
−10.199
−20.637
2.110
1.00
25.21
A

ATOM
789
CE
MET
A
704
−11.006
−21.626
0.904
1.00
26.56
A

ATOM
790
C
MET
A
704
−11.702
−16.055
0.226
1.00
18.73
A

ATOM
791
O
MET
A
704
−12.348
−16.027
−0.817
1.00
18.27
A

ATOM
792
N
ILE
A
705
−10.611
−15.327
0.446
1.00
16.10
A

ATOM
793
CA
ILE
A
705
−10.020
−14.433
−0.541
1.00
14.53
A

ATOM
794
CB
ILE
A
705
−10.033
−12.980
−0.079
1.00
14.98
A

ATOM
795
CG2
ILE
A
705
−9.219
−12.088
−1.066
1.00
13.23
A

ATOM
796
CG1
ILE
A
705
−11.473
−12.508
0.019
1.00
13.64
A

ATOM
797
CD1
ILE
A
705
−11.593
−11.116
0.560
1.00
15.61
A

ATOM
798
C
ILE
A
705
−8.588
−14.917
−0.598
1.00
14.95
A

ATOM
799
O
ILE
A
705
−7.921
−14.999
0.437
1.00
16.93
A

ATOM
800
N
ILE
A
706
−8.125
−15.247
−1.797
1.00
14.61
A

ATOM
801
CA
ILE
A
706
−6.776
−15.761
−1.995
1.00
14.22
A

ATOM
802
CB
ILE
A
706
−6.799
−17.054
−2.837
1.00
13.99
A

ATOM
803
CG2
ILE
A
706
−5.448
−17.747
−2.748
1.00
15.86
A

ATOM
804
CG1
ILE
A
706
−7.914
−17.987
−2.353
1.00
16.58
A

ATOM
805
CD1
ILE
A
706
−7.755
−18.443
−0.919
1.00
23.47
A

ATOM
806
C
ILE
A
706
−5.952
−14.726
−2.741
1.00
14.50
A

ATOM
807
O
ILE
A
706
−6.346
−14.300
−3.829
1.00
12.53
A

ATOM
808
N
THR
A
707
−4.806
−14.342
−2.179
1.00
12.32
A

ATOM
809
CA
THR
A
707
−3.930
−13.344
−2.810
1.00
14.46
A

ATOM
810
CB
THR
A
707
−3.926
−12.026
−2.005
1.00
16.58
A

ATOM
811
OG1
TER
A
707
−3.435
−12.288
−0.685
1.00
18.64
A

ATOM
812
CG2
THR
A
707
−5.334
−11.434
−1.895
1.00
15.55
A

ATOM
813
C
THR
A
707
−2.486
−13.847
−2.921
1.00
16.08
A

ATOM
814
O
THR
A
707
−2.153
−14.876
−2.337
1.00
15.13
A

ATOM
815
N
GLU
A
708
−1.631
−13.150
3.673
1.00
14.63
A

ATOM
816
CA
GLU
A
708
−0.240
−13.603
−3.798
1.00
16.45
A

ATOM
817
CB
GLU
A
708
0.576
−12.730
−4.779
1.00
16.75
A

ATOM
818
CG
GLU
A
708
0.855
−11.308
−4.315
1.00
17.77
A

ATOM
819
CD
GLU
A
708
1.522
−10.440
−5.399
1.00
18.66
A

ATOM
820
OE1
GLU
A
708
0.897
−9.447
−5.806
1.00
17.59
A

ATOM
821
OE2
GLU
A
708
2.670
−10.747
−5.833
1.00
18.27
A

ATOM
822
C
GLU
A
708
0.412
−13.574
−2.428
1.00
15.05
A

ATOM
823
O
GLU
A
708
0.091
−12.730
−1.582
1.00
12.21
A

ATOM
824
N
PHE
A
709
1.319
−14.516
−2.203
1.00
15.72
A

ATOM
825
CA
PHE
A
709
2.001
−14.599
−0.920
1.00
16.73
A

ATOM
826
CE
PHE
A
709
2.486
−16.035
−0.678
1.00
18.37
A

ATOM
827
CG
PHE
A
709
3.127
−16.238
0.661
1.00
20.59
A

ATOM
828
CD1
PHE
A
709
2.423
−15.975
1.829
1.00
21.91
A

ATOM
829
CD2
PHE
A
709
4.433
−16.690
0.756
1.00
23.57
A

ATOM
830
CE1
PHE
A
709
3.011
−16.160
3.074
1.00
24.66
A

ATOM
831
CE2
PHE
A
709
5.031
−16.881
1.997
1.00
23.90
A

ATOM
832
CZ
PHE
A
709
4.315
−16.614
3.160
1.00
25.45
A

ATOM
833
C
PHE
A
709
3.178
−13.630
−0.890
1.00
15.59
A

ATOM
834
O
PHE
A
709
3.928
−13.541
−1.853
1.00
16.82
A

ATOM
835
N
MET
A
710
3.316
−12.906
0.219
1.00
14.56
A

ATOM
836
CA
MET
A
710
4.393
−11.933
0.410
1.00
16.05
A

ATOM
837
CB
MET
A
710
3.793
−10.539
0.610
1.00
15.84
A

ATOM
838
CG
MET
A
710
2.896
−10.105
−0.547
1.00
16.83
A

ATOM
839
SD
MET
A
710
3.759
−9.890
−2.108
1.00
16.56
A

ATOM
840
CE
MET
A
710
4.355
−8.267
−1.864
1.00
17.49
A

ATOM
841
C
MET
A
710
5.198
−12.365
1.642
1.00
15.49
A

ATOM
842
O
MET
A
710
4.828
−12.075
2.774
1.00
13.99
A

ATOM
843
N
GLU
A
711
6.301
−13.065
1.400
1.00
17.57
A

ATOM
844
CA
GLU
A
711
7.130
−13.597
2.478
1.00
20.55
A

ATOM
845
CB
GLU
A
711
8.369
−14.275
1.906
1.00
23.66
A

ATOM
846
CG
GLU
A
711
8.052
−15.543
1.150
1.00
32.78
A

ATOM
847
CD
GLU
A
711
9.241
−16.479
1.065
1.00
35.60
A

ATOM
848
OE1
GLU
A
711
9.088
−17.579
0.478
1.00
37.62
A

ATOM
849
OE2
GLU
A
711
10.319
−16.112
1.589
1.00
36.45
A

ATOM
850
C
GLU
A
711
7.554
−12.624
3.551
1.00
20.06
A

ATOM
851
O
GLU
A
711
7.577
−12.970
4.728
1.00
20.45
A

ATOM
852
N
ASN
A
712
7.879
−11.403
3.165
1.00
16.84
A

ATOM
853
CA
ASN
A
712
8.322
−10.459
4.160
1.00
15.71
A

ATOM
854
CB
ASN
A
712
9.368
−9.544
3.547
1.00
17.27
A

ATOM
855
CG
ASN
A
712
10.627
−10.303
3.222
1.00
17.78
A

ATOM
856
OD1
ASN
A
712
11.034
−11.157
4.005
1.00
16.07
A

ATOM
857
ND2
ASN
A
712
11.236
−10.026
2.081
1.00
18.84
A

ATOM
858
C
ASN
A
712
7.237
−9.685
4.868
1.00
15.77
A

ATOM
859
O
ASN
A
712
7.515
−8.845
5.711
1.00
13.23
A

ATOM
860
N
GLY
A
713
5.991
−9.995
4.537
1.00
16.18
A

ATOM
861
CA
GLY
A
713
4.884
−9.350
5.207
1.00
13.90
A

ATOM
862
C
GLY
A
713
4.785
−7.846
5.109
1.00
14.53
A

ATOM
863
O
GLY
A
713
5.179
−7.237
4.108
1.00
13.01
A

ATOM
864
N
SER
A
714
4.258
−7.252
6.173
1.00
10.95
A

ATOM
865
CA
SER
A
714
4.068
−5.822
6.231
1.00
15.09
A

ATOM
866
CB
SER
A
714
3.195
−5.470
7.424
1.00
16.14
A

ATOM
867
OG
SER
A
714
1.949
−6.125
7.292
1.00
17.72
A

ATOM
868
C
SER
A
714
5.383
−5.075
6.288
1.00
15.86
A

ATOM
869
O
SER
A
714
6.290
−5.427
7.041
1.00
12.51
A

ATOM
870
N
LEU
A
715
5.457
−4.030
5.476
1.00
14.56
A

ATOM
871
CA
LEU
A
715
6.639
−3.206
5.352
1.00
15.61
A

ATOM
872
CB
LEU
A
715
6.399
−2.141
4.284
1.00
12.49
A

ATOM
873
CG
LEU
A
715
7.574
−1.209
4.012
1.00
15.37
A

ATOM
874
CD1
LEU
A
715
8.786
−2.009
3.583
1.00
12.76
A

ATOM
875
CD2
LEU
A
715
7.161
−0.200
2.907
1.00
12.52
A

ATOM
876
C
LEU
A
715
7.102
−2.540
6.637
1.00
14.75
A

ATOM
877
O
LEU
A
715
8.300
−2.500
6.905
1.00
17.56
A

ATOM
878
N
ASP
A
716
6.180
−2.018
7.444
1.00
15.82
A

ATOM
879
CA
ASP
A
716
6.631
−1.369
8.674
1.00
17.28
A

ATOM
880
CB
ASP
A
716
5.462
−0.652
9.389
1.00
16.92
A

ATOM
881
CG
ASP
A
716
4.361
−1.603
9.849
1.00
19.44
A

ATOM
882
OD1
ASP
A
716
3.995
−2.546
9.109
1.00
17.35
A

ATOM
883
OD2
ASP
A
716
3.850
−1.389
10.964
1.00
22.24
A

ATOM
884
C
ASP
A
716
7.317
−2.397
9.587
1.00
17.91
A

ATOM
885
O
ASP
A
716
8.431
−2.169
10.059
1.00
16.46
A

ATOM
886
N
SER
A
717
6.677
−3.542
9.802
1.00
17.08
A

ATOM
887
CA
SER
A
717
7.266
−4.593
10.654
1.00
17.05
A

ATOM
888
CB
SER
A
717
6.283
−5.747
10.807
1.00
18.18
A

ATOM
889
OG
SER
A
717
5.131
−5.296
11.484
1.00
24.48
A

ATOM
890
C
SER
A
717
8.568
−5.136
10.081
1.00
14.71
A

ATOM
891
O
SER
A
717
9.537
−5.363
10.807
1.00
12.74
A

ATOM
892
N
PHE
A
718
8.576
−5.340
8.766
1.00
13.61
A

ATOM
893
CA
PHE
A
718
9.742
−5.854
8.061
1.00
11.92
A

ATOM
894
CB
PHE
A
718
9.456
−5.913
6.566
1.00
12.48
A

ATOM
895
CG
PHE
A
718
10.624
−6.331
5.736
1.00
12.92
A

ATOM
896
CD1
PHE
A
718
11.172
−7.605
5.873
1.00
13.91
A

ATOM
897
CD2
PHE
A
718
11.155
−5.465
4.780
1.00
13.01
A

ATOM
898
CE1
PHE
A
718
12.229
−8.021
5.069
1.00
14.75
A

ATOM
899
CE2
PHE
A
718
12.209
−5.864
3.969
1.00
10.87
A

ATOM
900
CZ
PHE
A
718
12.752
−7.153
4.110
1.00
14.74
A

ATOM
901
C
PHE
A
718
10.947
−4.968
8.294
1.00
12.69
A

ATOM
902
O
PHE
A
718
12.044
−5.456
8.563
1.00
13.46
A

ATOM
903
N
LEU
A
719
10.736
−3.662
8.176
1.00
13.46
A

ATOM
904
CA
LEU
A
719
11.806
−2.698
8.358
1.00
16.37
A

ATOM
905
CB
LEU
A
719
11.357
−1.299
7.923
1.00
14.77
A

ATOM
906
CG
LEU
A
719
11.232
−1.091
6.407
1.00
18.77
A

ATOM
907
CD1
LEU
A
719
10.819
0.339
6.130
1.00
16.45
A

ATOM
908
CD2
LEU
A
719
12.556
−1.395
5.718
1.00
20.57
A

ATOM
909
C
LEU
A
719
12.280
−2.655
9.797
1.00
14.90
A

ATOM
910
O
LEU
A
719
13.465
−2.497
10.052
1.00
14.07
A

ATOM
911
N
ARG
A
720
11.360
−2.792
10.742
1.00
16.00
A

ATOM
912
CA
ARG
A
720
11.776
−2.759
12.137
1.00
16.42
A

ATOM
913
CB
ARG
A
720
10.563
−2.702
13.062
1.00
18.72
A

ATOM
914
CG
ARG
A
720
10.012
−1.296
13.208
1.00
19.57
A

ATOM
915
CD
ARG
A
720
8.967
−1.164
14.300
1.00
22.51
A

ATOM
916
NE
ARG
A
720
7.624
−1.499
13.843
1.00
28.80
A

ATOM
917
CZ
ARG
A
720
7.146
−2.734
13.738
1.00
31.29
A

ATOM
918
NH1
ARG
A
720
7.902
−3.777
14.063
1.00
33.67
A

ATOM
919
NH2
ARG
A
720
5.903
−2.922
13.311
1.00
31.71
A

ATOM
920
C
ARG
A
720
12.662
−3.951
12.463
1.00
17.27
A

ATOM
921
O
ARG
A
720
13.634
−3.826
13.215
1.00
18.44
A

ATOM
922
N
GLN
A
721
12.348
−5.098
11.870
1.00
16.09
A

ATOM
923
CA
GLN
A
721
13.116
−6.313
12.107
1.00
18.82
A

ATOM
924
CB
GLN
A
721
12.287
−7.532
11.709
1.00
20.62
A

ATOM
925
CG
GLN
A
721
10.946
−7.586
12.403
1.00
25.99
A

ATOM
926
CD
GLN
A
721
10.026
−8.623
11.801
1.00
29.22
A

ATOM
927
OE1
GLN
A
721
10.338
−9.231
10.768
1.00
31.08
A

ATOM
928
NE2
GLN
A
721
8.875
−8.827
12.435
1.00
30.60
A

ATOM
929
C
GLN
A
721
14.426
−6.339
11.332
1.00
18.64
A

ATOM
930
O
GLN
A
721
15.242
−7.261
11.481
1.00
17.28
A

ATOM
931
N
ASN
A
722
14.619
−5.326
10.499
1.00
17.52
A

ATOM
932
CA
ASN
A
722
15.813
−5.236
9.676
1.00
17.80
A

ATOM
933
CB
ASN
A
722
15.469
−5.642
8.241
1.00
17.53
A

ATOM
934
CG
ASN
A
722
15.262
−7.143
8.100
1.00
20.53
A

ATOM
935
OD1
ASN
A
722
16.227
−7.901
8.085
1.00
22.10
A

ATOM
936
ND2
ASN
A
722
13.997
−7.582
8.015
1.00
17.67
A

ATOM
937
C
ASN
A
722
16.347
−3.820
9.719
1.00
17.61
A

ATOM
938
O
ASN
A
722
16.752
−3.268
8.697
1.00
18.91
A

ATOM
939
N
ASP
A
723
16.361
−3.247
10.919
1.00
17.15
A

ATOM
940
CA
ASP
A
723
16.817
−1.887
11.101
1.00
19.13
A

ATOM
941
CB
ASP
A
723
16.702
−1.498
12.582
1.00
23.22
A

ATOM
942
CG
ASP
A
723
17.064
−0.043
12.844
1.00
26.94
A

ATOM
943
OD1
ASP
A
723
16.687
0.837
12.049
1.00
28.14
A

ATOM
944
OD2
ASP
A
723
17.728
0.220
13.869
1.00
30.54
A

ATOM
945
C
ASP
A
723
18.244
−1.685
10.569
1.00
19.80
A

ATOM
946
O
ASP
A
723
19.168
−2.433
10.906
1.00
15.82
A

ATOM
947
N
GLY
A
724
18.374
−0.682
9.698
1.00
16.08
A

ATOM
948
CA
GLY
A
724
19.644
−0.327
9.089
1.00
15.30
A

ATOM
949
C
CLY
A
724
20.264
−1.401
8.220
1.00
14.31
A

ATOM
950
O
GLY
A
724
21.430
−1.314
7.855
1.00
13.94
A

ATOM
951
N
GLN
A
725
19.481
−2.402
7.843
1.00
14.27
A

ATOM
952
CA
GLN
A
725
20.030
−3.503
7.064
1.00
14.66
A

ATOM
953
CB
GLN
A
725
19.286
−4.784
7.418
1.00
16.87
A

ATOM
954
CG
GLN
A
725
19.316
−5.104
8.912
1.00
18.98
A

ATOM
955
CD
GLN
A
725
20.744
−5.203
9.445
1.00
20.91
A

ATOM
956
OE1
GLN
A
725
21.198
−4.338
10.202
1.00
25.17
A

ATOM
957
NE2
GLN
A
725
21.456
−6.248
9.044
1.00
18.71
A

ATOM
958
C
GLN
A
725
20.092
−3.349
5.557
1.00
14.70
A

ATOM
959
O
GLN
A
725
20.722
−4.162
4.895
1.00
13.92
A

ATOM
960
N
PHE
A
726
19.454
−2.321
5.011
1.00
13.07
A

ATOM
961
CA
PHE
A
726
19.459
−2.151
3.561
1.00
15.29
A

ATOM
962
CB
PHE
A
726
18.016
−1.995
3.085
1.00
14.57
A

ATOM
963
CG
PHE
A
726
17.140
−3.143
3.477
1.00
15.22
A

ATOM
964
CD1
PHE
A
726
16.094
−2.966
4.375
1.00
14.78
A

ATOM
965
CD2
PHE
A
726
17.399
−4.419
2.984
1.00
14.88
A

ATOM
966
CE1
PHE
A
726
15.325
−4.042
4.776
1.00
13.36
A

ATOM
967
CE2
PHE
A
726
16.630
−5.505
3.386
1.00
17.90
A

ATOM
968
CZ
PHE
A
726
15.594
−5.314
4.285
1.00
14.76
A

ATOM
969
C
PHE
A
726
20.300
−0.997
3.050
1.00
13.71
A

ATOM
970
O
PHE
A
726
20.627
−0.070
3.794
1.00
16.24
A

ATOM
971
N
TEE
A
727
20.669
−1.066
1.776
1.00
17.14
A

ATOM
972
CA
THE
A
727
21.443
0.020
1.184
1.00
17.33
A

ATOM
973
CB
THR
A
727
22.177
−0.429
−0.095
1.00
17.31
A

ATOM
974
OG1
THR
A
727
21.233
−0.637
−1.148
1.00
16.76
A

ATOM
975
CG2
THR
A
727
22.934
−1.744
0.153
1.00
17.77
A

ATOM
976
C
THR
A
727
20.438
1.121
0.851
1.00
19.38
A

ATOM
977
O
THR
A
727
19.224
0.881
0.853
1.00
18.71
A

ATOM
978
N
VAL
A
728
20.941
2.326
0.601
1.00
17.57
A

ATOM
979
CA
VAL
A
728
20.090
3.465
0.257
1.00
18.19
A

ATOM
980
CB
VAL
A
728
20.924
4.776
0.134
1.00
19.55
A

ATOM
981
CG1
VAL
A
728
20.029
5.939
−0.320
1.00
20.05
A

ATOM
982
CG2
VAL
A
728
21.550
5.118
1.487
1.00
18.70
A

ATOM
983
C
VAL
A
728
19.367
3.184
−1.066
1.00
16.00
A

ATOM
984
O
VAL
A
728
18.181
3.482
−1.216
1.00
15.83
A

ATOM
985
N
ILE
A
729
20.085
2.603
−2.019
1.00
16.06
A

ATOM
986
CA
ILE
A
729
19.490
2.274
−3.306
1.00
16.24
A

ATOM
987
CB
ILE
A
729
20.565
1.765
−4.297
1.00
15.78
A

ATOM
988
CG2
ILE
A
729
19.949
0.856
−5.350
1.00
14.65
A

ATOM
989
CG1
ILE
A
729
21.272
2.962
−4.948
1.00
18.50
A

ATOM
990
CD1
ILE
A
729
20.387
3.784
−5.889
1.00
16.84
A

ATOM
991
C
ILE
A
729
18.375
1.247
−3.137
1.00
17.15
A

ATOM
992
O
ILE
A
729
17.377
1.274
−3.870
1.00
13.45
A

ATOM
993
N
GLN
A
730
18.521
0.342
−2.172
1.00
16.67
A

ATOM
994
CA
GLN
A
730
17.461
−0.649
−1.962
1.00
16.00
A

ATOM
995
CB
GLN
A
730
17.922
−1.763
−1.015
1.00
16.68
A

ATOM
996
CG
GLN
A
730
18.885
−2.733
−1.646
1.00
15.89
A

ATOM
997
CD
GLN
A
730
19.389
−3.777
−0.661
1.00
16.94
A

ATOM
998
OE1
GLN
A
730
19.843
−3.437
0.441
1.00
15.33
A

ATOM
999
NE2
GLN
A
730
19.312
−5.049
−1.052
1.00
13.76
A

ATOM
1000
C
GLN
A
730
16.202
0.008
−1.397
1.00
15.38
A

ATOM
1001
O
GLN
A
730
15.084
−0.284
−1.838
1.00
15.30
A

ATOM
1002
N
LEU
A
731
16.385
0.896
−0.424
1.00
14.09
A

ATOM
1003
CA
LEU
A
731
15.256
1.589
0.193
1.00
14.64
A

ATOM
1004
CB
LEU
A
731
15.721
2.437
1.377
1.00
13.73
A

ATOM
1005
CG
LEU
A
731
16.298
1.671
2.577
1.00
15.51
A

ATOM
1006
CD1
LEU
A
731
16.848
2.669
3.569
1.00
15.48
A

ATOM
1007
CD2
LEU
A
731
15.227
0.797
3.228
1.00
15.71
A

ATOM
1008
C
LEU
A
731
14.570
2.480
−0.828
1.00
15.58
A

ATOM
1009
O
LEU
A
731
13.341
2.582
−0.851
1.00
13.60
A

ATOM
1010
N
VAL
A
732
15.368
3.137
−1.665
1.00
14.16
A

ATOM
1011
CA
VAL
A
732
14.798
4.003
−2.687
1.00
13.56
A

ATOM
1012
CB
VAL
A
732
15.881
4.746
−3.497
1.00
12.54
A

ATOM
1013
CG1
VAL
A
732
15.225
5.497
−4.664
1.00
13.40
A

ATOM
1014
CG2
VAL
A
732
16.596
5.748
−2.598
1.00
12.18
A

ATOM
1015
C
VAL
A
732
13.980
3.137
−3.636
1.00
12.77
A

ATOM
1016
O
VAL
A
732
12.917
3.548
−4.097
1.00
16.70
A

ATOM
1017
N
GLY
A
733
14.479
1.932
−3.896
1.00
12.87
A

ATOM
1018
CA
GLY
A
733
13.796
1.003
−4.776
1.00
11.87
A

ATOM
1019
C
GLY
A
733
12.438
0.597
−4.231
1.00
12.83
A

ATOM
1020
O
GLY
A
733
11.485
0.367
−4.989
1.00
10.23
A

ATOM
1021
N
MET
A
734
12.347
0.499
−2.908
1.00
11.75
A

ATOM
1022
CA
MET
A
734
11.086
0.146
−2.263
1.00
11.65
A

ATOM
1023
CB
MET
A
734
11.298
−0.137
−0.773
1.00
11.18
A

ATOM
1024
CG
MET
A
734
12.101
−1.393
−0.479
1.00
13.32
A

ATOM
1025
SD
MET
A
734
12.561
−1.491
1.265
1.00
17.57
A

ATOM
1026
CE
MET
A
734
13.565
−2.978
1.245
1.00
14.96
A

ATOM
1027
C
MET
A
734
10.096
1.297
−2.419
1.00
12.17
A

ATOM
1028
O
MET
A
734
8.916
1.093
−2.732
1.00
12.13
A

ATOM
1029
N
LEU
A
735
10.590
2.510
−2.211
1.00
10.98
A

ATOM
1030
CA
LEU
A
735
9.751
3.692
−2.312
1.00
12.80
A

ATOM
1031
CB
LEU
A
735
10.509
4.919
−1.789
1.00
13.81
A

ATOM
1032
CG
LEU
A
735
10.854
4.931
−0.283
1.00
14.22
A

ATOM
1033
CD1
LEU
A
735
11.767
6.115
0.009
1.00
15.97
A

ATOM
1034
CD2
LEU
A
735
9.592
5.036
0.554
1.00
14.12
A

ATOM
1035
C
LEU
A
735
9.277
3.909
−3.756
1.00
12.46
A

ATOM
1036
O
LEU
A
735
8.162
4.387
−3.991
1.00
12.21
A

ATOM
1037
N
ARG
A
736
10.126
3.535
−4.707
1.00
11.75
A

ATOM
1038
CA
ARG
A
736
9.822
3.640
−6.141
1.00
13.68
A

ATOM
1039
CB
ARG
A
736
11.061
3.264
−6.961
1.00
15.62
A

ATOM
1040
CG
ARG
A
736
10.798
2.724
−8.368
1.00
22.11
A

ATOM
1041
CD
ARG
A
736
10.163
3.773
−9.223
1.00
21.46
A

ATOM
1042
NE
ARG
A
736
10.472
3.653
−10.651
1.00
25.32
A

ATOM
1043
CZ
ARG
A
736
9.737
3.010
−11.560
1.00
26.66
A

ATOM
1044
NE1
ARG
A
736
8.621
2.386
−11.212
1.00
27.03
A

ATOM
1045
NH2
ARG
A
736
10.092
3.040
−12.848
1.00
26.16
A

ATOM
1046
C
ARG
A
736
8.674
2.697
−6.484
1.00
14.97
A

ATOM
1047
O
ARG
A
736
7.713
3.078
−7.155
1.00
14.53
A

ATOM
1048
N
GLY
A
737
8.788
1.462
−6.010
1.00
14.21
A

ATOM
1049
CA
GLY
A
737
7.757
0.474
−6.256
1.00
13.57
A

ATOM
1050
C
GLY
A
737
6.422
0.906
−5.684
1.00
12.80
A

ATOM
1051
O
GLY
A
737
5.390
0.716
−6.314
1.00
11.94
A

ATOM
1052
N
ILE
A
738
6.437
1.492
−4.490
1.00
14.09
A

ATOM
1053
CA
ILE
A
738
5.208
1.943
−3.855
1.00
11.56
A

ATOM
1054
CB
ILE
A
738
5.467
2.392
−2.405
1.00
11.50
A

ATOM
1055
CG2
ILE
A
738
4.195
3.031
−1.825
1.00
9.80
A

ATOM
1056
CG1
ILE
A
738
5.917
1.175
−1.558
1.00
8.91
A

ATOM
1057
CD1
ILE
A
738
6.389
1.544
−0.153
1.00
8.26
A

ATOM
1058
C
ILE
A
738
4.584
3.110
−4.640
1.00
13.67
A

ATOM
1059
O
ILE
A
738
3.374
3.141
−4.871
1.00
11.58
A

ATOM
1060
N
ALA
A
739
5.416
4.055
−5.070
1.00
12.15
A

ATOM
1061
CA
ALA
A
739
4.918
5.207
−5.831
1.00
12.85
A

ATOM
1062
CB
ALA
A
739
6.054
6.219
−6.067
1.00
9.40
A

ATOM
1063
C
ALA
A
739
4.354
4.728
−7.170
1.00
10.80
A

ATOM
1064
O
ALA
A
739
3.374
5.277
−7.679
1.00
13.80
A

ATOM
1065
N
ALA
A
740
4.980
3.708
−7.736
1.00
8.81
A

ATOM
1066
CA
ALA
A
740
4.526
3.169
−9.009
1.00
11.12
A

ATOM
1067
CB
ALA
A
740
5.508
2.129
−9.514
1.00
9.71
A

ATOM
1068
C
ALA
A
740
3.151
2.532
−8.830
1.00
12.53
A

ATOM
1069
O
ALA
A
740
2.262
2.721
−9.655
1.00
9.41
A

ATOM
1070
N
GLY
A
741
2.992
1.748
−7.765
1.00
10.66
A

ATOM
1071
CA
GLY
A
741
1.700
1.138
−7.516
1.00
10.59
A

ATOM
1072
C
GLY
A
741
0.642
2.211
−7.310
1.00
10.74
A

ATOM
1073
O
GLY
A
741
−0.460
2.097
−7.838
1.00
11.88
A

ATOM
1074
N
MET
A
742
0.988
3.260
−6.564
1.00
9.32
A

ATOM
1075
CA
MET
A
742
0.057
4.342
−6.276
1.00
10.60
A

ATOM
1076
CE
MET
A
742
0.593
5.246
−5.170
1.00
9.60
A

ATOM
1077
CG
MET
A
742
0.530
4.658
−3.753
1.00
15.00
A

ATOM
1078
SD
MET
A
742
−1.113
4.092
−3.272
1.00
12.37
A

ATOM
1079
CE
MET
A
742
−1.973
5.605
−3.201
1.00
7.46
A

ATOM
1080
C
MET
A
742
−0.274
5.184
−7.506
1.00
11.90
A

ATOM
1081
O
MET
A
742
−1.396
5.681
−7.636
1.00
11.73
A

ATOM
1082
N
LYS
A
743
0.710
5.362
−8.382
1.00
12.27
A

ATOM
1083
CA
LYS
A
743
0.510
6.128
−9.606
1.00
14.85
A

ATOM
1084
CB
LYS
A
743
1.828
6.206
−10.386
1.00
14.51
A

ATOM
1085
CG
LYS
A
743
1.892
7.305
−11.431
1.00
17.54
A

ATOM
1086
CD
LYS
A
743
1.282
6.871
−12.720
1.00
18.67
A

ATOM
1087
CE
LYS
A
743
1.404
7.987
−13.780
1.00
21.38
A

ATOM
1088
NZ
LYS
A
743
0.863
7.526
−15.088
1.00
19.61
A

ATOM
1089
C
LYS
A
743
−0.554
5.375
−10.406
1.00
14.04
A

ATOM
1090
O
LYS
A
743
−1.503
5.971
−10.918
1.00
15.53
A

ATOM
1091
N
TYR
A
744
−0.401
4.055
−10.474
1.00
11.88
A

ATOM
1092
CA
TYR
A
744
−1.341
3.213
−11.194
1.00
11.73
A

ATOM
1093
CB
TYR
A
744
−0.884
1.747
−11.168
1.00
10.89
A

ATOM
1094
CG
TYR
A
744
−1.920
0.774
−11.699
1.00
12.71
A

ATOM
1095
CD1
TYR
A
744
−2.013
0.478
−13.063
1.00
11.56
A

ATOM
1096
CE1
TYR
A
744
−2.969
−0.441
−13.543
1.00
12.70
A

ATOM
1097
CD2
TYR
A
744
−2.807
0.144
−10.832
1.00
11.18
A

ATOM
1098
CE2
TYR
A
744
−3.756
−0.760
−11.299
1.00
12.54
A

ATOM
1099
CZ
TYR
A
744
−3.830
−1.054
−12.644
1.00
12.73
A

ATOM
1100
OH
TYR
A
744
−4.728
−2.010
−13.051
1.00
13.10
A

ATOM
1101
C
TYR
A
744
−2.761
3.333
−10.600
1.00
13.06
A

ATOM
1102
O
TYR
A
744
−3.725
3.511
−11.339
1.00
12.71
A

ATOM
1103
N
LEU
A
745
−2.892
3.239
−9.273
1.00
11.93
A

ATOM
1104
CA
LEU
A
745
−4.203
3.364
−8.634
1.00
9.55
A

ATOM
1105
CE
LEU
A
745
−4.098
3.129
−7.117
1.00
12.28
A

ATOM
1106
CG
LEU
A
745
−3.548
1.739
−6.716
1.00
10.40
A

ATOM
1107
CD1
LEU
A
745
−3.313
1.648
−5.206
1.00
11.47
A

ATOM
1108
CD2
LEU
A
745
−4.550
0.682
−7.149
1.00
13.02
A

ATOM
1109
C
LEU
A
745
−4.808
4.755
−8.898
1.00
12.15
A

ATOM
1110
O
LEU
A
745
−5.991
4.876
−9.231
1.00
11.72
A

ATOM
1111
N
ALA
A
746
−4.000
5.796
−8.732
1.00
13.75
A

ATOM
1112
CA
ALA
A
746
−4.447
7.159
−8.972
1.00
13.62
A

ATOM
1113
CB
ALA
A
746
−3.289
8.153
−8.704
1.00
14.55
A

ATOM
1114
C
ALA
A
746
−4.930
7.270
−10.425
1.00
14.12
A

ATOM
1115
O
ALA
A
746
−5.965
7.877
−10.694
1.00
16.20
A

ATOM
1116
N
ASP
A
747
−4.194
6.668
−11.356
1.00
13.55
A

ATOM
1117
CA
ASP
A
747
−4.583
6.692
−12.773
1.00
14.00
A

ATOM
1118
CB
ASP
A
747
−3.563
5.961
−13.641
1.00
14.56
A

ATOM
1119
CG
ASP
A
747
−2.358
6.820
−14.001
1.00
10.40
A

ATOM
1120
OD1
ASP
A
747
−2.345
8.018
−13.679
1.00
11.42
A

ATOM
1121
OD2
ASP
A
747
−1.438
6.263
−14.625
1.00
13.31
A

ATOM
1122
C
ASP
A
747
−5.939
6.023
−12.995
1.00
17.40
A

ATOM
1123
O
ASP
A
747
−6.680
6.391
−13.921
1.00
13.00
A

ATOM
1124
N
MET
A
748
−6.238
5.032
−12.151
1.00
15.80
A

ATOM
1125
CA
MET
A
748
−7.491
4.263
−12.210
1.00
17.56
A

ATOM
1126
CB
MET
A
748
−7.333
2.914
−11.491
1.00
18.98
A

ATOM
1127
CG
MET
A
748
−6.414
1.918
−12.149
1.00
24.30
A

ATOM
1128
SD
MET
A
748
−7.172
1.067
−13.532
1.00
32.75
A

ATOM
1129
CE
MET
A
748
−8.491
0.141
−12.704
1.00
26.32
A

ATOM
1130
C
MET
A
748
−8.593
5.022
−11.493
1.00
16.57
A

ATOM
1131
O
MET
A
748
−9.744
4.557
−11.402
1.00
17.25
A

ATOM
1132
N
ASN
A
749
−8.223
6.175
−10.954
1.00
15.84
A

ATOM
1133
CA
ASN
A
749
−9.148
7.007
−10.198
1.00
17.28
A

ATOM
1134
CB
ASN
A
749
−10.408
7.295
−11.017
1.00
22.09
A

ATOM
1135
CG
ASN
A
749
−11.210
8.431
−10.447
1.00
25.81
A

ATOM
1136
CD1
ASN
A
749
−10.647
9.371
−9.892
1.00
29.95
A

ATOM
1137
ND2
ASN
A
749
−12.528
8.363
−10.580
1.00
31.13
A

ATOM
1138
C
ASN
A
749
−9.528
6.329
−8.875
1.00
16.80
A

ATOM
1139
O
ASN
A
749
−10.660
6.444
−6.388
1.00
14.69
A

ATOM
1140
N
TYR
A
750
−8.579
5.608
−8.293
1.00
16.08
A

ATOM
1141
CA
TYR
A
750
−8.827
4.949
−7.007
1.00
13.01
A

ATOM
1142
CB
TYR
A
750
−8.345
3.498
−7.042
1.00
13.41
A

ATOM
1143
CG
TYR
A
750
−8.556
2.791
−5.721
1.00
13.96
A

ATOM
1144
CD1
TYR
A
750
−9.792
2.249
−5.402
1.00
11.49
A

ATOM
1145
CE1
TYR
A
750
−10.022
1.633
−4.175
1.00
13.84
A

ATOM
1146
CD2
TYR
A
750
−7.530
2.704
−4.774
1.00
13.22
A

ATOM
1147
CE2
TYR
A
750
−7.749
2.088
−3.534
1.00
13.77
A

ATOM
1148
CZ
TYR
A
750
−9.003
1.559
−3.251
1.00
14.92
A

ATOM
1149
OH
TYR
A
750
−9.262
0.963
−2.028
1.00
15.35
A

ATOM
1150
C
TYR
A
750
−8.039
5.699
−5.934
1.00
13.00
A

ATOM
1151
O
TYR
A
750
−6.814
5.785
−6.012
1.00
13.01
A

ATOM
1152
N
VAL
A
751
−8.743
6.256
−4.955
1.00
14.41
A

ATOM
1153
CA
VAL
A
751
−8.111
6.968
−3.843
1.00
15.71
A

ATOM
1154
CB
VAL
A
751
−8.968
8.151
−3.365
1.00
17.55
A

ATOM
1155
CG1
VAL
A
751
−8.324
8.792
−2.143
1.00
20.77
A

ATOM
1156
CG2
VAL
A
751
−9.123
9.181
−4.491
1.00
18.56
A

ATOM
1157
C
VAL
A
751
−8.027
5.946
−2.715
1.00
16.81
A

ATOM
1158
O
VAL
A
751
−9.058
5.449
−2.267
1.00
15.55
A

ATOM
1159
N
HIS
A
752
−6.814
5.643
−2.258
1.00
14.86
A

ATOM
1160
CA
HIS
A
752
−6.612
4.645
−1.209
1.00
13.56
A

ATOM
1161
CB
HIS
A
752
−5.123
4.316
−1.085
1.00
11.05
A

ATOM
1162
CG
HIS
A
752
−4.852
3.078
−0.290
1.00
8.71
A

ATOM
1163
CD2
HIS
A
752
−4.529
1.825
−0.681
1.00
10.79
A

ATOM
1164
ND1
HIS
A
752
−4.946
3.039
1.084
1.00
10.98
A

ATOM
1165
CE1
HIS
A
752
−4.688
1.614
1.505
1.00
11.77
A

ATOM
1166
NE2
HIS
A
752
−4.431
1.060
0.454
1.00
10.75
A

ATOM
1167
C
HIS
A
752
−7.149
5.044
0.161
1.00
14.58
A

ATOM
1168
O
HIS
A
752
−7.821
4.251
0.830
1.00
16.10
A

ATOM
1169
N
ARG
A
753
−6.825
6.269
0.573
1.00
15.29
A

ATOM
1170
CA
ARG
A
753
−7.251
6.827
1.855
1.00
17.04
A

ATOM
1171
CB
ARG
A
753
−8.755
6.598
2.068
1.00
21.60
A

ATOM
1172
CG
ARG
A
753
−9.654
7.287
1.056
1.00
25.77
A

ATOM
1173
CD
ARG
A
753
−11.110
7.212
1.484
1.00
31.38
A

ATOM
1174
NE
ARG
A
753
−11.969
8.087
0.685
1.00
33.80
A

ATOM
1175
CZ
ARG
A
753
−13.158
8.533
1.082
1.00
36.41
A

ATOM
1176
NH1
ARG
A
753
−13.637
8.187
2.271
1.00
35.80
A

ATOM
1177
NH2
ARG
A
753
−13.864
9.336
0.295
1.00
37.87
A

ATOM
1178
C
ARG
A
753
−6.503
6.326
3.097
1.00
16.08
A

ATOM
1179
O
ARG
A
753
−6.555
6.972
4.144
1.00
15.20
A

ATOM
1180
N
ASP
A
754
−5.819
5.189
3.008
1.00
14.97
A

ATOM
1181
CA
ASP
A
754
−5.101
4.667
4.180
1.00
16.09
A

ATOM
1182
CB
ASP
A
754
−5.941
3.555
4.826
1.00
18.24
A

ATOM
1183
CG
ASP
A
754
−5.413
3.098
6.188
1.00
23.23
A

ATOM
1184
OD1
ASP
A
754
−4.927
3.920
6.990
1.00
25.55
A

ATOM
1185
OD2
ASP
A
754
−5.515
1.881
6.469
1.00
28.99
A

ATOM
1186
C
ASP
A
754
−3.702
4.161
3.796
1.00
14.92
A

ATOM
1187
O
ASP
A
754
−3.280
3.078
4.201
1.00
13.06
A

ATOM
1188
N
LEU
A
755
−2.993
4.955
2.998
1.00
14.02
A

ATOM
1189
CA
LEU
A
755
−1.652
4.594
2.572
1.00
13.51
A

ATOM
1190
CB
LEU
A
755
−1.212
5.454
1.384
1.00
9.06
A

ATOM
1191
CD
LEU
A
755
0.216
5.254
0.884
1.00
10.93
A

ATOM
1192
CD1
LEU
A
755
0.452
3.797
0.483
1.00
9.97
A

ATOM
1193
CD2
LEU
A
755
0.458
6.186
−0.312
1.00
8.65
A

ATOM
1194
C
LEU
A
755
−0.705
4.772
3.758
1.00
13.01
A

ATOM
1195
O
LEU
A
755
−0.596
5.859
4.346
1.00
13.78
A

ATOM
1196
N
ALA
A
756
−0.043
3.675
4.110
1.00
12.68
A

ATOM
1197
CA
ALA
A
756
0.876
3.623
5.247
1.00
11.51
A

ATOM
1198
CB
ALA
A
756
0.068
3.568
6.560
1.00
9.75
A

ATOM
1199
C
ALA
A
756
1.732
2.361
5.094
1.00
10.18
A

ATOM
1200
O
ALA
A
756
1.303
1.398
4.465
1.00
7.97
A

ATOM
1201
N
ALA
A
757
2.930
2.346
5.671
1.00
7.85
A

ATOM
1202
CA
ALA
A
757
3.802
1.186
5.514
1.00
8.61
A

ATOM
1203
CB
ALA
A
757
5.153
1.430
6.239
1.00
7.83
A

ATOM
1204
C
ALA
A
757
3.148
−0.117
6.016
1.00
8.24
A

ATOM
1205
O
ALA
A
757
3.423
−1.189
5.490
1.00
10.34
A

ATOM
1206
N
ARG
A
758
2.279
−0.026
7.016
1.00
10.60
A

ATOM
1207
CA
ARG
A
758
1.607
−1.219
7.537
1.00
12.89
A

ATOM
1208
CB
ARG
A
758
0.806
−0.895
8.809
1.00
14.94
A

ATOM
1209
CG
ARG
A
758
−0.235
0.190
8.616
1.00
20.57
A

ATOM
1210
CD
ARG
A
758
−1.226
0.256
9.775
1.00
23.51
A

ATOM
1211
NE
ARG
A
758
−2.251
1.267
9.517
1.00
26.29
A

ATOM
1212
CZ
ARG
A
758
−2.017
2.574
9.514
1.00
26.89
A

ATOM
1213
NH1
ARG
A
758
−0.794
3.029
9.762
1.00
28.84
A

ATOM
1214
NH2
ARG
A
758
−2.999
3.425
9.259
1.00
29.97
A

ATOM
1215
C
ARG
A
758
0.667
−1.782
6.479
1.00
13.53
A

ATOM
1216
O
ARG
A
758
0.245
−2.942
6.555
1.00
11.71
A

ATOM
1217
N
ASN
A
759
0.348
−0.960
5.486
1.00
12.82
A

ATOM
1218
CA
ASN
A
759
−0.542
−1.401
4.421
1.00
14.76
A

ATOM
1219
CB
ASN
A
759
−1.659
−0.368
4.217
1.00
13.84
A

ATOM
1220
CD
ASN
A
759
−2.575
−0.315
5.409
1.00
17.87
A

ATOM
1221
OD1
ASN
A
759
−2.929
−1.366
5.947
1.00
14.41
A

ATOM
1222
ND2
ASN
A
759
−2.942
0.888
5.853
1.00
16.21
A

ATOM
1223
C
ASN
A
759
0.175
−1.727
3.117
1.00
14.12
A

ATOM
1224
O
ASN
A
759
−0.450
−1.885
2.067
1.00
16.83
A

ATOM
1225
N
ILE
A
760
1.499
−1.823
3.194
1.00
12.17
A

ATOM
1226
CA
ILE
A
760
2.316
−2.195
2.045
1.00
10.80
A

ATOM
1227
CB
ILE
A
760
3.503
−1.198
1.811
1.00
10.66
A

ATOM
1228
CG2
ILE
A
760
4.335
−1.671
0.629
1.00
7.31
A

ATOM
1229
CG1
ILE
A
760
2.992
0.233
1.571
1.00
9.62
A

ATOM
1230
CD1
ILE
A
760
2.035
0.369
0.357
1.00
10.93
A

ATOM
1231
C
ILE
A
760
2.905
−3.587
2.374
1.00
12.66
A

ATOM
1232
O
ILE
A
760
3.390
−3.801
3.496
1.00
14.01
A

ATOM
1233
N
LEU
A
761
2.846
−4.534
1.431
1.00
10.97
A

ATOM
1234
CA
LEU
A
761
3.409
−5.874
1.673
1.00
13.49
A

ATOM
1235
CB
LEU
A
761
2.452
−6.983
1.222
1.00
12.03
A

ATOM
1236
CG
LEU
A
761
1.135
−7.005
1.998
1.00
15.46
A

ATOM
1237
CD1
LEU
A
761
0.168
−8.010
1.371
1.00
15.87
A

ATOM
1238
CD2
LEU
A
761
1.418
−7.372
3.443
1.00
20.01
A

ATOM
1239
C
LEU
A
761
4.741
−6.004
0.936
1.00
12.83
A

ATOM
1240
O
LEU
A
761
4.955
−5.356
−0.085
1.00
11.29
A

ATOM
1241
N
VAL
A
762
5.623
−6.853
1.457
1.00
11.95
A

ATOM
1242
CA
VAL
A
762
6.957
−7.030
0.885
1.00
10.52
A

ATOM
1243
CB
VAL
A
762
8.017
−6.525
1.894
1.00
11.59
A

ATOM
1244
CG1
VAL
A
762
9.400
−6.502
1.267
1.00
10.27
A

ATOM
1245
CG2
VAL
A
762
7.627
−5.142
2.388
1.00
11.18
A

ATOM
1246
C
VAL
A
762
7.243
−8.488
0.525
1.00
11.69
A

ATOM
1247
O
VAL
A
762
7.014
−9.390
1.341
1.00
11.49
A

ATOM
1248
N
ASN
A
763
7.740
−8.734
−0.688
1.00
10.00
A

ATOM
1249
CA
ASN
A
763
8.028
−10.105
−1.082
1.00
13.20
A

ATOM
1250
CB
ASN
A
763
7.586
−10.369
−2.543
1.00
14.49
A

ATOM
1251
CG
ASN
A
763
8.550
−9.829
−3.587
1.00
15.38
A

ATOM
1252
OD1
ASN
A
763
9.605
−9.278
−3.274
1.00
17.52
A

ATOM
1253
ND2
ASN
A
763
8.186
−10.010
−4.855
1.00
17.84
A

ATOM
1254
C
ASN
A
763
9.487
−10.470
−0.842
1.00
14.94
A

ATOM
1255
O
ASN
A
763
10.259
−9.643
−0.358
1.00
15.44
A

ATOM
1256
N
SER
A
764
9.862
−11.711
−1.142
1.00
17.21
A

ATOM
1257
CA
SER
A
764
11.233
−12.153
−0.897
1.00
19.91
A

ATOM
1258
CB
SER
A
764
11.372
−13.649
−1.186
1.00
20.57
A

ATOM
1259
OG
SER
A
764
11.104
−13.926
−2.547
1.00
24.62
A

ATOM
1260
C
SER
A
764
12.274
−11.369
−1.687
1.00
21.35
A

ATOM
1261
O
SER
A
764
13.443
−11.319
−1.305
1.00
22.51
A

ATOM
1262
N
ASN
A
765
11.857
−10.746
−2.780
1.00
19.26
A

ATOM
1263
CA
ASN
A
765
12.790
−9.969
−3.571
1.00
19.42
A

ATOM
1264
CB
ASN
A
765
12.513
−10.164
−5.061
1.00
21.15
A

ATOM
1265
CG
ASN
A
765
12.836
−11.572
−5.524
1.00
24.10
A

ATOM
1266
OD1
ASN
A
765
13.910
−12.096
−5.210
1.00
27.80
A

ATOM
1267
ND2
ASN
A
765
11.917
−12.194
−6.271
1.00
26.16
A

ATOM
1268
C
ASN
A
765
12.738
−8.489
−3.198
1.00
19.25
A

ATOM
1269
O
ASN
A
765
13.266
−7.631
−3.915
1.00
19.32
A

ATOM
1270
N
LEU
A
766
12.125
−8.198
−2.053
1.00
15.23
A

ATOM
1271
CA
LEU
A
766
12.004
−6.830
−1.552
1.00
15.03
A

ATOM
1272
CB
LEU
A
766
13.386
−6.151
−1.451
1.00
14.51
A

ATOM
1273
CG
LEU
A
766
14.497
−6.907
−0.699
1.00
16.00
A

ATOM
1274
CD1
LEU
A
766
15.704
−5.956
−0.565
1.00
17.05
A

ATOM
1275
CD2
LEU
A
766
14.030
−7.346
0.691
1.00
17.26
A

ATOM
1276
C
LEU
A
766
11.054
−5.951
−2.376
1.00
13.94
A

ATOM
1277
O
LEU
A
766
11.021
−4.735
−2.196
1.00
13.56
A

ATOM
1278
N
VAL
A
767
10.289
−6.562
−3.279
1.00
12.87
A

ATOM
1279
CA
VAL
A
767
9.322
−5.808
−4.070
1.00
11.98
A

ATOM
1280
CB
VAL
A
767
8.714
−6.648
−5.202
1.00
9.61
A

ATOM
1281
CG1
VAL
A
767
7.574
−5.866
−5.857
1.00
9.58
A

ATOM
1282
CG2
VAL
A
767
9.783
−6.976
−6.240
1.00
9.18
A

ATOM
1283
C
VAL
A
767
8.190
−5.409
−3.130
1.00
13.34
A

ATOM
1284
O
VAL
A
767
7.635
−6.265
−2.436
1.00
12.17
A

ATOM
1285
N
CYS
A
768
7.860
−4.118
−3.099
1.00
12.87
A

ATOM
1286
CA
CYS
A
768
6.786
−3.594
−2.255
1.00
12.04
A

ATOM
1287
CB
CYS
A
768
7.213
−2.257
−1.649
1.00
12.99
A

ATOM
1288
SG
CYS
A
768
8.599
−2.417
−0.477
1.00
15.84
A

ATOM
1289
C
CYS
A
768
5.475
−3.421
−3.027
1.00
13.36
A

ATOM
1290
O
CYS
A
768
5.454
−2.834
−4.116
1.00
13.26
A

ATOM
1291
N
LYS
A
769
4.376
−3.894
−2.440
1.00
12.25
A

ATOM
1292
CA
LYS
A
769
3.073
−3.814
−3.101
1.00
12.28
A

ATOM
1293
CB
LYS
A
769
2.659
−5.209
−3.562
1.00
9.87
A

ATOM
1294
CG
LYS
A
769
3.672
−5.857
−4.462
1.00
13.00
A

ATOM
1295
CD
LYS
A
769
3.170
−7.163
−5.105
1.00
14.19
A

ATOM
1296
CE
LYS
A
769
4.242
−7.757
−6.022
1.00
16.77
A

ATOM
1297
NZ
LYS
A
769
3.710
−8.749
−6.980
1.00
16.50
A

ATOM
1298
C
LYS
A
769
1.978
−3.202
−2.245
1.00
13.01
A

ATOM
1299
O
LYS
A
769
1.844
−3.531
−1.063
1.00
14.29
A

ATOM
1300
N
VAL
A
770
1.184
−2.309
−2.835
1.00
12.10
A

ATOM
1301
CA
VAL
A
770
0.101
−1.677
−2.089
1.00
11.10
A

ATOM
1302
CB
VAL
A
770
−0.512
−0.499
−2.889
1.00
10.58
A

ATOM
1303
CG1
VAL
A
770
−1.644
0.112
−2.100
1.00
5.30
A

ATOM
1304
CG2
VAL
A
770
0.570
0.551
−3.186
1.00
6.99
A

ATOM
1305
C
VAL
A
770
−1.000
−2.696
−1.796
1.00
11.89
A

ATOM
1306
O
VAL
A
770
−1.353
−3.491
−2.662
1.00
12.00
A

ATOM
1307
N
SER
A
771
−1.527
−2.667
−0.576
1.00
11.80
A

ATOM
1308
CA
SER
A
771
−2.590
−3.578
−0.150
1.00
14.70
A

ATOM
1309
CB
SER
A
771
−2.002
−4.666
0.765
1.00
17.32
A

ATOM
1310
OG
SER
A
771
−2.990
−5.600
1.144
1.00
19.91
A

ATOM
1311
C
SER
A
771
−3.655
−2.795
0.630
1.00
14.68
A

ATOM
1312
O
SER
A
771
−3.709
−1.562
0.544
1.00
15.55
A

ATOM
1313
N
ASP
A
772
−4.508
−3.504
1.378
1.00
14.81
A

ATOM
1314
CA
ASP
A
772
−5.530
−2.865
2.212
1.00
16.39
A

ATOM
1315
CB
ASP
A
772
−4.852
−1.845
3.134
1.00
19.62
A

ATOM
1316
CG
ASP
A
772
−5.816
−1.172
4.110
1.00
22.71
A

ATOM
1317
OD1
ASP
A
772
−5.738
0.078
4.232
1.00
26.71
A

ATOM
1318
0D2
ASP
A
772
−6.622
−1.863
4.762
1.00
20.60
A

ATOM
1319
C
ASP
A
772
−6.633
−2.184
1.391
1.00
15.48
A

ATOM
1320
O
ASP
A
772
−6.867
−0.983
1.528
1.00
15.52
A

ATOM
1321
N
PHE
A
773
−7.302
−2.962
0.546
1.00
16.06
A

ATOM
1322
CA
PHE
A
773
−8.376
−2.449
−0.293
1.00
17.22
A

ATOM
1323
CB
PHE
A
773
−8.295
−3.087
−1.679
1.00
16.56
A

ATOM
1324
CG
PHE
A
773
−7.062
−2.696
−2.441
1.00
14.03
A

ATOM
1325
CD1
PHE
A
773
−6.005
−3.593
−2.611
1.00
12.33
A

ATOM
1326
CD2
PHE
A
773
−6.927
−1.396
−2.927
1.00
12.51
A

ATOM
1327
CE1
PHE
A
773
−4.824
−3.188
−3.251
1.00
14.35
A

ATOM
1328
CE2
PHE
A
773
−5.757
−0.989
−3.563
1.00
13.76
A

ATOM
1329
CZ
PHE
A
773
−4.706
−1.885
−3.723
1.00
11.63
A

ATOM
1330
C
PHE
A
773
−9.748
−2.693
0.325
1.00
21.10
A

ATOM
1331
O
PHE
A
773
−10.023
−3.784
0.824
1.00
25.25
A

ATOM
1332
N
PRO
A
797
−4.563
6.529
12.016
1.00
37.73
A

ATOM
1333
CD
PRO
A
797
−5.935
5.995
12.099
1.00
39.20
A

ATOM
1334
CA
PRO
A
797
−4.285
7.010
10.661
1.00
36.62
A

ATOM
1335
CB
PRO
A
797
−5.445
6.428
9.856
1.00
37.71
A

ATOM
1336
CG
PRO
A
797
−6.572
6.522
10.818
1.00
37.28
A

ATOM
1337
C
PRO
A
797
−4.206
8.537
10.558
1.00
34.49
A

ATOM
1338
O
PRO
A
797
−3.764
9.071
9.543
1.00
33.47
A

ATOM
1339
N
ILE
A
798
−4.629
9.236
11.609
1.00
32.85
A

ATOM
1340
CA
ILE
A
798
−4.588
10.695
11.606
1.00
29.61
A

ATOM
1341
CB
ILE
A
798
−4.905
11.269
12.997
1.00
30.95
A

ATOM
1342
CG2
ILE
A
798
−4.587
12.766
13.042
1.00
29.66
A

ATOM
1343
CG1
ILE
A
798
−6.373
11.020
13.327
1.00
29.77
A

ATOM
1344
CD1
ILE
A
798
−6.754
11.463
14.701
1.00
32.23
A

ATOM
1345
C
ILE
A
798
−3.241
11.252
11.157
1.00
28.44
A

ATOM
1346
O
ILE
A
798
−3.192
12.111
10.282
1.00
28.15
A

ATOM
1347
N
ARG
A
799
−2.154
10.768
11.752
1.00
26.63
A

ATOM
1348
CA
ARG
A
799
−0.818
11.247
11.403
1.00
26.62
A

ATOM
1349
CB
ARG
A
799
0.193
10.798
12.466
1.00
28.00
A

ATOM
1350
CG
ARG
A
799
0.416
9.294
12.553
1.00
28.23
A

ATOM
1351
CD
ARG
A
799
1.120
8.966
13.857
1.00
29.46
A

ATOM
1352
NE
ARG
A
799
0.298
9.380
14.993
1.00
30.72
A

ATOM
1353
CZ
ARG
A
799
0.771
9.878
16.129
1.00
30.22
A

ATOM
1354
NH1
ARG
A
799
2.074
10.036
16.303
1.00
28.37
A

ATOM
1355
NE2
ARG
A
799
−0.066
10.228
17.093
1.00
31.49
A

ATOM
1356
C
ARG
A
799
−0.350
10.789
10.018
1.00
24.48
A

ATOM
1357
O
ARG
A
799
0.781
11.053
9.622
1.00
24.93
A

ATOM
1358
N
TRP
A
800
−1.226
10.102
9.292
1.00
23.10
A

ATOM
1359
CA
TRP
A
800
−0.924
9.619
7.943
1.00
22.60
A

ATOM
1360
CB
TRP
A
800
−1.212
8.118
7.836
1.00
21.43
A

ATOM
1361
CG
TRP
A
800
−0.067
7.225
8.213
1.00
21.86
A

ATOM
1362
CD2
TRP
A
800
0.187
6.640
9.499
1.00
24.24
A

ATOM
1363
CE2
TRP
A
800
1.379
5.894
9.388
1.00
23.31
A

ATOM
1364
CE3
TRP
A
800
−0.477
6.673
10.734
1.00
24.72
A

ATOM
1365
CD1
TRP
A
800
0.953
6.822
7.404
1.00
20.84
A

ATOM
1366
NE1
TRP
A
800
1.824
6.023
8.099
1.00
21.54
A

ATOM
1367
CZ2
TRP
A
800
1.924
5.186
10.464
1.00
25.57
A

ATOM
1368
CZ3
TRP
A
800
0.067
5.966
11.807
1.00
26.76
A

ATOM
1369
CH2
TRP
A
800
1.255
5.234
11.662
1.00
25.91
A

ATOM
1370
C
TRP
A
800
−1.818
10.349
6.950
1.00
22.35
A

ATOM
1371
O
TRP
A
800
−1.623
10.276
5.741
1.00
22.75
A

ATOM
1372
N
THR
A
801
−2.805
11.054
7.480
1.00
22.40
A

ATOM
1373
CA
THR
A
801
−3.781
11.739
6.653
1.00
21.32
A

ATOM
1374
CE
THR
A
801
−5.177
11.517
7.242
1.00
20.93
A

ATOM
1375
OG1
THR
A
801
−5.356
10.114
7.520
1.00
19.55
A

ATOM
1376
CG2
THR
A
801
−6.253
11.971
6.264
1.00
19.42
A

ATOM
1377
C
THR
A
801
−3.521
13.224
6.483
1.00
22.83
A

ATOM
1378
O
THR
A
801
−3.073
13.901
7.405
1.00
24.26
A

ATOM
1379
N
ALA
A
802
−3.806
13.719
5.285
1.00
23.67
A

ATOM
1380
CA
ALA
A
802
−3.616
15.125
4.961
1.00
24.60
A

ATOM
1381
CB
ALA
A
802
−3.827
15.349
3.472
1.00
21.86
A

ATOM
1382
C
ALA
A
802
−4.567
16.006
5.755
1.00
25.32
A

ATOM
1383
O
ALA
A
802
−5.664
15.586
6.121
1.00
26.16
A

ATOM
1384
N
PRO
A
803
−4.155
17.250
6.032
1.00
26.55
A

ATOM
1385
CD
PRO
A
803
−2.823
17.825
5.768
1.00
25.55
A

ATOM
1386
CA
PRO
A
803
−4.991
18.183
6.791
1.00
27.54
A

ATOM
1387
CE
PRO
A
803
−4.158
19.462
6.789
1.00
29.43
A

ATOM
1388
CG
PRO
A
803
−2.743
18.934
6.788
1.00
29.14
A

ATOM
1389
C
PRO
A
803
−6.384
18.396
6.188
1.00
28.40
A

ATOM
1390
O
PRO
A
803
−7.387
18.318
6.897
1.00
28.43
A

ATOM
1391
N
GLU
A
804
−6.445
18.658
4.884
1.00
29.91
A

ATOM
1392
CA
GLU
A
804
−7.730
18.897
4.231
1.00
31.54
A

ATOM
1393
CB
GLU
A
804
−7.536
19.320
2.764
1.00
29.69
A

ATOM
1394
CG
GLU
A
804
−7.162
18.217
1.779
1.00
29.36
A

ATOM
1395
CD
GLU
A
804
−5.671
17.936
1.723
1.00
26.95
A

ATOM
1396
OE1
GLU
A
804
−4.921
18.476
2.563
1.00
26.91
A

ATOM
1397
OE2
GLU
A
804
−5.254
17.164
0.838
1.00
27.14
A

ATOM
1398
C
GLU
A
804
−8.634
17.671
4.318
1.00
32.75
A

ATOM
1399
O
GLU
A
804
−9.857
17.793
4.406
1.00
32.85
A

ATOM
1400
N
ALA
A
805
−8.022
16.492
4.314
1.00
34.37
A

ATOM
1401
CA
ALA
A
805
−8.768
15.244
4.397
1.00
36.04
A

ATOM
1402
CB
ALA
A
805
−7.859
14.069
4.054
1.00
36.25
A

ATOM
1403
C
ALA
A
805
−9.364
15.050
5.787
1.00
37.08
A

ATOM
1404
O
ALA
A
805
−10.422
14.444
5.942
1.00
37.44
A

ATOM
1405
N
ILE
A
806
−8.687
15.564
6.802
1.00
38.80
A

ATOM
1406
CA
ILE
A
806
−9.180
15.425
8.162
1.00
40.65
A

ATOM
1407
CB
ILE
A
806
−8.035
15.591
9.179
1.00
40.28
A

ATOM
1408
CG2
ILE
A
806
−8.582
15.572
10.601
1.00
39.56
A

ATOM
1409
CG1
ILE
A
806
−7.019
14.463
8.988
1.00
39.83
A

ATOM
1410
CD1
ILE
A
806
−5.798
14.585
9.870
1.00
41.52
A

ATOM
1411
C
ILE
A
806
−10.267
16.449
8.457
1.00
42.79
A

ATOM
1412
O
ILE
A
806
−11.265
16.137
9.108
1.00
42.39
A

ATOM
1413
N
GLN
A
807
−10.080
17.665
7.952
1.00
44.65
A

ATOM
1414
CA
GLN
A
807
−11.032
18.746
8.183
1.00
46.54
A

ATOM
1415
CB
GLN
A
807
−10.345
20.091
7.959
1.00
48.25
A

ATOM
1416
CG
GLN
A
807
−11.184
21.287
8.352
1.00
51.25
A

ATOM
1417
CD
GLN
A
807
−10.330
22.490
8.710
1.00
52.70
A

ATOM
1418
OE1
GLN
A
807
−9.503
22.940
7.914
1.00
52.66
A

ATOM
1419
NE2
GLN
A
807
−10.524
23.015
9.917
1.00
53.69
A

ATOM
1420
C
GLN
A
807
−12.306
18.679
7.348
1.00
47.43
A

ATOM
1421
O
GLN
A
807
−13.407
18.637
7.899
1.00
48.05
A

ATOM
1422
N
TYR
A
808
−12.161
18.673
6.025
1.00
48.14
A

ATOM
1412
CA
TYR
A
808
−13.319
18.630
5.134
1.00
47.86
A

ATOM
1424
CB
TYR
A
808
−13.151
19.628
3.981
1.00
49.76
A

ATOM
1425
CG
TYR
A
808
−12.690
21.004
4.407
1.00
51.92
A

ATOM
1426
CD1
TYR
A
808
−11.335
21.288
4.555
1.00
52.77
A

ATOM
1427
CE1
TYR
A
808
−10.905
22.540
4.976
1.00
54.43
A

ATOM
1428
CD2
TYR
A
808
−13.610
22.013
4.689
1.00
53.07
A

ATOM
1429
CE2
TYR
A
808
−13.191
23.271
5.113
1.00
54.30
A

ATOM
1430
CZ
TYR
A
808
−11.836
23.527
5.256
1.00
54.97
A

ATOM
1431
OH
TYR
A
808
−11.404
24.760
5.693
1.00
55.34
A

ATOM
1432
C
TYR
A
808
−13.556
17.244
4.547
1.00
46.87
A

ATOM
1433
O
TYR
A
808
−14.334
17.092
3.608
1.00
45.84
A

ATOM
1434
N
ARG
A
809
−12.884
16.240
5.103
1.00
45.55
A

ATOM
1435
CA
ARG
A
809
−13.004
14.863
4.632
1.00
44.26
A

ATOM
1436
CB
ARG
A
809
−14.346
14.259
5.060
1.00
45.12
A

ATOM
1437
CG
ARG
A
809
−14.499
14.086
6.563
0.00
45.61
A

ATOM
1438
CD
ARG
A
809
−15.764
13.313
6.903
0.00
46.19
A

ATOM
1439
NE
ARG
A
809
−15.851
13.007
8.328
0.00
46.63
A

ATOM
1440
CZ
ARG
A
809
−16.810
12.268
8.878
0.00
46.86
A

ATOM
1441
NH1
ARG
A
809
−17.771
11.756
8.122
0.00
47.00
A

ATOM
1442
NH2
ARG
A
809
−16.806
12.038
10.184
0.00
47.00
A

ATOM
1443
C
ARG
A
809
−12.849
14.761
3.115
1.00
42.45
A

ATOM
1444
O
ARG
A
809
−13.488
13.928
2.469
1.00
42.37
A

ATOM
1445
N
LYS
A
810
−11.992
15.610
2.557
1.00
40.09
A

ATOM
1446
CA
LYS
A
810
−11.737
15.617
1.120
1.00
38.13
A

ATOM
1447
CB
LYS
A
810
−11.373
17.030
0.649
1.00
39.28
A

ATOM
1448
CG
LYS
A
810
−12.443
18.075
0.917
1.00
41.84
A

ATOM
1449
CD
LYS
A
810
−12.071
19.415
0.293
1.00
43.21
A

ATOM
1450
CE
LYS
A
810
−13.210
20.414
0.425
1.00
43.84
A

ATOM
1451
NZ
LYS
A
810
−12.950
21.652
−0.356
1.00
44.49
A

ATOM
1452
C
LYS
A
810
−10.594
14.665
0.770
1.00
35.30
A

ATOM
1453
O
LYS
A
810
−9.429
15.050
0.826
1.00
34.59
A

ATOM
1454
N
PHE
A
811
−10.928
13.427
0.417
1.00
32.42
A

ATOM
1455
CA
PHE
A
811
−9.912
12.445
0.056
1.00
29.29
A

ATOM
1456
CB
PHE
A
811
−10.296
11.053
0.539
1.00
29.39
A

ATOM
1457
CG
PHE
A
811
−10.238
10.888
2.023
1.00
29.16
A

ATOM
1458
CD1
PHE
A
811
−11.317
11.258
2.822
1.00
30.55
A

ATOM
1459
CD2
PHE
A
811
−9.110
10.342
2.624
1.00
28.31
A

ATOM
1460
CE1
PHE
A
811
−11.273
11.078
4.202
1.00
31.51
A

ATOM
1461
CE2
PHE
A
811
−9.051
10.157
3.998
1.00
29.55
A

ATOM
1462
CZ
PHE
A
811
−10.135
10.526
4.792
1.00
31.35
A

ATOM
1463
C
PHE
A
811
−9.705
12.400
−1.444
1.00
27.52
A

ATOM
1464
O
PHE
A
811
−10.646
12.167
−2.198
1.00
26.39
A

ATOM
1465
N
THR
A
812
−8.462
12.617
−1.862
1.00
23.72
A

ATOM
1466
CA
THR
A
812
−8.089
12.617
−3.271
1.00
23.05
A

ATOM
1467
CB
THR
A
812
−7.970
14.054
−3.790
1.00
23.97
A

ATOM
1468
OG1
THR
A
812
−6.932
14.727
−3.067
1.00
23.73
A

ATOM
1469
CG2
THR
A
812
−9.283
14.818
−3.564
1.00
25.01
A

ATOM
1470
C
THR
A
812
−6.721
11.964
−3.424
1.00
21.01
A

ATOM
1471
O
THR
A
812
−6.102
11.577
−2.437
1.00
20.62
A

ATOM
1472
N
SER
A
813
−6.237
11.850
−4.655
1.00
20.38
A

ATOM
1473
CA
SER
A
813
−4.918
11.270
−4.859
1.00
20.11
A

ATOM
1474
CB
SER
A
813
−4.616
11.071
−6.350
1.00
18.76
A

ATOM
1475
OG
SER
A
813
−5.445
10.061
−6.898
1.00
18.07
A

ATOM
1476
C
SER
A
813
−3.872
12.185
−4.241
1.00
20.46
A

ATOM
1477
O
SER
A
813
−2.818
11.719
−3.809
1.00
19.84
A

ATOM
1478
N
ALA
A
814
−4.171
13.482
−4.178
1.00
19.76
A

ATOM
1479
CA
ALA
A
814
−3.239
14.450
−3.600
1.00
20.26
A

ATOM
1480
CB
ALA
A
814
−3.710
15.882
−3.875
1.00
19.22
A

ATOM
1481
C
ALA
A
814
−3.182
14.195
−2.110
1.00
19.65
A

ATOM
1482
O
ALA
A
814
−2.178
14.478
−1.446
1.00
18.61
A

ATOM
1483
N
SER
A
815
−4.289
13.691
−1.579
1.00
19.76
A

ATOM
1484
CA
SER
A
815
−4.355
13.364
−0.164
1.00
19.99
A

ATOM
1485
CB
SER
A
815
−5.803
13.086
0.244
1.00
19.81
A

ATOM
1486
OG
SER
A
815
−5.869
12.896
1.640
1.00
27.92
A

ATOM
1487
C
SER
A
815
−3.490
12.114
0.062
1.00
18.44
A

ATOM
1488
O
SER
A
815
−2.786
12.010
1.065
1.00
19.10
A

ATOM
1489
N
ASP
A
816
−3.544
11.169
−0.875
1.00
17.07
A

ATOM
1490
CA
ASP
A
816
−2.734
9.947
−0.774
1.00
15.75
A

ATOM
1491
CB
ASP
A
816
−3.041
8.953
−1.901
1.00
13.79
A

ATOM
1492
CG
ASP
A
816
−4.343
8.184
−1.691
1.00
12.11
A

ATOM
1493
OD1
ASP
A
816
−4.823
8.097
−0.543
1.00
14.33
A

ATOM
1494
OD2
ASP
A
816
−4.864
7.646
−2.685
1.00
13.38
A

ATOM
1495
C
ASP
A
816
−1.259
10.307
−0.887
1.00
16.60
A

ATOM
1496
O
ASP
A
816
−0.399
9.571
−0.392
1.00
14.48
A

ATOM
1497
N
VAL
A
817
−0.961
11.419
−1.557
1.00
13.18
A

ATOM
1498
CA
VAL
A
817
0.422
11.826
−1.721
1.00
13.34
A

ATOM
1499
CB
VAL
A
817
0.566
12.960
−2.788
1.00
11.51
A

ATOM
1500
CG1
VAL
A
817
1.944
13.589
−2.704
1.00
9.74
A

ATOM
1501
CG2
VAL
A
817
0.376
12.363
−4.179
1.00
11.50
A

ATOM
1502
C
VAL
A
817
1.005
12.286
−0.395
1.00
12.99
A

ATOM
1503
O
VAL
A
817
2.172
12.046
−0.108
1.00
13.15
A

ATOM
1504
N
TRP
A
818
0.189
12.938
0.421
1.00
14.23
A

ATOM
1505
CA
TRP
A
818
0.654
13.378
1.726
1.00
15.45
A

ATOM
1506
CB
TRP
A
818
−0.447
14.188
2.410
1.00
15.49
A

ATOM
1507
CG
TRP
A
818
−0.133
14.556
3.801
1.00
19.74
A

ATOM
1508
CD2
TRP
A
818
0.180
15.863
4.295
1.00
20.03
A

ATOM
1509
CE2
TRP
A
818
0.436
15.733
5.676
1.00
21.03
A

ATOM
1510
CE3
TRP
A
818
0.269
17.131
3.706
1.00
21.31
A

ATOM
1511
CD1
TRP
A
818
−0.060
13.715
4.869
1.00
19.62
A

ATOM
1512
NE1
TRP
A
818
0.283
14.410
5.998
1.00
21.47
A

ATOM
1513
CZ2
TRP
A
818
0.774
16.823
6.481
1.00
20.05
A

ATOM
1514
CZ3
TRP
A
818
0.606
18.217
4.506
1.00
19.00
A

ATOM
1515
CH2
TRP
A
818
0.854
18.055
5.878
1.00
20.15
A

ATOM
1516
C
TRP
A
818
0.996
12.121
2.531
1.00
15.99
A

ATOM
1517
O
TRP
A
818
2.033
12.048
3.210
1.00
15.51
A

ATOM
1518
N
SER
A
819
0.118
11.130
2.428
1.00
14.95
A

ATOM
1519
CA
SER
A
819
0.281
9.855
3.122
1.00
15.25
A

ATOM
1520
CB
SER
A
819
−0.929
8.950
2.852
1.00
14.79
A

ATOM
1521
OG
SER
A
819
−2.112
9.536
3.348
1.00
16.39
A

ATOM
1522
C
SER
A
819
1.550
9.181
2.629
1.00
14.02
A

ATOM
1523
O
SER
A
819
2.325
8.640
3.414
1.00
16.48
A

ATOM
1524
N
TYR
A
820
1.765
9.229
1.320
1.00
13.71
A

ATOM
1525
CA
TYR
A
820
2.956
8.636
0.726
1.00
14.58
A

ATOM
1526
CB
TYR
A
820
2.956
8.842
−0.799
1.00
13.45
A

ATOM
1527
CG
TYR
A
820
4.197
8.297
−1.453
1.00
12.89
A

ATOM
1528
CD1
TYR
A
820
4.336
6.931
−1.732
1.00
15.07
A

ATOM
1529
CE1
TYR
A
820
5.541
6.419
−2.219
1.00
12.78
A

ATOM
1530
CD2
TYR
A
820
5.285
9.128
−1.690
1.00
12.38
A

ATOM
1531
CE2
TYR
A
820
6.466
8.633
−2.166
1.00
11.32
A

ATOM
1532
CZ
TYR
A
820
6.600
7.288
−2.426
1.00
12.29
A

ATOM
1533
OH
TYR
A
820
7.814
6.840
−2.859
1.00
10.71
A

ATOM
1534
C
TYR
A
820
4.210
9.280
1.338
1.00
13.53
A

ATOM
1535
O
TYR
A
820
5.223
8.616
1.545
1.00
13.71
A

ATOM
1536
N
GLY
A
821
4.137
10.574
1.628
1.00
14.63
A

ATOM
1537
CA
GLY
A
821
5.275
11.245
2.218
1.00
13.52
A

ATOM
1538
C
GLY
A
821
5.563
10.662
3.585
1.00
14.77
A

ATOM
1539
O
GLY
A
821
6.719
10.454
3.959
1.00
13.85
A

ATOM
1540
N
ILE
A
822
4.509
10.408
4.347
1.00
15.25
A

ATOM
1541
CA
ILE
A
822
4.690
9.820
5.666
1.00
17.12
A

ATOM
1542
CB
ILE
A
822
3.344
9.674
6.420
1.00
17.21
A

ATOM
1543
CG2
ILE
A
822
3.569
9.002
7.768
1.00
17.52
A

ATOM
1544
CG1
ILE
A
822
2.689
11.050
6.577
1.00
17.04
A

ATOM
1545
OD1
ILE
A
822
3.528
12.069
7.309
1.00
15.74
A

ATOM
1546
C
ILE
A
822
5.326
8.443
5.495
1.00
15.76
A

ATOM
1547
O
ILE
A
822
6.174
8.059
6.293
1.00
17.81
A

ATOM
1548
N
VAL
A
823
4.920
7.710
4.457
1.00
13.84
A

ATOM
1549
CA
VAL
A
823
5.459
6.375
4.199
1.00
12.67
A

ATOM
1550
CB
VAL
A
823
4.738
5.711
2.989
1.00
12.77
A

ATOM
1551
CG1
VAL
A
823
5.403
4.386
2.633
1.00
10.23
A

ATOM
1552
CG2
VAL
A
823
3.271
5.494
3.328
1.00
13.23
A

ATOM
1553
C
VAL
A
823
6.961
6.454
3.918
1.00
14.16
A

ATOM
1554
O
VAL
A
823
7.745
5.594
4.339
1.00
12.18
A

ATOM
1555
N
MET
A
824
7.352
7.488
3.188
1.00
12.40
A

ATOM
1556
CA
MET
A
824
8.755
7.710
2.883
1.00
12.87
A

ATOM
1557
CB
MET
A
824
8.938
8.982
2.070
1.00
13.48
A

ATOM
1558
CG
MET
A
824
8.457
8.904
0.641
1.00
11.06
A

ATOM
1559
SD
MET
A
824
8.815
10.500
−0.170
1.00
15.45
A

ATOM
1560
CE
MET
A
824
10.625
10.460
−0.293
1.00
14.21
A

ATOM
1561
C
MET
A
824
9.506
7.876
4.181
1.00
11.22
A

ATOM
1562
O
MET
A
824
10.632
7.411
4.318
1.00
12.72
A

ATOM
1563
N
TRP
A
825
8.882
8.550
5.135
1.00
13.43
A

ATOM
1564
CA
TRP
A
825
9.532
8.760
6.416
1.00
16.39
A

ATOM
1565
CB
TRP
A
825
8.733
9.758
7.251
1.00
17.69
A

ATOM
1566
CG
TRP
A
825
9.485
10.300
8.429
1.00
21.01
A

ATOM
1567
CD2
TRP
A
825
9.404
9.836
9.783
1.00
22.74
A

ATOM
1568
CE2
TRP
A
825
10.267
10.646
10.556
1.00
24.73
A

ATOM
1569
CE3
TRP
A
825
8.686
8.816
10.417
1.00
24.55
A

ATOM
1570
CD1
TRP
A
825
10.372
11.338
8.433
1.00
22.10
A

ATOM
1571
NE1
TRP
A
825
10.842
11.554
9.708
1.00
23.09
A

ATOM
1572
CZ2
TRP
A
825
10.429
10.469
11.937
1.00
23.91
A

ATOM
1573
CZ3
TRP
A
825
8.848
8.641
11.791
1.00
24.02
A

ATOM
1574
CH2
TRP
A
825
9.713
9.464
12.532
1.00
24.58
A

ATOM
1575
C
TRP
A
825
9.651
7.415
7.143
1.00
16.44
A

ATOM
1576
O
TRP
A
825
10.718
7.074
7.648
1.00
15.71
A

ATOM
1577
N
GLU
A
826
8.564
6.645
7.185
1.00
15.79
A

ATOM
1578
CA
GLU
A
826
8.597
5.338
7.854
1.00
15.47
A

ATOM
1579
CB
GLU
A
826
7.266
4.609
7.685
1.00
15.67
A

ATOM
1580
CG
GLU
A
826
6.066
5.409
8.128
1.00
17.54
A

ATOM
1581
CD
GLU
A
826
4.797
4.603
8.027
1.00
17.43
A

ATOM
1582
OE1
GLU
A
826
4.561
3.760
8.918
1.00
17.18
A

ATOM
1583
OE2
GLU
A
826
4.047
4.793
7.054
1.00
19.83
A

ATOM
1584
C
GLU
A
826
9.698
4.459
7.297
1.00
15.72
A

ATOM
1585
O
GLU
A
826
10.430
3.809
8.043
1.00
14.73
A

ATOM
1586
N
VAL
A
827
9.807
4.448
5.973
1.00
14.75
A

ATOM
1587
CA
VAL
A
827
10.812
3.652
5.293
1.00
13.80
A

ATOM
1588
CB
VAL
A
827
10.605
3.688
3.776
1.00
13.84
A

ATOM
1589
CG1
VAL
A
827
11.864
3.166
3.064
1.00
15.75
A

ATOM
1590
CG2
VAL
A
827
9.395
2.845
3.404
1.00
12.52
A

ATOM
1591
C
VAL
A
827
12.241
4.107
5.598
1.00
15.22
A

ATOM
1592
O
VAL
A
827
13.114
3.282
5.855
1.00
15.68
A

ATOM
1593
N
MET
A
828
12.488
5.411
5.571
1.00
15.64
A

ATOM
1594
CA
MET
A
828
13.840
5.885
5.830
1.00
16.82
A

ATOM
1595
CB
MET
A
828
14.020
7.304
5.282
1.00
16.69
A

ATOM
1596
CG
MET
A
828
13.812
7.400
3.764
1.00
15.12
A

ATOM
1597
SD
MET
A
828
14.617
6.077
2.862
1.00
17.16
A

ATOM
1598
CE
MET
A
828
16.377
6.500
3.177
1.00
16.75
A

ATOM
1599
C
MET
A
828
14.182
5.815
7.319
1.00
17.74
A

ATOM
1600
O
MET
A
828
15.353
5.818
7.697
1.00
17.30
A

ATOM
1601
N
SER
A
829
13.146
5.731
8.146
1.00
16.25
A

ATOM
1602
CA
SER
A
829
13.287
5.639
9.602
1.00
19.49
A

ATOM
1603
CS
SER
A
829
12.129
6.360
10.296
1.00
19.11
A

ATOM
1604
OG
SER
A
829
12.153
7.747
10.028
1.00
26.80
A

ATOM
1605
C
SER
A
829
13.263
4.186
10.069
1.00
17.50
A

ATOM
1606
O
SER
A
829
13.340
3.921
11.264
1.00
19.93
A

ATOM
1607
N
TYR
A
830
13.157
3.265
9.118
1.00
15.75
A

ATOM
1608
CA
TYR
A
830
13.053
1.837
9.382
1.00
15.79
A

ATOM
1609
CB
TYR
A
830
14.364
1.250
9.930
1.00
15.94
A

ATOM
1610
CG
TYR
A
830
15.392
0.987
8.861
1.00
15.43
A

ATOM
1611
CD1
TYR
A
830
16.370
1.925
8.566
1.00
19.30
A

ATOM
1612
CE1
TYR
A
830
17.330
1.682
7.577
1.00
17.62
A

ATOM
1613
CD2
TYR
A
830
15.387
−0.202
8.141
1.00
15.96
A

ATOM
1614
CE2
TYR
A
830
16.332
−0.454
7.156
1.00
17.55
A

ATOM
1615
CZ
TYR
A
830
17.304
0.494
6.883
1.00
16.47
A

ATOM
1616
OH
TYR
A
830
18.260
0.225
5.930
1.00
15.91
A

ATOM
1617
C
TYR
A
830
11.889
1.459
10.298
1.00
16.77
A

ATOM
1618
O
TYR
A
830
12.051
0.715
11.273
1.00
14.45
A

ATOM
1619
N
GLY
A
831
10.707
1.979
9.979
1.00
14.09
A

ATOM
1620
CA
GLY
A
831
9.521
1.635
10.742
1.00
17.12
A

ATOM
1621
C
GLY
A
831
9.224
2.407
12.003
1.00
19.41
A

ATOM
1622
O
GLY
A
831
8.410
1.977
12.827
1.00
19.28
A

ATOM
1623
N
GLU
A
832
9.883
3.545
12.171
1.00
21.71
A

ATOM
1624
CA
GLU
A
832
9.633
4.365
13.340
1.00
23.50
A

ATOM
1625
CB
GLU
A
832
10.627
5.522
13.384
1.00
25.11
A

ATOM
1626
CG
GLU
A
832
10.396
6.521
14.496
1.00
26.95
A

ATOM
1627
CD
GLU
A
832
10.411
5.869
15.874
1.00
32.36
A

ATOM
1628
OE1
GLU
A
832
9.344
5.380
16.326
1.00
31.18
A

ATOM
1629
OE2
GLU
A
832
11.497
5.834
16.500
1.00
32.72
A

ATOM
1630
C
GLU
A
832
8.210
4.897
13.216
1.00
24.54
A

ATOM
1631
O
GLU
A
832
7.668
4.981
12.111
1.00
22.49
A

ATOM
1632
N
ARG
A
833
7.608
5.249
14.347
1.00
24.48
A

ATOM
1633
CA
ARG
A
833
6.252
5.780
14.351
1.00
26.11
A

ATOM
1634
CB
ARG
A
833
5.597
5.564
15.720
1.00
28.74
A

ATOM
1635
CG
ARG
A
833
4.146
6.014
15.794
1.00
31.71
A

ATOM
1636
CD
ARG
A
833
3.443
5.386
16.985
1.00
34.54
A

ATOM
1637
NE
ARG
A
833
2.023
5.716
17.036
1.00
36.70
A

ATOM
1638
CZ
ARG
A
833
1.538
6.853
17.521
1.00
38.93
A

ATOM
1639
NH1
ARG
A
833
2.360
7.776
17.998
1.00
40.22
A

ATOM
1640
NH2
ARG
A
833
0.230
7.063
17.538
1.00
39.85
A

ATOM
1641
C
ARG
A
833
6.280
7.266
14.019
1.00
26.85
A

ATOM
1642
O
ARG
A
833
7.007
8.038
14.641
1.00
28.24
A

ATOM
1643
N
PRO
A
834
5.495
7.683
13.018
1.00
25.28
A

ATOM
1644
CD
PRO
A
834
4.678
6.857
12.112
1.00
26.97
A

ATOM
1645
CA
PRO
A
834
5.450
9.090
12.624
1.00
24.70
A

ATOM
1646
CB
PRO
A
834
4.381
9.108
11.534
1.00
25.64
A

ATOM
1647
CG
PRO
A
834
4.533
7.759
10.896
1.00
24.55
A

ATOM
1648
C
PRO
A
834
5.086
9.984
13.802
1.00
25.05
A

ATOM
1649
O
PRO
A
834
4.091
9.742
14.487
1.00
23.06
A

ATOM
1650
N
TYR
A
835
5.902
11.013
14.023
1.00
26.38
A

ATOM
1651
CA
TYR
A
835
5.704
11.977
15.102
1.00
26.19
A

ATOM
1652
CD
TYR
A
835
4.303
12.584
15.002
1.00
26.82
A

ATOM
1653
CG
TYR
A
835
4.015
13.202
13.648
1.00
27.32
A

ATOM
1654
CD1
TYR
A
835
4.405
14.511
13.358
1.00
26.52
A

ATOM
1655
CE1
TYR
A
835
4.175
15.069
12.101
1.00
26.25
A

ATOM
1656
CD2
TYR
A
835
3.387
12.464
12.647
1.00
25.35
A

ATOM
1657
CE2
TYR
A
835
3.153
13.008
11.386
1.00
25.46
A

ATOM
1658
CZ
TYR
A
835
3.550
14.314
11.116
1.00
26.18
A

ATOM
1659
OH
TYR
A
835
3.325
14.859
9.867
1.00
25.65
A

ATOM
1660
C
TYR
A
835
5.933
11.349
16.477
1.00
27.36
A

ATOM
1661
O
TYR
A
835
5.554
11.912
17.505
1.00
28.19
A

ATOM
1662
N
TRP
A
836
6.554
10.174
16.475
1.00
26.91
A

ATOM
1663
CA
TRP
A
836
6.885
9.454
17.699
1.00
28.58
A

ATOM
1664
CB
TRP
A
836
8.097
10.114
18.356
1.00
27.99
A

ATOM
1665
CG
TRP
A
836
9.233
10.282
17.406
1.00
29.96
A

ATOM
1666
CD2
TRP
A
836
9.533
11.449
16.631
1.00
29.72
A

ATOM
1667
CE2
TRP
A
836
10.666
11.149
15.847
1.00
30.62
A

ATOM
1668
CE3
TRP
A
836
8.953
12.722
16.525
1.00
31.28
A

ATOM
1669
CD1
TRP
A
836
10.169
9.346
17.069
1.00
29.72
A

ATOM
1670
NE1
TRP
A
836
11.034
9.859
16.133
1.00
29.17
A

ATOM
1671
CZ2
TRP
A
836
11.235
12.076
14.965
1.00
30.51
A

ATOM
1672
CZ3
TRP
A
836
9.520
13.646
15.648
1.00
30.96
A

ATOM
1673
CH2
TRP
A
836
10.649
13.315
14.882
1.00
31.70
A

ATOM
1674
C
TRP
A
836
5.727
9.370
18.688
1.00
29.62
A

ATOM
1675
O
TRP
A
836
4.627
8.945
18.328
1.00
29.84
A

ATOM
1676
N
ASP
A
837
5.977
9.789
19.927
1.00
31.12
A

ATOM
1677
CA
ASP
A
837
4.972
9.743
20.992
1.00
34.13
A

ATOM
1678
CB
ASP
A
837
5.659
9.720
22.365
1.00
34.74
A

ATOM
1679
CG
ASP
A
837
6.593
8.536
22.533
0.00
35.23
A

ATOM
1680
OD1
ASP
A
837
6.132
7.384
22.389
0.00
35.57
A

ATOM
1681
OD2
ASP
A
837
7.790
8.758
22.811
0.00
35.57
A

ATOM
1682
C
ASP
A
837
3.954
10.879
20.969
1.00
35.08
A

ATOM
1683
O
ASP
A
837
3.129
10.998
21.875
1.00
35.37
A

ATOM
1684
N
MET
A
838
4.009
11.722
19.946
1.00
36.64
A

ATOM
1685
CA
MET
A
838
3.056
12.821
19.845
1.00
37.58
A

ATOM
1686
CB
MET
A
838
3.315
13.648
18.594
1.00
37.74
A

ATOM
1687
CG
MET
A
838
4.207
14.831
18.800
1.00
37.49
A

ATOM
1688
SD
MET
A
838
4.120
15.864
17.349
1.00
40.87
A

ATOM
1689
CE
MET
A
838
5.651
15.422
16.541
1.00
37.80
A

ATOM
1690
C
MET
A
838
1.638
12.295
19.771
1.00
38.85
A

ATOM
1691
O
MET
A
838
1.392
11.235
19.196
1.00
38.49
A

ATOM
1692
N
THR
A
839
0.708
13.052
20.345
1.00
40.77
A

ATOM
1693
CA
THR
A
839
−0.707
12.691
20.332
1.00
42.26
A

ATOM
1694
CB
THR
A
839
−1.470
13.372
21.493
1.00
42.77
A

ATOM
1695
OG1
THR
A
839
−1.599
14.773
21.224
0.00
42.95
A

ATOM
1696
CG2
THR
A
839
−0.719
13.188
22.806
0.00
42.95
A

ATOM
1697
C
THR
A
839
−1.289
13.184
19.007
1.00
42.46
A

ATOM
1698
O
THR
A
839
−0.688
14.030
18.348
1.00
42.49
A

ATOM
1699
N
ASN
A
840
−2.450
12.660
18.616
1.00
43.26
A

ATOM
1700
CA
ASN
A
840
−3.078
13.075
17.363
1.00
44.27
A

ATOM
1701
CB
ASN
A
840
−4.419
12.360
17.154
1.00
43.58
A

ATOM
1702
CG
ASN
A
840
−4.256
10.885
16.855
1.00
43.42
A

ATOM
1703
OD1
ASN
A
840
−3.230
10.460
16.328
1.00
42.91
A

ATOM
1704
ND2
ASN
A
840
−5.278
10.097
17.172
1.00
44.00
A

ATOM
1705
C
ASN
A
840
−3.299
14.585
17.327
1.00
44.84
A

ATOM
1706
O
ASN
A
840
−3.017
15.236
16.321
1.00
45.54
A

ATOM
1707
N
GLN
A
841
−3.798
15.141
18.427
1.00
45.95
A

ATOM
1708
CA
GLN
A
841
−4.046
16.573
18.497
1.00
46.24
A

ATOM
1709
CB
GLN
A
841
−4.718
16.947
19.818
1.00
47.54
A

ATOM
1710
CG
GLN
A
841
−5.283
18.356
19.818
1.00
48.27
A

ATOM
1711
CD
GLN
A
841
−6.207
18.594
18.638
1.00
49.43
A

ATOM
1712
OE1
GLN
A
841
−7.199
17.884
18.460
1.00
49.85
A

ATOM
1713
NE2
GLN
A
841
−5.882
19.591
17.820
1.00
49.98
A

ATOM
1714
C
GLN
A
841
−2.744
17.347
18.350
1.00
46.35
A

ATOM
1715
O
GLN
A
841
−2.702
18.373
17.673
1.00
46.78
A

ATOM
1716
N
ASP
A
842
−1.682
16.861
18.984
1.00
45.35
A

ATOM
1717
CA
ASP
A
842
−0.396
17.531
18.878
1.00
45.11
A

ATOM
1718
CB
ASP
A
842
0.651
16.836
19.745
1.00
47.46
A

ATOM
1719
CG
ASP
A
842
0.324
16.913
21.224
1.00
49.71
A

ATOM
1720
OD1
ASP
A
842
−0.182
17.971
21.667
1.00
50.47
A

ATOM
1721
OD2
ASP
A
842
0.583
15.923
21.942
1.00
50.96
A

ATOM
1722
C
ASP
A
842
0.059
17.535
17.424
1.00
43.89
A

ATOM
1723
O
ASP
A
842
0.540
18.549
16.923
1.00
43.19
A

ATOM
1724
N
VAL
A
843
−0.099
16.397
16.752
1.00
42.58
A

ATOM
1725
CA
VAL
A
843
0.285
16.281
15.351
1.00
41.17
A

ATOM
1726
CB
VAL
A
843
−0.012
14.869
14.795
1.00
41.26
A

ATOM
1727
CG1
VAL
A
843
0.278
14.828
13.308
1.00
39.84
A

ATOM
1728
CG2
VAL
A
843
0.831
13.829
15.530
1.00
41.01
A

ATOM
1729
C
VAL
A
843
−0.481
17.301
14.518
1.00
40.78
A

ATOM
1730
O
VAL
A
843
0.103
18.029
13.714
1.00
38.98
A

ATOM
1731
N
ILE
A
844
−1.795
17.340
14.717
1.00
40.52
A

ATOM
1732
CA
ILE
A
844
−2.657
18.269
14.003
1.00
41.21
A

ATOM
1733
CB
ILE
A
844
−4.123
18.129
14.470
1.00
40.73
A

ATOM
1734
CG2
ILE
A
844
−4.995
19.190
13.800
1.00
40.70
A

ATOM
1735
CG1
ILE
A
844
−4.625
16.716
14.160
1.00
40.24
A

ATOM
1736
CD1
ILE
A
844
−6.004
16.413
14.694
1.00
39.42
A

ATOM
1737
C
ILE
A
844
−2.196
19.706
14.237
1.00
42.20
A

ATOM
1738
O
ILE
A
844
−2.035
20.478
13.289
1.00
42.32
A

ATOM
1739
N
ASN
A
845
−1.988
20.059
15.502
1.00
42.47
A

ATOM
1740
CA
ASN
A
845
−1.550
21.405
15.845
1.00
43.99
A

ATOM
1741
CB
ASN
A
845
−1.525
21.606
17.361
1.00
45.43
A

ATOM
1742
CG
ASN
A
845
−2.885
21.417
17.998
1.00
46.85
A

ATOM
1743
OD1
ASN
A
845
−3.903
21.851
17.458
1.00
47.77
A

ATOM
1744
ND2
ASN
A
845
−2.908
20.779
19.163
1.00
48.25
A

ATOM
1745
C
ASN
A
845
−0.162
21.664
15.286
1.00
43.59
A

ATOM
1746
O
ASN
A
845
0.123
22.750
14.784
1.00
43.60
A

ATOM
1747
N
ALA
A
846
0.702
20.660
15.376
1.00
42.78
A

ATOM
1748
CA
ALA
A
846
2.057
20.793
14.870
1.00
42.00
A

ATOM
1749
CB
ALA
A
846
2.829
19.497
15.092
1.00
41.98
A

ATOM
1750
C
ALA
A
846
2.021
21.142
13.386
1.00
41.78
A

ATOM
1751
O
ALA
A
846
2.662
22.100
12.954
1.00
41.69
A

ATOM
1752
N
ILE
A
847
1.269
20.364
12.609
1.00
41.74
A

ATOM
1753
CA
ILE
A
847
1.155
20.600
11.173
1.00
41.99
A

ATOM
1754
CB
ILE
A
847
0.250
19.540
10.492
1.00
41.47
A

ATOM
1755
CG2
ILE
A
847
0.083
19.863
9.013
1.00
41.65
A

ATOM
1756
CG1
ILE
A
847
0.869
18.148
10.641
1.00
41.58
A

ATOM
1757
CD1
ILE
A
847
2.243
18.014
10.006
1.00
41.53
A

ATOM
1758
C
ILE
A
847
0.584
21.990
10.906
1.00
42.01
A

ATOM
1759
O
ILE
A
847
1.032
22.685
9.996
1.00
41.61
A

ATOM
1760
N
GLU
A
848
−0.402
22.388
11.704
1.00
42.81
A

ATOM
1761
CA
GLU
A
848
−1.022
23.700
11.563
1.00
43.79
A

ATOM
1762
CB
GLU
A
848
−2.158
23.866
12.576
1.00
43.97
A

ATOM
1763
CG
GLU
A
848
−3.380
23.013
12.281
0.00
44.75
A

ATOM
1764
CD
GLU
A
848
−4.518
23.276
13.247
0.00
45.09
A

ATOM
1765
OE1
GLU
A
848
−4.985
24.432
13.317
0.00
45.34
A

ATOM
1766
OE2
GLU
A
848
−4.947
22.326
13.935
0.00
45.34
A

ATOM
1767
C
GLU
A
848
0.011
24.806
11.760
1.00
44.25
A

ATOM
1768
O
GLU
A
848
−0.033
25.830
11.082
1.00
44.97
A

ATOM
1769
N
GLN
A
849
0.939
24.588
12.689
1.00
43.53
A

ATOM
1770
CA
GLN
A
849
1.997
25.552
12.974
1.00
43.30
A

ATOM
1771
CE
GLN
A
849
2.554
25.328
14.380
1.00
43.26
A

ATOM
1772
CG
GLN
A
849
1.549
25.528
15.492
0.00
44.28
A

ATOM
1773
CD
GLN
A
849
1.042
26.951
15.557
0.00
44.64
A

ATOM
1774
OE1
GLN
A
849
1.816
27.889
15.745
0.00
44.94
A

ATOM
1775
NE2
GLN
A
849
−0.264
27.121
15.399
0.00
44.94
A

ATOM
1776
C
GLN
A
849
3.126
25.402
11.959
1.00
43.40
A

ATOM
1777
O
GLN
A
849
4.252
25.842
12.199
1.00
43.15
A

ATOM
1778
N
ASP
A
850
2.814
24.770
10.830
1.00
43.13
A

ATOM
1779
CA
ASP
A
850
3.780
24.545
9.760
1.00
42.79
A

ATOM
1780
CE
ASP
A
850
4.287
25.888
9.222
1.00
43.87
A

ATOM
1781
CG
ASP
A
850
3.260
26.586
8.350
1.00
45.00
A

ATOM
1782
OD1
ASP
A
850
2.980
26.080
7.245
1.00
47.53
A

ATOM
1783
OD2
ASP
A
850
2.724
27.634
8.767
1.00
45.98
A

ATOM
1784
C
ASP
A
850
4.957
23.652
10.162
1.00
41.93
A

ATOM
1785
O
ASP
A
850
6.064
23.796
9.651
1.00
42.38
A

ATOM
1786
N
TYR
A
851
4.709
22.718
11.073
1.00
40.80
A

ATOM
1787
CA
TYR
A
851
5.752
21.795
11.512
1.00
39.73
A

ATOM
1788
CE
TYR
A
851
5.481
21.311
12.930
1.00
39.91
A

ATOM
1789
CG
TYR
A
851
6.348
20.139
13.323
1.00
39.84
A

ATOM
1790
CD1
TYR
A
851
7.674
20.327
13.710
1.00
40.12
A

ATOM
1791
CE1
TYR
A
851
8.478
19.249
14.062
1.00
39.79
A

ATOM
1792
CD2
TYR
A
851
5.847
18.837
13.294
1.00
40.19
A

ATOM
1793
CE2
TYR
A
851
6.643
17.751
13.642
1.00
39.38
A

ATOM
1794
CZ
TYR
A
851
7.955
17.964
14.027
1.00
39.62
A

ATOM
1795
OH
TYR
A
851
8.742
16.899
14.388
1.00
36.75
A

ATOM
1796
C
TYR
A
851
5.811
20.577
10.592
1.00
37.96
A

ATOM
1797
O
TYR
A
851
4.778
20.053
10.185
1.00
39.15
A

ATOM
1798
N
ARG
A
852
7.018
20.127
10.273
1.00
35.12
A

ATOM
1799
CA
ARG
A
852
7.185
18.961
9.419
1.00
33.58
A

ATOM
1800
CE
ARG
A
852
7.568
19.394
8.003
1.00
32.00
A

ATOM
1801
CG
ARG
A
852
6.478
20.173
7.287
1.00
31.55
A

ATOM
1802
CD
ARG
A
852
5.271
19.293
7.006
1.00
29.23
A

ATOM
1803
NE
ARC
A
852
4.254
19.976
6.206
1.00
30.58
A

ATOM
1804
CZ
ARG
A
852
3.358
20.835
6.688
1.00
28.76
A

ATOM
1805
NE1
ARG
A
852
3.336
21.132
7.980
1.00
27.10
A

ATOM
1806
NH2
ARC
A
852
2.476
21.393
5.876
1.00
28.74
A

ATOM
1807
C
ARG
A
852
8.253
18.033
9.992
1.00
32.81
A

ATOM
1808
O
ARG
A
852
9.260
18.490
10.524
1.00
33.32
A

ATOM
1809
N
LEU
A
853
8.025
16.729
9.881
1.00
31.16
A

ATOM
1810
CA
LEU
A
853
8.963
15.741
10.392
1.00
29.31
A

ATOM
1811
CB
LEU
A
853
8.493
14.330
10.040
1.00
28.71
A

ATOM
1812
CG
LEU
A
853
7.220
13.837
10.730
1.00
31.23
A

ATOM
1813
CD1
LEU
A
853
6.775
12.514
10.090
1.00
29.70
A

ATOM
1814
CD2
LEU
A
853
7.472
13.662
12.227
1.00
31.09
A

ATOM
1815
C
LEU
A
853
10.360
15.959
9.832
1.00
29.46
A

ATOM
1816
O
LEU
A
853
10.534
16.191
8.633
1.00
27.80
A

ATOM
1817
N
PRO
A
854
11.379
15.878
10.700
1.00
29.14
A

ATOM
1818
CD
PRO
A
854
11.277
15.586
12.144
1.00
29.31
A

ATOM
1819
CA
PRO
A
854
12.775
16.063
10.302
1.00
29.67
A

ATOM
1820
CB
PRO
A
854
13.487
16.207
11.641
1.00
29.68
A

ATOM
1821
CG
PRO
A
854
12.715
15.259
12.514
1.00
29.75
A

ATOM
1822
C
PRO
A
854
13.278
14.861
9.511
1.00
29.68
A

ATOM
1823
O
PRO
A
854
12.659
13.803
9.520
1.00
29.00
A

ATOM
1824
N
PRO
A
855
14.415
15.008
8.820
1.00
30.63
A

ATOM
1825
CD
PRO
A
855
15.242
16.220
8.668
1.00
30.11
A

ATOM
1826
CA
PRO
A
855
14.965
13.897
8.040
1.00
30.85
A

ATOM
1827
CB
PRO
A
855
16.089
14.561
7.246
1.00
31.70
A

ATOM
1828
CG
PRO
A
855
16.548
15.653
8.163
1.00
29.94
A

ATOM
1829
C
PRO
A
855
15.479
12.757
8.919
1.00
32.90
A

ATOM
1830
O
PRO
A
855
16.201
12.990
9.886
1.00
33.00
A

ATOM
1831
N
PRO
A
856
15.092
11.508
8.602
1.00
33.31
A

ATOM
1832
CD
PRO
A
856
14.065
11.113
7.620
1.00
32.70
A

ATOM
1833
CA
PRO
A
856
15.542
10.348
9.380
1.00
34.07
A

ATOM
1834
CM
PRO
A
856
14.894
9.172
8.649
1.00
33.02
A

ATOM
1835
CG
PRO
A
856
13.617
9.767
8.158
1.00
33.24
A

ATOM
1836
C
PRO
A
856
17.073
10.258
9.374
1.00
34.59
A

ATOM
1837
O
PRO
A
856
17.732
10.793
8.484
1.00
34.67
A

ATOM
1838
N
MET
A
857
17.633
9.571
10.363
1.00
35.13
A

ATOM
1839
CA
MET
A
857
19.081
9.429
10.467
1.00
34.34
A

ATOM
1840
CM
MET
A
857
19.432
8.523
11.650
1.00
34.60
A

ATOM
1841
CG
MET
A
857
20.891
8.620
12.117
1.00
36.07
A

ATOM
1842
SD
MET
A
857
21.222
7.511
13.500
0.00
36.17
A

ATOM
1843
CE
MET
A
857
20.786
8.554
14.888
0.00
36.58
A

ATOM
1844
C
MET
A
857
19.673
8.865
9.178
1.00
33.94
A

ATOM
1845
O
MET
A
857
19.195
7.858
8.643
1.00
32.79
A

ATOM
1846
N
ASP
A
858
20.713
9.533
8.685
1.00
32.95
A

ATOM
1847
CA
ASP
A
858
21.398
9.120
7.463
1.00
32.12
A

ATOM
1848
CB
ASP
A
858
21.957
7.697
7.631
1.00
33.12
A

ATOM
1849
CG
ASP
A
858
22.948
7.591
8.778
0.00
33.41
A

ATOM
1850
OD1
ASP
A
858
23.981
8.292
8.740
0.00
33.74
A

ATOM
1851
OD2
ASP
A
858
22.694
6.809
9.719
0.00
33.74
A

ATOM
1852
C
ASP
A
858
20.500
9.181
6.227
1.00
30.87
A

ATOM
1853
O
ASP
A
858
20.787
8.546
5.212
1.00
31.21
A

ATOM
1854
N
CYS
A
859
19.420
9.951
6.299
1.00
29.14
A

ATOM
1855
CA
CYS
A
859
18.514
10.056
5.158
1.00
28.23
A

ATOM
1856
CM
CYS
A
859
17.091
10.370
5.620
1.00
27.03
A

ATOM
1857
SG
CYS
A
859
15.950
10.675
4.219
1.00
25.20
A

ATOM
1858
C
CYS
A
859
18.951
11.128
4.172
1.00
26.49
A

ATOM
1859
O
CYS
A
859
19.063
12.297
4.535
1.00
27.20
A

ATOM
1860
N
PRO
A
860
19.187
10.747
2.905
1.00
25.74
A

ATOM
1861
CD
PRO
A
860
19.019
9.402
2.329
1.00
25.44
A

ATOM
1862
CA
PRO
A
860
19.610
11.703
1.877
1.00
25.49
A

ATOM
1863
CB
PRO
A
860
19.450
10.911
0.584
1.00
26.82
A

ATOM
1864
CG
PRO
A
860
19.754
9.518
1.013
1.00
25.74
A

ATOM
1865
C
PRO
A
860
18.750
12.965
1.875
1.00
25.80
A

ATOM
1866
O
PRO
A
860
17.528
12.887
2.037
1.00
23.12
A

ATOM
1867
N
SER
A
861
19.383
14.123
1.685
1.00
24.04
A

ATOM
1868
CA
SER
A
861
18.651
15.390
1.661
1.00
24.93
A

ATOM
1869
CB
SER
A
861
19.597
16.579
1.437
1.00
24.17
A

ATOM
1870
OG
SER
A
861
20.464
16.754
2.533
1.00
28.72
A

ATOM
1871
C
SER
A
861
17.596
15.405
0.558
1.00
22.46
A

ATOM
1872
O
SER
A
861
16.491
15.887
0.765
1.00
23.97
A

ATOM
1873
N
ALA
A
862
17.937
14.886
−0.612
1.00
22.99
A

ATOM
1874
CA
ALA
A
862
16.990
14.879
−1.715
1.00
23.51
A

ATOM
1875
CB
ALA
A
862
17.626
14.254
−2.962
1.00
25.30
A

ATOM
1876
C
ALA
A
862
15.720
14.121
−1.327
1.00
23.45
A

ATOM
1877
O
ALA
A
862
14.619
14.549
−1.667
1.00
22.65
A

ATOM
1878
N
LEU
A
863
15.870
13.004
−0.615
1.00
21.36
A

ATOM
1879
CA
LEU
A
863
14.703
12.232
−0.186
1.00
20.64
A

ATOM
1880
CB
LEU
A
863
15.110
10.907
0.470
1.00
17.21
A

ATOM
1881
CG
LEU
A
863
15.473
9.807
−0.522
1.00
17.62
A

ATOM
1882
CD1
LEU
A
863
15.995
8.573
0.220
1.00
19.11
A

ATOM
1883
CD2
LEU
A
863
14.236
9.465
−1.353
1.00
16.32
A

ATOM
1884
C
LEU
A
863
13.821
13.010
0.774
1.00
19.07
A

ATOM
1885
O
LEU
A
863
12.595
13.005
0.641
1.00
20.62
A

ATOM
1886
N
HIS
A
864
14.434
13.683
1.741
1.00
19.85
A

ATOM
1887
CA
HIS
A
864
13.643
14.448
2.686
1.00
18.66
A

ATOM
1888
CB
HIS
A
864
14.496
14.954
3.847
1.00
19.81
A

ATOM
1889
CG
HIS
A
864
13.700
15.626
4.922
1.00
19.98
A

ATOM
1890
CD2
HIS
A
864
12.826
15.127
5.828
1.00
20.76
A

ATOM
1891
ND1
HIS
A
864
13.735
16.988
5.133
1.00
21.33
A

ATOM
1892
CE1
HIS
A
864
12.919
17.299
6.125
1.00
22.47
A

ATOM
1893
NE2
HIS
A
864
12.355
16.187
6.564
1.00
22.76
A

ATOM
1894
C
HIS
A
864
12.968
15.619
1.983
1.00
19.62
A

ATOM
1895
O
HIS
A
864
11.897
16.052
2.366
1.00
19.61
A

ATOM
1896
N
GLN
A
865
13.641
16.143
0.952
1.00
19.53
A

ATOM
1897
CA
GLN
A
865
13.039
17.259
0.238
1.00
19.79
A

ATOM
1898
CB
GLN
A
865
13.977
17.808
−0.837
1.00
19.80
A

ATOM
1899
CG
GLN
A
865
13.379
18.989
−1.567
1.00
21.23
A

ATOM
1900
CD
GLN
A
865
13.013
20.114
−0.618
1.00
22.98
A

ATOM
1901
OE1
GLN
A
865
13.868
20.627
0.105
1.00
26.38
A

ATOM
1902
NE2
GLN
A
865
11.739
20.504
−0.613
1.00
21.44
A

ATOM
1903
C
GLN
A
865
11.766
16.750
−0.416
1.00
19.00
A

ATOM
1904
O
GLN
A
865
10.735
17.419
−0.396
1.00
19.67
A

ATOM
1905
N
LEU
A
866
11.841
15.559
−0.997
1.00
19.26
A

ATOM
1906
CA
LEU
A
866
10.663
14.988
−1.632
1.00
18.45
A

ATOM
1907
CB
LEU
A
866
11.014
13.649
−2.283
1.00
18.07
A

ATOM
1908
CG
LEU
A
866
9.887
12.930
−3.021
1.00
19.78
A

ATOM
1909
CD1
LEU
A
866
9.168
13.903
−3.967
1.00
18.91
A

ATOM
1910
CD2
LEU
A
866
10.467
11.754
−3.775
1.00
20.97
A

ATOM
1911
C
LEU
A
866
9.546
14.824
−0.589
1.00
18.47
A

ATOM
1912
O
LEU
A
866
8.367
15.026
−0.890
1.00
17.51
A

ATOM
1913
N
MET
A
867
9.921
14.476
0.641
1.00
18.09
A

ATOM
1914
CA
MET
A
867
8.946
14.318
1.711
1.00
18.01
A

ATOM
1915
CB
MET
A
867
9.632
13.865
3.005
1.00
19.66
A

ATOM
1916
CG
MET
A
867
10.077
12.419
2.991
1.00
20.12
A

ATOM
1917
SD
MET
A
867
10.968
11.976
4.495
1.00
22.40
A

ATOM
1918
CE
MET
A
867
12.235
10.954
3.814
1.00
22.54
A

ATOM
1919
C
MET
A
867
8.228
15.640
1.965
1.00
18.98
A

ATOM
1920
O
MET
A
867
6.996
15.686
2.058
1.00
17.83
A

ATOM
1921
N
LEU
A
868
9.007
16.712
2.089
1.00
18.92
A

ATOM
1922
CA
LEU
A
868
8.447
18.042
2.326
1.00
20.03
A

ATOM
1923
CB
LEU
A
868
9.566
19.083
2.455
1.00
21.62
A

ATOM
1924
CG
LEU
A
868
10.538
18.913
3.628
1.00
22.08
A

ATOM
1925
CD1
LEU
A
868
11.607
20.012
3.578
1.00
21.68
A

ATOM
1926
CD2
LEU
A
868
9.772
18.989
4.942
1.00
18.97
A

ATOM
1927
C
LEU
A
868
7.506
18.444
1.191
1.00
19.33
A

ATOM
1928
O
LEU
A
868
6.501
19.111
1.420
1.00
20.63
A

ATOM
1929
N
ASP
A
869
7.836
18.037
−0.031
1.00
17.79
A

ATOM
1930
CA
ASP
A
869
7.001
18.360
−1.183
1.00
18.56
A

ATOM
1931
CB
ASP
A
869
7.707
17.971
−2.484
1.00
17.65
A

ATOM
1932
CG
ASP
A
869
8.988
18.762
−2.703
1.00
18.80
A

ATOM
1933
OD1
ASP
A
869
9.175
19.790
−2.021
1.00
16.81
A

ATOM
1934
OD2
ASP
A
869
9.799
18.364
−3.557
1.00
18.79
A

ATOM
1935
C
ASP
A
869
5.664
17.651
−1.072
1.00
20.40
A

ATOM
1936
O
ASP
A
869
4.631
18.197
−1.460
1.00
21.71
A

ATOM
1937
N
CYS
A
870
5.682
16.439
−0.524
1.00
18.78
A

ATOM
1938
CA
CYS
A
870
4.456
15.670
−0.354
1.00
19.36
A

ATOM
1939
CB
CYS
A
870
4.769
14.201
−0.039
1.00
15.21
A

ATOM
1940
SO
CYS
A
870
5.477
13.278
−1.422
1.00
17.93
A

ATOM
1941
C
CYS
A
870
3.616
16.256
0.769
1.00
16.15
A

ATOM
1942
O
CYS
A
870
2.390
16.090
0.785
1.00
20.18
A

ATOM
1943
N
TRP
A
871
4.259
16.956
1.702
1.00
19.04
A

ATOM
1944
CA
TRP
A
871
3.529
17.534
2.833
1.00
20.40
A

ATOM
1945
CB
TRP
A
871
4.306
17.335
4.151
1.00
20.01
A

ATOM
1946
CG
TRP
A
871
4.663
15.890
4.480
1.00
21.12
A

ATOM
1947
CD2
TRP
A
871
5.874
15.424
5.095
1.00
19.64
A

ATOM
1948
CE2
TRP
A
871
5.778
14.014
5.204
1.00
18.64
A

ATOM
1949
CE3
TRP
A
871
7.029
16.060
5.563
1.00
18.54
A

ATOM
1950
CD1
TRP
A
871
3.897
14.770
4.254
1.00
21.26
A

ATOM
1951
NE1
TRP
A
871
4.567
13.642
4.685
1.00
19.40
A

ATOM
1952
CZ2
TRP
A
871
6.797
13.232
5.762
1.00
18.78
A

ATOM
1953
CZ3
TRP
A
871
8.045
15.282
6.120
1.00
19.69
A

ATOM
1954
CH2
TRP
A
871
7.920
13.878
6.214
1.00
19.97
A

ATOM
1955
C
TRP
A
871
3.168
19.012
2.669
1.00
20.88
A

ATOM
1956
O
TRP
A
871
2.999
19.736
3.654
1.00
21.06
A

ATOM
1957
N
GLN
A
872
3.055
19.459
1.424
1.00
22.74
A

ATOM
1958
CA
GLN
A
872
2.680
20.846
1.157
1.00
25.25
A

ATOM
1959
CB
GLN
A
872
2.768
21.158
−0.337
1.00
23.93
A

ATOM
1960
CG
GLN
A
872
4.174
21.383
−0.816
1.00
29.27
A

ATOM
1961
CD
GLN
A
872
4.857
22.495
−0.047
1.00
32.45
A

ATOM
1962
OE1
GLN
A
872
4.377
23.628
−0.015
1.00
34.77
A

ATOM
1963
NE2
GLN
A
872
5.979
22.178
0.579
1.00
35.29
A

ATOM
1964
C
GLN
A
872
1.260
21.073
1.639
1.00
25.69
A

ATOM
1965
O
GLN
A
872
0.351
20.310
1.301
1.00
26.55
A

ATOM
1966
N
LYS
A
873
1.081
22.112
2.445
1.00
26.47
A

ATOM
1967
CA
LYS
A
873
0.224
22.463
2.990
1.00
27.95
A

ATOM
1968
CB
LYS
A
873
0.139
23.849
3.639
1.00
28.61
A

ATOM
1969
CG
LYS
A
873
1.390
24.304
4.360
1.00
29.84
A

ATOM
1970
CD
LYS
A
873
1.188
25.685
4.968
0.00
30.40
A

ATOM
1971
CE
LYS
A
873
2.412
26.130
5.752
0.00
30.90
A

ATOM
1972
NZ
LYS
A
873
2.223
27.477
6.360
0.00
31.23
A

ATOM
1973
C
LYS
A
873
1.281
22.446
1.886
1.00
29.41
A

ATOM
1974
O
LYS
A
873
2.320
21.800
2.022
1.00
28.03
A

ATOM
1975
N
ASP
A
874
1.008
23.156
0.793
1.00
30.47
A

ATOM
1976
CA
ASP
A
874
1.930
23.211
−0.340
1.00
32.13
A

ATOM
1977
CB
ASP
A
874
1.585
24.399
−1.247
1.00
34.07
A

ATOM
1978
CD
ASP
A
874
2.640
24.656
−2.304
1.00
36.22
A

ATOM
1979
OD1
ASP
A
874
3.160
23.678
−2.887
1.00
36.33
A

ATOM
1980
OD2
ASP
A
874
2.944
25.844
−2.560
1.00
38.96
A

ATOM
1981
C
ASP
A
874
1.806
21.919
−1.146
1.00
30.91
A

ATOM
1982
O
ASP
A
874
0.758
21.653
−1.727
1.00
30.73
A

ATOM
1983
N
ARG
A
875
2.876
21.134
−1.207
1.00
30.76
A

ATOM
1984
CA
ARG
A
875
2.837
19.870
−1.939
1.00
30.91
A

ATOM
1985
CB
ARG
A
875
4.177
19.138
−1.832
1.00
32.82
A

ATOM
1986
CG
ARG
A
875
5.316
19.755
−2.635
1.00
35.08
A

ATOM
1987
CD
ARG
A
875
6.413
18.722
−2.844
1.00
40.15
A

ATOM
1988
NE
ARG
A
875
7.555
19.234
−3.603
1.00
43.73
A

ATOM
1989
CZ
ARG
A
875
8.448
20.094
−3.125
1.00
45.20
A

ATOM
1990
NH1
ARG
A
875
8.334
20.544
−1.883
1.00
45.87
A

ATOM
1991
NH2
ARG
A
875
9.457
20.502
−3.885
1.00
47.30
A

ATOM
1992
C
ARG
A
875
2.477
20.028
−3.412
1.00
28.33
A

ATOM
1993
O
ARG
A
875
1.965
19.100
−4.033
1.00
26.80
A

ATOM
1994
N
ASN
A
876
2.756
21.199
−3.972
1.00
27.63
A

ATOM
1995
CA
ASN
A
876
2.463
21.457
−5.375
1.00
27.11
A

ATOM
1996
CB
ASN
A
876
3.223
22.703
−5.845
1.00
29.67
A

ATOM
1997
CG
ASN
A
876
4.663
22.396
−6.237
1.00
32.23
A

ATOM
1998
OD1
ASN
A
876
5.558
23.221
−6.060
1.00
36.30
A

ATOM
1999
ND2
ASP
A
876
4.887
21.212
−6.786
1.00
33.53
A

ATOM
2000
C
ASN
A
876
0.972
21.621
−5.632
1.00
25.93
A

ATOM
2001
O
ASN
A
876
0.500
21.396
−6.746
1.00
26.98
A

ATOM
2002
N
HIS
A
877
0.235
21.996
−4.592
1.00
24.62
A

ATOM
2003
CA
HIS
A
877
1.206
22.203
−4.690
1.00
23.95
A

ATOM
2004
CB
HIS
A
877
1.666
23.185
−3.605
1.00
25.91
A

ATOM
2005
CG
HIS
A
877
1.103
24.566
−3.758
1.00
28.62
A

ATOM
2006
CD2
HIS
A
877
0.390
25.134
−4.760
1.00
29.02
A

ATOM
2007
ND1
HIS
A
877
1.268
25.548
−2.803
1.00
30.92
A

ATOM
2008
CE1
HIS
A
877
0.682
26.659
−3.212
1.00
32.07
A

ATOM
2009
NE2
HIS
A
877
0.142
26.434
−4.396
1.00
30.42
A

ATOM
2010
C
HIS
A
877
1.987
20.893
−4.555
1.00
24.02
A

ATOM
2011
O
HIS
A
877
3.169
20.821
−4.919
1.00
21.47
A

ATOM
2012
N
ARG
A
878
1.342
19.860
−4.017
1.00
22.45
A

ATOM
2013
CA
ARG
A
878
2.022
18.574
−3.858
1.00
20.77
A

ATOM
2014
CE
ARG
A
878
1.196
17.618
−2.995
1.00
18.58
A

ATOM
2015
CG
ARG
A
878
0.878
18.140
−1.609
1.00
17.53
A

ATOM
2016
CD
AEG
A
878
0.163
17.267
−0.922
1.00
17.61
A

ATOM
2017
NE
ARG
A
878
0.678
17.923
0.273
1.00
15.99
A

ATOM
2018
CZ
ARG
A
878
1.898
17.746
0.763
1.00
17.79
A

ATOM
2019
NH1
ARG
A
878
2.743
16.913
0.165
1.00
18.10
A

ATOM
2020
NH2
ARG
A
878
2.282
18.440
1.830
1.00
20.88
A

ATOM
2021
C
ARG
A
878
2.264
17.922
−5.208
1.00
20.33
A

ATOM
2022
O
ARG
A
878
1.473
18.058
−6.137
1.00
21.44
A

ATOM
2023
N
PRO
A
879
3.377
17.200
−5.334
1.00
19.46
A

ATOM
2024
CD
PRO
A
879
4.395
16.893
−4.307
1.00
20.47
A

ATOM
2025
CA
PRO
A
879
3.687
16.530
−6.592
1.00
18.32
A

ATOM
2026
CB
PRO
A
879
5.116
16.050
−6.374
1.00
19.77
A

ATOM
2027
CG
PRO
A
879
5.106
15.696
−4.907
1.00
18.07
A

ATOM
2028
C
PRO
A
879
2.727
15.362
−6.801
1.00
18.71
A

ATOM
2029
O
PRO
A
879
2.135
14.846
−5.849
1.00
17.95
A

ATOM
2030
N
LYS
A
880
2.569
14.957
−8.051
1.00
18.17
A

ATOM
2031
CA
LYS
A
880
1.705
13.835
−8.373
1.00
19.50
A

ATOM
2032
CB
LYS
A
880
1.118
14.000
−9.775
1.00
20.53
A

ATOM
2033
CG
LYS
A
880
0.082
15.125
−9.888
1.00
23.92
A

ATOM
2034
CD
LYS
A
880
0.316
15.362
−11.334
1.00
26.32
A

ATOM
2035
CB
LYS
A
880
1.444
16.384
−11.434
1.00
29.77
A

ATOM
2036
NZ
LYS
A
880
1.732
16.761
−12.845
1.00
33.61
A

ATOM
2037
C
LYS
A
880
2.545
12.572
−8.315
1.00
18.71
A

ATOM
2038
O
LYS
A
880
3.775
12.637
−8.369
1.00
15.94
A

ATOM
2039
N
PHE
A
881
1.887
11.421
−8.207
1.00
19.36
A

ATOM
2040
CA
PHE
A
881
2.625
10.174
−8.148
1.00
18.63
A

ATOM
2041
CB
PHE
A
881
1.679
8.980
−7.961
1.00
18.14
A

ATOM
2042
CG
PHE
A
881
1.154
8.856
−6.561
1.00
15.85
A

ATOM
2043
CD1
PHE
A
681
0.182
9.091
−6.281
1.00
15.19
A

ATOM
2044
CD2
PHE
A
881
2.017
8.573
−5.503
1.00
14.91
A

ATOM
2045
CE1
PHE
A
881
0.661
9.055
−4.974
1.00
14.06
A

ATOM
2046
CE2
PHE
A
881
1.545
8.536
−4.186
1.00
14.78
A

ATOM
2047
CZ
PHE
A
881
0.201
8.780
−3.923
1.00
16.56
A

ATOM
2048
C
PHE
A
881
3.500
9.975
−9.365
1.00
18.17
A

ATOM
2049
O
PHE
A
881
4.570
9.381
−9.266
1.00
17.98
A

ATOM
2050
N
GLY
A
882
3.065
10.476
−10.517
1.00
17.95
A

ATOM
2051
CA
GLY
A
882
3.879
10.329
−11.710
1.00
17.50
A

ATOM
2052
C
GLY
A
882
5.173
11.112
−11.559
1.00
17.01
A

ATOM
2053
O
GLY
A
882
6.256
10.660
−11.953
1.00
15.84
A

ATOM
2054
N
GLN
A
883
5.066
12.295
−10.974
1.00
16.68
A

ATOM
2055
CA
GLN
A
883
6.240
13.129
−10.776
1.00
18.91
A

ATOM
2056
CE
GLN
A
883
5.813
14.551
−10.391
1.00
19.76
A

ATOM
2057
CG
GLN
A
883
4.850
15.188
−11.387
1.00
25.47
A

ATOM
2058
CD
GLN
A
883
4.415
16.571
−10.949
1.00
26.44
A

ATOM
2059
OE1
GLN
A
883
3.799
16.739
−9.896
1.00
24.34
A

ATOM
2060
NE2
GLN
A
883
4.748
17.577
−11.756
1.00
28.51
A

ATOM
2061
C
GLN
A
883
7.116
12.519
−9.677
1.00
17.08
A

ATOM
2062
O
GLN
A
883
8.336
12.625
−9.719
1.00
16.89
A

ATOM
2063
N
ILE
A
884
6.484
11.872
−8.700
1.00
16.63
A

ATOM
2064
CA
ILE
A
884
7.221
11.240
−7.612
1.00
15.31
A

ATOM
2065
CB
ILE
A
884
6.267
10.698
−6.516
1.00
16.28
A

ATOM
2066
CG2
ILE
A
884
7.001
9.708
−5.594
1.00
15.49
A

ATOM
2067
CG1
ILE
A
884
5.700
11.869
−5.714
1.00
15.76
A

ATOM
2068
CD1
ILE
A
884
4.588
11.471
−4.743
1.00
19.15
A

ATOM
2069
C
ILE
A
884
8.058
10.102
−8.166
1.00
14.47
A

ATOM
2070
O
ILE
A
884
9.229
9.988
−7.839
1.00
14.80
A

ATOM
2071
N
VAL
A
885
7.466
9.275
−9.024
1.00
13.59
A

ATOM
2072
CA
VAL
A
885
8.223
8.177
−9.601
1.00
16.17
A

ATOM
2073
CB
VAL
A
885
7.352
7.284
−10.502
1.00
16.96
A

ATOM
2074
CG1
VAL
A
885
8.216
6.248
−11.178
1.00
15.86
A

ATOM
2075
CG2
VAL
A
885
6.252
6.588
−9.676
1.00
16.66
A

ATOM
2076
C
VAL
A
885
9.414
8.683
−10.416
1.00
19.15
A

ATOM
2077
O
VAL
A
885
10.479
8.062
−10.417
1.00
16.03
A

ATOM
2078
N
ASN
A
886
9.241
9.808
−11.110
1.00
19.66
A

ATOM
2079
CA
ASN
A
886
10.320
10.343
−11.936
1.00
21.37
A

ATOM
2080
CB
ASN
A
886
9.819
11.509
−12.793
1.00
23.10
A

ATOM
2081
CD
ASN
A
886
8.698
11.102
−13.734
1.00
26.10
A

ATOM
2082
OD1
ASN
A
886
8.773
10.056
−14.398
1.00
26.24
A

ATOM
2083
ND2
ASN
A
886
7.654
11.926
−13.804
1.00
27.46
A

ATOM
2084
C
ASN
A
886
11.500
10.807
−11.097
1.00
20.89
A

ATOM
2085
O
ASN
A
886
12.655
10.562
−11.446
1.00
21.26
A

ATOM
2086
N
THR
A
887
11.189
11.479
−9.995
1.00
19.17
A

ATOM
2087
CA
THR
A
887
12.180
11.991
−9.069
1.00
20.28
A

ATOM
2088
CE
THR
A
887
11.500
12.789
−7.939
1.00
22.23
A

ATOM
2089
OG1
THR
A
887
10.751
13.872
−8.504
1.00
24.34
A

ATOM
2090
CG2
THR
A
887
12.525
13.327
−6.968
1.00
22.10
A

ATOM
2091
C
THR
A
887
12.961
10.838
−8.450
1.00
20.51
A

ATOM
2092
O
THR
A
887
14.182
10.906
−8.339
1.00
20.72
A

ATOM
2093
N
LEU
A
888
12.259
9.778
−8.055
1.00
17.54
A

ATOM
2094
CA
LEU
A
888
12.926
8.619
−7.458
1.00
16.74
A

ATOM
2095
CB
LEU
A
888
11.906
7.629
−6.882
1.00
13.99
A

ATOM
2096
CG
LEU
A
888
11.102
8.102
−5.660
1.00
16.09
A

ATOM
2097
CD1
LEU
A
888
9.998
7.118
−5.344
1.00
12.23
A

ATOM
2098
CD2
LEU
A
888
12.037
8.236
−4.444
1.00
15.84
A

ATOM
2099
C
LEU
A
888
13.791
7.939
−8.507
1.00
17.83
A

ATOM
2100
O
LEU
A
888
14.914
7.509
−8.215
1.00
16.99
A

ATOM
2101
N
ASP
A
889
13.278
7.846
−9.731
1.00
18.45
A

ATOM
2102
CA
ASP
A
889
14.047
7.231
−10.809
1.00
19.88
A

ATOM
2103
CB
ASP
A
889
13.216
7.089
−12.092
1.00
20.92
A

ATOM
2104
CG
ASP
A
889
12.212
5.954
−12.031
1.00
24.40
A

ATOM
2105
OD1
ASP
A
889
12.496
4.901
−11.417
1.00
23.95
A

ATOM
2106
OD2
ASP
A
889
11.127
6.106
−12.620
1.00
26.43
A

ATOM
2107
C
ASP
A
889
15.303
8.053
−11.128
1.00
20.04
A

ATOM
2108
O
ASP
A
889
16.341
7.491
−11.459
1.00
20.12
A

ATOM
2109
N
LYS
A
890
15.221
9.377
−11.035
1.00
20.98
A

ATOM
2110
CA
LYS
A
890
16.398
10.193
−11.320
1.00
24.03
A

ATOM
2111
CB
LYS
A
890
16.042
11.679
−11.353
1.00
23.49
A

ATOM
2112
CD
LYS
A
890
15.102
12.062
−12.480
0.00
24.55
A

ATOM
2113
CD
LYS
A
890
14.792
13.544
−12.453
0.00
25.01
A

ATOM
2114
CE
LYS
A
890
13.901
13.935
−13.615
0.00
25.39
A

ATOM
2115
NZ
LYS
A
890
13.587
15.388
−13.598
0.00
25.67
A

ATOM
2116
C
LYS
A
890
17.455
9.935
−10.255
1.00
23.54
A

ATOM
2117
O
LYS
A
890
18.644
9.911
−10.555
1.00
23.88
A

ATOM
2118
N
MET
A
891
17.014
9.744
−9.012
1.00
23.82
A

ATOM
2119
CA
MET
A
891
17.939
9.457
−7.913
1.00
22.98
A

ATOM
2120
CB
MET
A
891
17.192
9.425
−6.573
1.00
22.51
A

ATOM
2121
CD
MET
A
891
16.487
10.746
−6.206
1.00
19.86
A

ATOM
2122
SD
MET
A
891
15.428
10.557
−4.742
1.00
22.11
A

ATOM
2123
CE
MET
A
891
15.002
12.246
−4.390
1.00
18.78
A

ATOM
2124
C
MET
A
891
18.639
8.120
−8.163
1.00
23.81
A

ATOM
2125
O
MET
A
891
19.853
8.009
−7.996
1.00
23.38
A

ATOM
2126
N
ILE
A
892
17.874
7.113
−8.581
1.00
22.60
A

ATOM
2127
CA
ILE
A
892
18.451
5.806
−8.853
1.00
23.72
A

ATOM
2128
CS
ILE
A
892
17.362
4.765
−9.203
1.00
22.69
A

ATOM
2129
CG2
ILE
A
892
18.001
3.453
−9.665
1.00
21.79
A

ATOM
2130
CG1
ILE
A
892
16.501
4.487
−7.969
1.00
21.70
A

ATOM
2131
CD1
ILE
A
892
15.347
3.569
−8.236
1.00
21.58
A

ATOM
2132
C
ILE
A
892
19.454
5.892
−10.004
1.00
25.56
A

ATOM
2133
O
ILE
A
892
20.508
5.264
−9.964
1.00
24.56
A

ATOM
2134
N
ARG
A
893
19.126
6.672
−11.029
1.00
27.68
A

ATOM
2135
CA
ARG
A
893
20.006
6.826
−12.183
1.00
28.93
A

ATOM
2136
CS
ARG
A
893
19.246
7.486
−13.338
1.00
29.77
A

ATOM
2137
CG
ARG
A
893
18.033
6.702
−13.801
0.00
30.52
A

ATOM
2138
CD
ARG
A
893
17.167
7.518
−14.745
0.00
31.20
A

ATOM
2139
NE
ARG
A
893
16.022
6.747
−15.220
0.00
31.76
A

ATOM
2140
CZ
ARG
A
893
15.065
7.231
−16.006
0.00
32.04
A

ATOM
2141
NH1
ARG
A
893
15.109
8.492
−16.413
0.00
32.22
A

ATOM
2142
NH2
ARG
A
893
14.065
6.449
−16.388
0.00
32.22
A

ATOM
2143
C
ARG
A
893
21.237
7.659
−11.828
1.00
28.29
A

ATOM
2144
O
ARG
A
893
22.304
7.472
−12.410
1.00
30.73
A

ATOM
2145
N
ASN
A
894
21.090
8.574
−10.873
1.00
27.77
A

ATOM
2146
CA
ASN
A
894
22.206
9.421
−10.454
1.00
28.66
A

ATOM
2147
CS
ASN
A
894
21.928
10.883
−10.791
1.00
32.18
A

ATOM
2148
CG
ASN
A
894
21.446
11.064
−12.204
1.00
35.93
A

ATOM
2149
OD2
ASN
A
894
20.309
10.708
−12.536
1.00
38.24
A

ATOM
2150
ND2
ASN
A
894
22.305
11.613
−13.056
1.00
37.07
A

ATOM
2151
C
ASN
A
894
22.444
9.295
−8.960
1.00
27.18
A

ATOM
2152
O
ASN
A
894
22.256
10.257
−8.205
1.00
24.38
A

ATOM
2153
N
PRO
A
895
22.878
8.102
−8.517
1.00
27.24
A

ATOM
2154
CD
PRO
A
895
23.287
6.996
−9.402
1.00
27.14
A

ATOM
2155
CA
PRO
A
895
23.162
7.774
−7.116
1.00
27.82
A

ATOM
2156
CS
PRO
A
895
23.990
6.496
−7.222
1.00
27.28
A

ATOM
2157
CG
PRO
A
895
23.441
5.847
−8.435
1.00
29.74
A

ATOM
2158
C
PRO
A
895
23.888
8.859
−6.324
1.00
27.80
A

ATOM
2159
O
PRO
A
895
23.705
8.974
−5.112
1.00
26.04
A

ATOM
2160
N
ASN
A
896
24.718
9.655
−6.988
1.00
29.72
A

ATOM
2161
CA
ASN
A
896
25.426
10.694
−6.257
1.00
31.84
A

ATOM
2162
CB
ASN
A
896
26.449
11.392
−7.152
1.00
35.12
A

ATOM
2163
CG
ASN
A
896
27.619
10.495
−7.491
1.00
37.45
A

ATOM
2164
OD1
ASN
A
896
28.189
9.842
−6.610
1.00
37.32
A

ATOM
2165
ND2
ASN
A
896
27.988
10.455
−8.769
1.00
38.75
A

ATOM
2166
C
ASN
A
896
24.471
11.715
−5.652
1.00
31.58
A

ATOM
2167
O
ASN
A
896
24.789
12.337
−4.642
1.00
32.05
A

ATOM
2168
N
SER
A
897
23.295
11.876
−6.250
1.00
31.96
A

ATOM
2169
CA
SER
A
897
22.322
12.828
−5.724
1.00
31.92
A

ATOM
2170
CS
SER
A
897
21.100
12.903
−6.639
1.00
32.67
A

ATOM
2171
OG
SER
A
897
20.427
11.657
−6.699
1.00
32.92
A

ATOM
2172
C
SER
A
897
21.872
12.442
−4.320
1.00
32.25
A

ATOM
2173
O
SER
A
897
21.288
13.253
−3.601
1.00
32.16
A

ATOM
2174
N
LEU
A
898
22.150
11.200
−3.939
1.00
32.39
A

ATOM
2175
CA
LEU
A
898
21.767
10.684
−2.629
1.00
33.14
A

ATOM
2176
CS
LEU
A
898
21.338
9.223
−2.755
1.00
31.76
A

ATOM
2177
CG
LEU
A
898
20.167
8.938
−3.702
1.00
30.43
A

ATOM
2178
CD1
LEU
A
898
20.050
7.442
−3.949
1.00
28.81
A

ATOM
2179
CD2
LEU
A
898
18.886
9.489
−3.101
1.00
29.32
A

ATOM
2180
C
LEU
A
898
22.915
10.791
−1.633
1.00
35.96
A

ATOM
2181
O
LEU
A
898
22.780
10.410
−0.466
1.00
35.50
A

ATOM
2182
N
LYS
A
899
24.049
11.304
−2.100
1.00
37.16
A

ATOM
2183
CA
LYS
A
899
25.218
11.453
−1.245
1.00
39.32
A

ATOM
2184
CB
LYS
A
899
26.458
11.783
−2.083
1.00
39.91
A

ATOM
2185
CG
LYS
A
899
26.875
10.675
−3.035
1.00
40.95
A

ATOM
2186
CD
LYS
A
899
27.176
9.392
−2.279
1.00
42.25
A

ATOM
2187
CE
LYS
A
899
27.521
8.254
−3.230
1.00
43.26
A

ATOM
2188
NZ
LYS
A
899
27.655
6.962
−2.498
1.00
43.52
A

ATOM
2189
C
LYS
A
899
24.996
12.540
−0.204
1.00
39.53
A

ATOM
2190
O
LYS
A
899
25.502
12.451
0.913
1.00
39.99
A

ATOM
2191
N
ALA
A
900
24.238
13.566
−0.574
1.00
40.31
A

ATOM
2192
CA
ALA
A
900
23.952
14.672
0.332
1.00
41.45
A

ATOM
2193
CB
ALA
A
900
23.297
15.816
−0.436
1.00
42.71
A

ATOM
2194
C
ALA
A
900
23.047
14.225
1.477
1.00
42.06
A

ATOM
2195
O
ALA
A
900
23.462
14.222
2.638
1.00
43.54
A

ATOM
2196
O
HOH
A
1
14.457
−2.301
−3.629
1.00
11.91
A

ATOM
2197
O
HOH
A
2
−4.397
11.098
3.237
1.00
16.01
A

ATOM
2198
O
HOH
A
3
−4.918
7.667
−5.486
1.00
16.02
A

ATOM
2199
O
HOH
A
4
−18.623
−6.192
−4.002
1.00
15.98
A

ATOM
2200
O
HOH
A
5
−1.021
11.333
−8.745
1.00
17.20
A

ATOM
2201
O
HOH
A
6
2.429
1.976
9.154
1.00
11.10
A

ATOM
2202
O
HOH
A
7
9.183
−2.818
−7.644
1.00
12.10
A

ATOM
2203
O
HOH
A
8
−2.277
−6.184
−9.377
1.00
16.99
A

ATOM
2204
O
HOH
A
9
−3.892
7.522
2.129
1.00
11.77
A

ATOM
2205
O
HOH
A
10
−24.765
−1.312
−3.431
1.00
11.31
A

ATOM
2206
O
HOH
A
11
−18.960
−7.652
−1.790
1.00
14.87
A

ATOM
2207
O
HOH
A
12
1.251
−7.943
−7.973
1.00
15.91
A

ATOM
2208
O
HOH
A
13
−5.135
9.220
4.569
1.00
25.85
A

ATOM
2209
O
HOH
A
14
0.868
13.830
8.869
1.00
19.70
A

ATOM
2210
O
HOH
A
15
−4.469
−6.600
−8.030
1.00
17.02
A

ATOM
2211
O
HOH
A
16
−23.949
2.424
−2.051
1.00
16.20
A

ATOM
2212
O
HOH
A
17
16.570
−0.194
−6.147
1.00
20.96
A

ATOM
2213
O
HOH
A
18
0.212
10.493
−11.249
1.00
32.03
A

ATOM
2214
O
HOH
A
19
18.352
−5.806
−3.576
1.00
21.26
A

ATOM
2215
O
HOH
A
20
−1.189
18.011
−6.297
1.00
21.54
A

ATOM
2216
O
HOH
A
21
−2.174
−6.079
3.946
1.00
21.58
A

ATOM
2217
O
HOH
A
22
2.660
2.991
−12.285
1.00
10.78
A

ATOM
2218
O
HOH
A
23
6.194
0.599
12.618
1.00
26.49
A

ATOM
2219
O
HOH
A
24
6.270
2.873
10.787
1.00
20.59
A

ATOM
2220
O
HOH
A
25
−30.350
−2.848
−5.086
1.00
21.38
A

ATOM
2221
O
HOH
A
26
−5.973
10.069
1.056
1.00
21.49
A

ATOM
2222
O
HOH
A
27
7.351
−13.578
−1.294
1.00
24.35
A

ATOM
2223
O
HOH
A
28
−16.655
−18.205
6.142
1.00
32.77
A

ATOM
2224
O
HOH
A
29
−23.065
−6.820
10.522
1.00
24.13
A

ATOM
2225
O
HOH
A
30
14.170
15.934
−4.146
1.00
22.96
A

ATOM
2226
O
HOH
A
31
−5.570
−1.784
−15.428
1.00
26.29
A

ATOM
2227
O
HOH
A
32
−12.382
−1.657
−13.385
1.00
22.13
A

ATOM
2228
O
HOH
A
33
11.773
−2.825
−3.978
1.00
17.40
A

ATOM
2229
O
HOH
A
34
24.033
2.469
1.013
1.00
28.57
A

ATOM
2230
O
HOH
A
35
5.376
16.007
8.519
1.00
24.65
A

ATOM
2231
O
HOH
A
36
−9.608
−13.304
−9.571
1.00
27.67
A

ATOM
2232
O
HOH
A
37
−5.225
−8.904
−9.144
1.00
18.25
A

ATOM
2233
O
HOH
A
38
11.257
−0.407
−7.511
1.00
29.30
A

ATOM
2234
O
HOH
A
39
0.499
−15.307
6.874
1.00
26.00
A

ATOM
2235
O
HOH
A
40
−11.598
−8.243
3.065
1.00
18.53
A

ATOM
2236
O
HOH
A
41
2.939
23.776
3.343
1.00
30.46
A

ATOM
2237
O
HOH
A
42
−10.147
−9.966
7.681
1.00
26.73
A

ATOM
2238
O
HOH
A
43
9.258
−2.252
−4.749
1.00
18.60
A

ATOM
2239
O
HOH
A
44
−7.990
1.495
0.072
1.00
32.79
A

ATOM
2240
O
HOH
A
45
−21.791
−2.653
3.395
1.00
30.17
A

ATOM
2241
O
HOH
A
46
−12.258
8.515
−1.865
1.00
32.59
A

ATOM
2242
O
HOH
A
47
17.934
−29.751
44.878
1.00
37.91
A

ATOM
2243
O
HOH
A
48
−5.849
14.783
−6.753
1.00
25.08
A

ATOM
2244
O
HOH
A
49
−0.415
−15.909
−10.041
1.00
30.96
A

ATOM
2245
O
HOH
A
50
2.276
−3.698
10.859
1.00
31.49
A

ATOM
2246
O
HOH
A
51
13.707
−1.745
14.947
1.00
26.81
A

ATOM
2247
O
HOH
A
52
−11.484
5.994
−4.734
1.00
28.94
A

ATOM
2248
O
HOH
A
53
15.597
8.025
12.778
1.00
25.87
A

ATOM
2249
O
HOH
A
54
−12.859
−0.373
−16.318
1.00
38.15
A

ATOM
2250
O
HOH
A
55
−21.136
−9.246
−1.882
1.00
23.94
A

ATOM
2251
O
HOH
A
56
10.996
16.960
15.947
1.00
36.67
A

ATOM
2252
O
HOH
A
57
−6.591
9.869
−9.281
1.00
26.49
A

ATOM
2253
O
HOH
A
58
13.911
−11.174
2.291
1.00
30.38
A

ATOM
2254
O
HOH
A
59
−9.562
−10.942
−10.916
1.00
27.88
A

ATOM
2255
O
HOH
A
60
4.745
−19.626
−5.080
1.00
29.55
A

ATOM
2256
O
HOH
A
61
−1.717
−8.359
−10.786
1.00
24.62
A

ATOM
2257
O
HOH
A
62
−10.559
−3.268
−13.949
1.00
24.21
A

ATOM
2258
O
HOH
A
63
−0.660
15.194
−5.517
1.00
22.53
A

ATOM
2259
O
HOH
A
64
9.037
−0.135
−9.783
1.00
36.19
A

ATOM
2260
O
HOH
A
65
−23.460
−16.827
8.631
1.00
29.91
A

ATOM
2261
O
HOH
A
66
−24.192
−1.276
5.295
1.00
27.96
A

ATOM
2262
O
HOH
A
67
−16.353
4.624
−11.962
1.00
32.05
A

ATOM
2263
O
HOH
A
68
−17.396
3.801
−9.450
1.00
28.58
A

ATOM
2264
O
HOH
A
69
10.752
−11.093
7.051
1.00
37.06
A

ATOM
2265
O
HOH
A
70
1.620
−15.965
−4.603
1.00
24.63
A

ATOM
2266
O
HOH
A
71
−8.238
12.427
−6.673
1.00
33.82
A

ATOM
2267
O
HOH
A
72
−16.577
−14.805
−9.290
1.00
29.62
A

ATOM
2268
O
HOH
A
73
9.083
−13.189
−4.221
1.00
35.45
A

ATOM
2269
O
HOH
A
74
10.287
7.919
−14.356
1.00
33.56
A

ATOM
2270
O
HOH
A
75
−20.538
−21.060
2.242
1.00
45.67
A

ATOM
2271
O
HOH
A
76
−1.640
14.388
9.613
1.00
30.93
A

ATOM
2272
O
HOH
A
77
40.049
−7.010
13.782
1.00
30.14
A

ATOM
2273
O
HOH
A
78
18.418
12.809
−8.404
1.00
37.11
A

ATOM
2274
O
HOH
A
79
−25.942
−2.805
−10.608
1.00
34.88
A

ATOM
2275
0
HOH
A
80
16.293
−4.489
17.257
1.00
25.34
A

ATOM
2276
O
HOH
A
81
−16.209
−8.340
11.406
1.00
40.96
A

ATOM
2277
O
HOH
A
82
11.439
19.084
12.127
1.00
33.B2
A

ATOM
2278
O
HOH
A
83
20.159
2.221
5.570
1.00
28.54
A

ATOM
2279
O
HOH
A
84
−13.713
5.569
−10.421
1.00
29.53
A

ATOM
2280
O
HOH
A
85
−7.262
16.174
−0.684
1.00
29.54
A

ATOM
2281
O
HOH
A
86
9.742
−10.617
−7.415
1.00
25.62
A

ATOM
2282
O
HOH
A
87
−20.632
0.152
7.971
1.00
39.94
A

ATOM
2283
O
HOH
A
88
1.339
18.421
−9.397
1.00
43.65
A

ATOM
2284
0
HOH
A
89
−4.943
13.752
20.778
1.00
37.74
A

ATOM
2285
O
HOH
A
90
−3.157
10.534
20.505
1.00
40.33
A

ATOM
2286
O
HOH
A
91
20.471
14.004
−1.018
1.00
22.59
A

ATOM
2287
O
HOH
A
92
−3.126
−6.909
11.806
1.00
31.06
A

ATOM
2288
O
HOH
A
93
−14.587
−14.560
24.267
1.00
48.31
A

ATOM
2289
O
HOH
A
94
4.029
−14.349
32.347
1.00
56.74
A

ATOM
2290
O
HOH
A
95
7.949
−15.761
−2.076
1.00
40.76
A

ATOM
2291
O
HOH
A
96
−2.357
13.362
−6.866
1.00
25.37
A

ATOM
2292
O
HOH
A
97
0.273
12.223
−13.491
1.00
30.12
A

ATOM
2293
O
HOH
A
98
23.889
−3.917
11.357
1.00
25.67
A

ATOM
2294
0
HOH
A
99
−4.748
−9.020
−11.939
1.00
46.38
A

ATOM
2295
O
HOH
A
100
−1.430
−10.359
−13.316
1.00
31.41
A

ATOM
2296
O
HOH
A
101
10.739
−23.422
−9.199
1.00
39.50
A

ATOM
2297
O
HOH
A
102
−3.937
14.980
−8.334
1.00
24.93
A

ATOM
2298
O
HOH
A
103
−7.054
−10.787
−10.296
1.00
33.68
A

ATOM
2299
O
HOH
A
104
13.492
0.660
13.579
1.00
31.04
A

ATOM
2300
O
HOH
A
105
−6.920
−14.447
−11.281
1.00
45.39
A

ATOM
2301
O
HOH
A
106
13.348
22.708
2.254
1.00
36.30
A

ATOM
2302
O
HOH
A
107
5.408
−11.711
−5.405
1.00
31.10
A

ATOM
2303
O
HOH
A
108
18.256
−2.341
15.534
1.00
28.95
A

ATOM
2304
O
HOH
A
109
−8.503
0.787
3.249
1.00
41.16
A

ATOM
2305
O
HOH
A
110
14.317
3.040
−11.205
1.00
42.00
A

ATOM
2306
O
HOH
A
111
11.881
17.271
−4.308
1.00
37.83
A

ATOM
2307
O
HOH
A
112
19.020
15.966
5.172
1.00
45.54
A

ATOM
2308
O
HOH
A
113
0.998
−12.806
−8.453
1.00
43.81
A

ATOM
2309
O
HOH
A
114
13.315
−10.545
8.002
1.00
33.76
A

ATOM
2310
O
HOH
A
115
−10.798
3.629
0.360
1.00
29.35
A

ATOM
2311
O
HOH
A
116
26.244
9.778
1.025
1.00
38.48
A

ATOM
2312
O
HOH
A
117
−18.933
−5.540
7.951
1.00
21.69
A

ATOM
2313
O
HOH
A
118
−2.346
9.089
14.123
1.00
28.55
A

ATOM
2314
O
HOH
A
119
12.331
−3.683
−6.285
1.00
28.50
A

ATOM
2315
O
HOH
A
120
17.652
7.204
6.269
1.00
33.34
A

ATOM
2316
O
HOH
A
121
−20.972
−5.394
10.153
1.00
29.22
A

ATOM
2317
O
HOH
A
122
1.126
−29.138
3.663
1.00
54.55
A

ATOM
2318
O
HOH
A
123
19.859
5.269
4.518
1.00
48.81
A

ATOM
2319
O
HOH
A
124
16.235
−4.760
13.356
1.00
24.74
A

ATOM
2320
O
HOH
A
125
−1.781
27.895
−1.429
1.00
49.94
A

ATOM
2321
O
HOH
A
126
0.978
3.103
−14.170
1.00
33.01
A

ATOM
2322
O
HOH
A
127
23.932
8.429
−15.415
1.00
43.20
A

ATOM
2323
O
HOH
A
128
−15.975
−7.260
14.283
1.00
35.46
A

ATOM
2324
O
HOH
A
129
−5.663
−12.681
1.284
1.00
25.44
A

ATOM
2325
O
HOH
A
130
−9.888
−12.927
7.395
1.00
35.96
A

ATOM
2326
O
HOH
A
131
25.472
3.261
−1.345
1.00
31.42
A

ATOM
2327
O
HOH
A
132
0.225
−4.710
9.057
1.00
40.52
A

ATOM
2328
O
HOH
A
133
1.153
−8.592
−10.392
1.00
41.49
A

ATOM
2329
O
HOH
A
134
0.318
−2.638
−15.320
1.00
36.09
A

ATOM
2330
O
HOH
A
135
−6.483
17.186
−5.700
1.00
31.77
A

ATOM
2331
O
HOH
A
136
−11.747
5.541
−2.116
1.00
37.63
A

ATOM
2332
O
HOH
A
137
3.033
5.741
−16.256
1.00
60.91
A

ATOM
2333
O
HOH
A
138
15.857
−9.756
3.393
1.00
42.40
A

ATOM
2334
O
HOH
A
139
1.315
24.812
0.302
1.00
37.92
A

ATOM
2335
O
HOH
A
140
18.429
−8.563
6.665
1.00
40.06
A

ATOM
2336
O
HOH
A
141
8.361
17.182
−5.869
1.00
35.77
A

ATOM
2337
O
HOH
A
142
−3.830
−1.160
8.545
1.00
30.75
A

ATOM
2338
O
HOH
A
143
−11.080
−0.626
−21.190
1.00
42.20
A

ATOM
2339
O
HOH
A
144
−25.620
−5.418
−10.124
1.00
24.22
A

ATOM
2340
N9
ANE
A
400
−0.759
−10.830
5.271
1.00
36.71
A

ATOM
2341
C8
ANE
A
400
−1.712
−10.252
4.469
1.00
37.40
A

ATOM
2342
N7
ANE
A
400
−1.529
−10.440
3.198
1.00
37.33
A

ATOM
2343
Cs
ANE
A
400
−0.400
−11.181
3.134
1.00
37.65
A

ATOM
2344
C6
ANE
A
400
0.318
−11.704
2.059
1.00
38.63
A

ATOM
2345
N6
ANE
A
400
−0.086
−11.507
0.797
1.00
39.54
A

ATOM
2346
M1
ANE
A
400
1.443
−12.423
2.375
1.00
37.38
A

ATOM
2347
C2
ANE
A
400
1.792
−12.580
3.654
1.00
38.46
A

ATOM
2348
N3
ANE
A
400
1.207
−12.139
4.749
1.00
39.20
A

ATOM
2349
C4
ANE
A
400
0.099
−11.435
4.400
1.00
37.67
A

END

LIST OF REFERENCES

[0445] Abrahams, J. P. and Leslie, A. G. W. (1996). Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Cryst. D52, 30-42.

[0446] Andersson L. C. (1998) c-kit gain-of-function mutations and human tumors. Japn. J. of Cancer Res., 89:1.

[0447] Adams, R. H., Wilkinson, G. A., Weiss, C., Diella, F., Gale, N. W., Deutsch, U., Risau, W., and Klein, R (1999) Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis. Genes Dev. 13, 295-306.

[0448] Barker, S. C., Kassel D. B., Weigl, D., Huang, X., Luther, M. A., and Knight, W. B. (1995). Characterization of pp60c-src tyrosine kinase activities using a continuous assay: autoactivation of the enzyme is an intermolecular autophosphorylation process. Biochem. 34, 14843-14851.

[0449] Baxter, R. M., Secrist, J. P., Vaillancourt, R. R., and Kazlauskas, A. (1998). Full activation of the platelet-derived growth factor β-receptor kinase involves multiple events. J. Biol. Chem. 273, 17050-17055.

[0450] Binns, K. L., Taylor, P. P., Sicheri, F., Pawson, T., and Holland, S. J. (2000). Phosphorylation of tyrosine residues in the kinase domain and juxtamembrane region regulates the biological and catalytic activities of Eph receptors. Mol. Cell. Biol. 20, 4791-4805.

[0451] Brown, A., Yates, P. A., Burrola, P., Ortuno, D., Vaidya, A., Jessell, T. M., Pfaff, S. L., O'Leary, D. D., and Lemke, G. (2000). Topographic mapping from the retina to the midbrain is controlled by relative but not absolute levels of EphA receptor signaling. Cell 102, 77-88.

[0452] Bruckner, K., Pasquale, E. B., and Klein, R (1997). Tyrosine phosphorylation of transmembrane ligands for Eph receptors. Science 275, 1640-1643.

[0453] Brunger, A. T., Adams, P. D., Clore, G. M., Gros, P., Grosse-Kuntsleve, R. W., Jiang, J. -S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., and et al. (1998). Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D54, 905-921.

[0454] Cann, A. D., Bishop, S. M., Ablooglu, A. J., and Kohanski, R. A. (2000). Partial activation of the insulin receptor kinase domain by juxtamembrane autophosphorylation. Biochem. 37, 11289-11300.

[0455] Carson, M. (1991). Ribbons 2.0. J. Appl. Crystallgr. 24, 958-961.

[0456] Collaborative Computational Project, Number 4. (1994). The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D50, 760-763.

[0457] Chin-Sang, I. D., George, S. E., Ding, M., Moseley, S. L., Lynch, A. S., and Chisholm, A. D. (1999). The ephrin VAB-2/EFN-1 functions in neuronal signaling to regulate epidermal morphogenesis in C. elegans. Cell 99, 781-790.

[0458] Choi, S. and Park, S. (1999). Phosphorylation at Tyr-838 in the kinase domain of EphA8 modulates Fyn binding to the Tyr-615 site by enhancing tyrosine kinase activity. Oncogene 18, 5413-5422.

[0459] Connor, R. J. and Pasquale, E. B. (1995). Genomic organization and alternatively processed forms of Cek5, a receptor protein-tyrosine kinase of the Eph family. Oncogene 11, 2429-2438.

[0460] Dodelet, V. C., Pazzagli, C., Zisch, A. H., Hauser, C. A., and Pasquale, E. B. (1999). A novel signaling intermediate, SHEP1, directly couples Eph receptors to R-Ras and Rap1A. J. Biol. Chem. 274, 31941-31946.

[0461] Drescher, U., Kremoser, C., Handwerker, C. J. L., Noda, M., and Bonhoeffer, F. (1995). In vitro guidance of retinal ganglion cell exons by RAGS, a 25 kDa tectal protein related to ligands for Eph receptor tyrosine kinases. Cell 82, 359-370.

[0462] Ellis, C., Kasmi F., Ganju, P., Walls E., and Panayotou (1996). A juxtamembrane autophosphorylation site in the Eph family receptor tyrosine kinase, Sek, mediates high affinity interaction with p59fyn. Oncogene 12, 1727-1736.

[0463] Eph Nomenclature Committee (1997). Unified nomenclature for Eph family receptors and their ligands, the ephrins. Cell 90, 403-404.

[0464] Gale, N. W., Holland, S. J., Valenzuela, D. M., Flenniken, A., Pan, L., Ryan, T. E., Henkemeyer, M., Strebhardt, K., Hirai, H., Wilkinson, D. G., Pawson, T., Davis, S., and Yancopoulos, G. D. (1996). Eph receptors and ligands comprise two major specificity subclasses and are reciprocally compartmentalized during embryogenesis. Neuron 17, 9-19.

[0465] George, S. E., Simokat K., Hardin, J., and Chisholm, A. D. (1998). The VAB-1 Eph receptor tyrosine kinase functions in neural and epithelial morphogenesis in C. elegans. Cell 92, 633-643.

[0466] Gerety, S. S., Wang, H. U., Chen, Z. F., and Anderson, D. J. (1999). Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development. Mol. Cell 4, 403-414.

[0467] Hanks, S. K., Quinn, A. M., and Hunter, T. (1988). The protein kinase family: Conserved features and deduced phylogeny of the catalytic domains. Science 241, 42-52.

[0468] Hayakawa F., Towatari M., Kiyoi H., Tanimoto M., Kitamura T., Saito H. and Naoe T. (2000) Tandem-duplicated Flt3 constitutively activates STAT5 and MAP kinase and introduces autonomous cell growth in IL-3 dependent cell lines. Oncogene, 19:624-631.

[0469] Heldin, C. H. (1995). Dimerization of cell surface receptors in signal transduction. Cell 80, 213-223.

[0470] Henkemeyer, M., Marengere, L. E. M., McGlade, J., Olivier, J. P., Conlon, R. A., Holmyard, D. P., Letwin, K, and Pawson, T. (1994). Immunolocalization of the Nuk receptor tyrosine kinase suggests roles in segmental patterning of the brain and axonogenesis. Oncogene 9, 1001-1014.

[0471] Himanen, J. P., Henkemeyer, M., and Nikolov, D. B. (1998). Crystal structure of the ligand-binding domain of the receptor tyrosine kinase EphB2. Nature 396, 486-491.

[0472] Hirota, S., Isozaki, K., Moriyama, Y., Hashimoto, K., Nishida, T., Ishiguro, S., Kawano, K., Hanada, M., Kurata, A., Takeda, M., Tunio, G. M., Matsuzawa, Y., Kanakura, Y., Shinomura, Y., and Kitamura, Y. (1998). Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science 279, 577-580.

[0473] Hock, B., Bohme, B., Karn, T., Yamamoto, T., Kaibuchi, K., Holtrich, U., Holland, S., Pawson, T., Rubsamen-Waigmann, H., and Strebhardt, K. (1998). PDZ-domain-mediated interaction of the Eph-related receptor tyrosine kinase EphB3 and the ras-binding protein AF6 depends of the kinase activity of the receptor. Proc. Natl. Acad. Sci. USA 95, 9779-9784.

[0474] Holder, N. and Klein, R (1999). Eph receptors and ephrins: effectors of morphogenesis. Development 126, 2033-2044.

[0475] Holland, S. J., Gale, N. W., Gish, G. D., Roth, R. A., Songyang, Z., Cantley, L. C., Henkemeyer, M., Yancopoulos, G. D., and Pawson, T. (1997). Juxtamembrane tyrosine residues couple the Eph family receptor Eph B2/Nuk to specific SH2 domain proteins in neuronal cells. EMBO J. 16, 3877-3888.

[0476] Holland, S. J., Gale, N. W., Mbamalu, G., Yancopoulos, G. D., Henkemeyer, M., and Pawson, T. (1996). Bi-directional signalling through the Eph family receptor Nuk and its transmembrane ligands. Nature 383, 722-725.

[0477] Holland, S. J., Peles, E., Pawson, T., and Schlessinger, J. (1998). Cell-contact-dependent signalling in axon growth and guidance: Eph receptor tyrosine kinases and receptor protein tyrosine phosphatase □. Curr. Opin. Neurobiol. 8, 117-127.

[0478] Hubbard, S. R., Wei L., Ellis L., and Hendrickson, W. A. (1994). Crystal structure of the tyrosine kinase domain of the human insulin receptor. Nature 372, 746-754.

[0479] Hubbard, S. R. (1997). Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog. EMBO J. 16, 5572-5581.

[0480] Hubbard, S. R. and Till, J. H. (2000). Protein tyrosine kinase structure and function. Annu. Rev. Biochem. 69, 373-398.

[0481] Huse, M., Chen, Y. G., Massague, J., and Kuriyan, J. (1999). Crystal structure of the cytoplasmic domain of the type I TGF β receptor in complex with FKBP12. Cell 96, 425-436.

[0482] Irusta, P. M. and DiMaio, D. (1998). A single amino acid substitution in a WW-like domain of diverse members of the PDGF receptor subfamily of tyrosine kinases causes constitutive receptor activation. EMBO J. 17, 6912-6923.

[0483] Johnson, L. N., Noble, M. E., and Owen, D. J. (1996). Active and inactive protein kinases: structural basis for regulation. Cell 85, 149-158.

[0484] Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991). Improved methods for binding protein models in electron density maps and the localization of errors in these models. Acta Cryst. A47, 110-119.

[0485] Kalo, M. S. and Pasquale, E. B. (1999). Multiple in vivo tyrosine phosphorylation sites in EphB receptors. Biochem. 38, 14396-14408.

[0486] Kitayama, H., Kanakura, Y., Furitsu, T., Tsujimura, T., Oritani, K., Ikeda, H., Sugahara, H., Mitsui, H., Kanayama, Y., Kitamura, Y., and Matsuzawa, Y. (1995). Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines. Blood 85, 790-798.

[0487] Knighton, D. R., Zheng, J., Ten Eyck L. F., Ashford, V. A., Xuong, N. -H., Taylor, S. S., and Sowadski, J. M. (1991). Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 253, 407-414.

[0488] Krull, C. E., Lansford, R., Gale, N. W., Collazo A., Marcelle, C., Yancopoulos, G. D., Fraser, S. E., and Bronner-Fraser, M. (1997). Interactions of Eph-related receptors and ligands confer rostrocaudal pattern to trunk neural crest migration. Curr. Biol. 7, 571-580.

[0489] Kuriyan, J. and Cowburn, D. (1997). Modular peptide recognition domains in eukaryotic signaling. Annu. Rev. Biophys. Biomol. Struct. 26, 259-288.

[0490] La Fortelle, E. de. and Bricogne, G. (1997). Maximum-likelihood heavy-atom parameter in the MIR and MAD methods. Methods Enzymol. 276, 472-494.

[0491] Labrador, J. P., Brambilla, R., and Klein, R. (1997). The N-terminal globular domain of Eph receptors is sufficient for ligand binding and receptor signaling. EMBO J. 16, 3889-3897.

[0492] Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993). PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283-291.

[0493] Mellitzer, G., Xu, Q., and Wilkinson, D. G. (1999). Eph receptors and ephrins restrict cell intermingling and communication. Nature 400, 77-81.

[0494] Miller, R., Gallo, S. M., Khalak, H. G., and Weeks, C. M. (1994). SnB: crystal structure determination via Shake-and-Bake. J. Appl. Crystl. 27, 613-621.

[0495] Mohammadi, M., Schlessinger, J., and Hubbard, S. R. (1996). Structure of the FGF receptor tyrosine kinase domain reveals a novel autoinhibitory mechanism. Cell 86, 577-587.

[0496] Myles, G. M., Brandt, C. S., Carlberg, K., and Rohrschneider, L. R. (1994). Tyrosine 569 in the c-Fms juxtamembrane domain is essential for kinase activity and macrophage colony-stimulating factor-dependent internalization. Mol. Cell Biol. 14, 4843-4854.

[0497] Nakahara M., Isozaki K., Hirota S., Miyagawa J., Hase-Sawada N., Taniguchi M., Nishida T., Kanayama S., Kitamura Y., Shinomura Y., and Matsuzawa Y. (1998) A novel gain-of-function mutation of c-kit in gastrointestinal stromal tumors. Gastroenterology, 115:1090-1095.

[0498] Nakao M., Yokota S., Iwai T., Kaneko H., Horiike S., Kashima K., Sonoda Y., Fujimoto T., and Misaw S. (1996) Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia 10: 1911-1918.

[0499] Nakamoto, M., Cheng, H. J., Friedman, G. C., McLaughlin, T., Hansen, M. J., Yoon, C. H., O'Leary, D. D., and Flanagan, J. G. (1996). Topographically specific effects of ELF-1 on retinal axon guidance in vitro and retinal axon mapping in vivo. Cell 86, 755-766.

[0500] Navaza, J. (1994). Automated Package for Molecular Replacement. Acta Cryst. A50, 157-183.

[0501] Nicholls, A., Sharp, K. A., and Honig, B. (1991). Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281-296.

[0502] Otwinowski, Z. and Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307-326.

[0503] Pasquale, E. B. (1991). Identification of chicken embryo kinase 5, a developmentally regulated receptor-type tyrosine kinase of the Eph family. Cell Regul 2, 523-534.

[0504] Pawson, T. and Scott, J. D. (1997). Signaling through scaffold, anchoring, and adaptor proteins. Science 278, 2075-2080.

[0505] Remy, I., Wilson, I. A., and Michnick, S. W. (1999). Erythropoietin receptor activation by a ligand-induced conformation change. Science 283, 990-993.

[0506] Schindler, T., Bornmann, W., Pellicena, P., Miller, W. T., Clarkson, B., and Kuriyan, J. (2000). Structural mechanism for STI-571 inhibition of abelson tyrosine kinase. Science 289, 1938-1942.

[0507] Schlessinger, J. (2000). Cell signaling by receptor tyrosine kinases. Cell 103, 211-225.

[0508] Shewchuk, L. M., Hassell, A. M., Ellis, B., Holmes, W. D., Davis, R., Horne E. L., Kadwell, S. H., McKee, D. D., and Moore, J. T. (2000). Structure of the Tie2 RTK domain. Self-inhibition by the nucleotide binding loop, activation loop, and C-terminal tail. Structure Fold Des 8, 1105-1113.

[0509] Sicheri, F., Moarefi, I., and Kuriyan, J. (1997). Crystal structure of the Src family tyrosine kinase Hck. Nature 385, 602-609.

[0510] Stapleton, D., Balan, I., Pawson, T., and Sicheri, F. (1999). The crystal structure of an Eph receptor SAM domain reveals a mechanism for modular dimerization. Nature Struct Biol. 6, 44-49.

[0511] Thanos, C. D., Goodwill, K. E., and Bowie, J. U. (1999). Oligomeric structure of the human EphB2 receptor SAM domain. Science 283, 833-836.

[0512] Torres, R., Firestein, B. L., Dong, H., Staudinger, J., Olson, E. N., Huganir, R. L., Bredt, D. S., Gale, N. W., and Yancopoulos, G. D. (1998). PDZ proteins bind, cluster, and synaptically colocalize with Eph receptors and their ephrin ligands. Neuron 21, 1453-1463.

[0513] Tsujimura, T., Morimoto, M., Hashinoto, K., Moriyama, Y., Kitayama, H., Matsuzawa, Y., Kitamura, Y., and Kanakura, Y. (1996). Constitutive activation of c-kit in FMA3 murine mastocytoma cells caused be deletion of seven amino acids at the juxtamembrane domain. Blood 87, 273-283.

[0514] van der Geer, P. and Hunter, T. (1994). Receptor protein-tyosine kinases and their signal transduction pathways. Ann. Rev. Cell Biol. 10, 251-337.

[0515] Wang, H. U. and Anderson, D. J. (1997). Eph family transmembrane ligands can mediate repulsive guidance of trunk neural crest migration and motor axon outgrowth. Neuron 18, 383-396.

[0516] Wang, H. U., Chen, Z. F., and Anderson, D. J. (1999b). Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell 93, 741-753.

[0517] Wang, X., Roy, P. J., Holland, S., Zhang, L. W., Culotti J. G., and Pawson, T. (1999a). Multiple ephrins control cell organization in C. elegans through kinase-dependent and kinase-independent functions of the VAB-1 Eph receptor. Molecular Cell 4, 903-913.

[0518] Weinmaster, G., Zoller, M. J., Smith, M., Hinze, E., and Pawson, T. (1984). Mutagenesis of fujinami sarcoma virus: evidence that tyrosine phosphorylation of p130pag-fps modulates its biological activity. Cell 37, 559-568.

[0519] Xu, Q., Mellitzer, G., Robinson, V., and Wilkinson, D. G. (1999). In vivo cell sorting in complementary segmental domains mediated by Eph receptors and ephrins. Nature 399, 267-271.

[0520] Xu, W., Harrison, S. C., and Eck, M. J. (1997). Three-dimensional structure of the tyrosine kinase c-Src. Nature 385, 595-602.

[0521] Yokota S., Kiyoi H., Nakao M., Iwai T., Misawa S., Okuda T., Sonoda Y., Abe T., Kabsima K, Matsu Y., Naoe T. (1997) Internal tandem duplication of the FLT3 gene is preferentially seen in acute myeloid leukemia and myelodysplastic syndrome among various hematological malignancies. A study on a large series of patients and cell lines. Leukemia, 11:1605-1609.

[0522] Zisch, A. H., Kalo, M. S., Chong, L. D., and Pasquale E. B. (1998). Complex formation between EphB2 and Src requires phosphorylation of tyrosine 611 in the EphB2 juxtamembrane region. Oncogene 16, 2657-2670.

[0523] Zisch, A. H., Pazzagli, C., Freeman, A. L., Schneller, M., Hadman, M., Smith, J. W., Ruoslahti, E., and Pasquale, E. B. (2000). Replacing two conserved tyrosines of the EphB2 receptors with glutamic acid prevents binding of SH2 domains without abrogating kinase activity and biological responses. Oncogene 19, 177-187.

Compositions and methods for regulating the kinase domain of receptor tyrosine kinases

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