CRYSTAL STRUCTURE OF FLT3 LIGAND-RECEPTOR COMPLEX

TECHNICAL FIELD

The present invention generally relates to structural studies of the Flt3 receptor tyrosine kinase. In particular, the present invention relates to the crystal structure of Flt3 in complex with its cognate ligand FL. The present invention also relates to the applicability in modulating Flt3 activity. Methods for the identification as well as the rational design of agonistic or antagonistic modulators of Flt3 signaling are disclosed.

BACKGROUND

Hematopoiesis is a finely regulated process during which diverse cell types originating from a limited and self-renewing population of hematopoietic stem cells (HSC) are stimulated to proliferate and differentiate to create the cellular repertoire that sustains the mammalian hematopoietic and immune systems (Metcalf, 2008). The hematopoietic pathway is orchestrated by intracellular signaling pathways, which are initiated via the activation of hematopoietic receptors by their cognate cytokine ligands at the cell surface (Bryder 2006; Li and Li, 2006; Metcalf, 2007; Ross and Li, 2006).

The Fms-like tyrosine kinase receptor 3 (Flt3), is the most recent addition to the diverse family of hematopoietic receptors (Matthews 1991; Rosnet 1991). Flt3 is activated on HSC and early myeloid and lymphoid progenitors by its cognate ligand (FL) (Lyman, 1993; Hannum, 1994), to initiate downstream signaling via the PI3K/AKT and the RAS/RAF/MEK/ERK pathways (Parcells, 2006; Stirewalt, 2003). Consistent with the narrow expression profile of Flt3 in the bone marrow environment, signaling via the Flt3 ligand/receptor complex primarily impacts early hematopoiesis, particularly the proliferation and development of HSC and B-cell progenitors (Stirewalt, 2003; Kikushige, 2008). In recent years Flt3 and FL emerged as potent regulators of dendritic cell (DC) development and homeostasis (Waskow, 2008; Onai, 2007; Liu, 2009; Liu and Nussenweig, 2010; Schmid, 2010), and DC-mediated natural killer cell activation (Eidenschenk, 2010; Guimond, 2010), thereby gaining an important role at the interface of innate and acquired immunity and in cancer immunotherapy (Antonysamy and Thomson, 2000; Dong, 2002; Fong, 2001; Karsunky, 2003; Wu and Liu, 2007). Notably, Flt3/FL-driven DC generation yields both classical- and plasmacytoid DC from bone-marrow progenitors regardless of myeloid or lymphoid commitment, a property that is currently unmatched by any other receptor/cytokine system relevant for DC physiology (Schmid, 2010).

Flt3 is together with the prototypic platelet-derived growth factor receptor (PDGFR), colony-stimulating factor 1 receptor (CSF-1R), and KIT (Robinson, 2000; Grassot, 2006) a class III receptor tyrosine kinase III (RTKIII). Thus, Flt3 has been predicted to be organized into a modular structure featuring an extracellular segment with 5 immunoglobulin (Ig)-like domains (residues 27-543), a single transmembrane helix (TM, residues 544-563), a cytoplasmic juxtamembrane domain (JM, residues 572-603) and a split intracellular kinase module (residues 604-958). The RTKIII family is closely related to the RTKV family of vascular endothelial growth factor receptors (VEGFR), which have 7 extracellular Ig-like domains. The hallmark of RTKIII/V signaling lies in the dimerization of the extracellular receptor segments upon binding of their respective cytokine ligands, followed by intermolecular autophosphorylation and activation of the intracellular kinase domains (Turner, 1996; Kiyoi, 1998; Hubbard and Miller, 2007; Lemmon and Schlessinger, 2010).

Besides the outspoken role of Flt3 signaling in hematopoiesis and immune system development, overexpression of wild type or oncogenic forms of Flt3 have been implicated in a number hematopoietic malignancies (Stirewalt and Radich, 2003; Sanz, 2010), and inflammatory disorders (Dehlin, 2008). In particular, internal tandem duplication (ITD) in the JM region or point mutations in the kinase activation loop occur in 35% of patients with Acute Myeloid Leukemia (AML) resulting in constitutive activation of the receptor and uncontrolled proliferation of hematopoietic precursors (Kiyoi, 1998; Stirewalt and Radich, 2003; Reindl, 2006; Parcells, 2006; Frohling, 2007). Such mutation fingerprints have established Flt3 as the predominant prognostic factor in AML cases (Eklund, 2010), and have rationalized targeting of Flt3 in a clinical setting (Sanz, 2009; Parcells, 2006; Sternberg and Licht, 2005; Stirewalt, 2003; Kindler, 2010).

Although the cellular and physiological role of the Flt3 ligand-receptor interaction has been featured prominently in the biomedical literature over the last two decades, the Flt3 signaling complex has remained uncharacterized at the molecular and structural level. Such insights are the missing link in exposing the structural and functional diversity of RTKIII/V extracellular complexes, and would help provide a nearly complete picture of the entire Flt3 signaling complex given the available structure of the Flt3 intracellular kinase domains (Griffith, 2004). A recent flurry of studies of RTKIII/V extracellular complexes led to a structural paradigm for RTKIII/V activation, whereby the receptors bind via their N-terminal Ig-like domains to the activating dimeric cytokine and concomitantly make homotypic contacts between their membrane-proximal domains. A universal feature of all characterized RTKIII/V complexes thus far is that the cytokine-binding epitope is distributed equally between extracellular domains 2 and 3 covering ˜2000 Å²of surface area, and that homotypic receptor-receptor interactions are mediated by a few but well-conserved residues found in the membrane-proximal domains (domain 4 in RTKIII and domain 7 in RTKV). Nonetheless, Flt3 appears to be an outlier among RTKIII/V receptors due to several unique features in its extracellular segment (Lyman, 1993; Maroc, 1993), thus raising the question whether the current structural paradigm could be extrapolated to Flt3. Notably, Flt3 exhibits intragenic homology relating extracellular domains 1 and 4, and domains 2 and 5, indicative of an ancient internal duplication event during evolution. Furthermore, Flt3 has an N-terminal sequence of 50 amino acids preceding ectodomain 1 that shows no similarity to other proteins, and contains 12 additional cysteines that are not present in any of the homologous receptors.

Rational drug design for modulating Flt3-mediated signaling is hampered by the lack of structural information of the Flt3-receptor, in particular the Flt3 ligand-receptor interaction. It is therefore an object of the present invention to provide such structural information. In particular, identification of the binding site of Flt3 for its cognate ligand FL is instructive in screening, identifying and designing for ligands of Flt3 and FL which can be used to modulate Flt3 signaling.

SUMMARY

The present inventors have resolved the crystal structure of Flt3 bound to its cognate ligand. Surprisingly, and contrary to expectations, the inventors have identified a particular compact Flt3/FL binding interface. Flt3 employs a single and very compact ligand-binding epitope contributed exclusively by Ig-like domain 3 (D3), without engaging in homotypic interactions with its tandem receptor in the complex. This combination of features is completely unexpected because it deviates drastically from the current paradigm for extracellular activation of RTKIII receptors. More specifically, it was expected that Flt3 would collectively employ ectodomains D1-D3 to bind to its cognate cytokine, and that this interaction would be accompanied by homotypic interactions in the membrane-proximal domains D4-D5. The resolved crystal structure proves otherwise. As such, the Flt3 receptor is the only helical cytokine receptor that does not use more than one interaction site to bind its cognate ligand. In addition, FL is identified as the only helical cytokine that does not use any helix-helix groove to engage its receptor. Moreover, FL uses a preformed binding epitope to bind to the receptor subregion of the extracellular Flt3 domain.

Previous predictions identified a much larger region of the extracellular signaling complex as crucial for ligand binding and Flt3 activation. This hampered rational design of novel drugs targeting this large domain as it was not clear which regions were the most important. With the new data set, the binding epitope has been identified and turns out to be compact making it an interesting target for drug design. Also, it is clear now how FL interacts with this epitope, making blocking strategies of the ligand also a possibility, next to blocking its extracellular epitope (receptor blocking strategy versus ligand blocking strategy).

Aberrant Flt3 signaling is caused by oncogenic forms of the receptor or by overexpression of the wild type receptor. Furthermore, autocrine signaling loops seem to play an important role in leukogenesis (Zheng, 2004). Currently known strategies to modulate Flt3 signaling are mainly focused on targeting and inhibiting the intracellular tyrosine kinase domain with the use of tyrosine kinsase inhibitors (TKI). However, primary and secondary acquired resistance severely compromise long-term and durable efficacy of these inhibitors as a therapeutic strategy. Therefore, a major contribution of the present invention over the art includes the identification of a compact Flt3/FL binding interface, making it a very attractive target useful for protein-based therapeutic strategies aiming at blocking the binding of the cognate ligand FL to the Flt3 extracellular domain, or alternatively activating Flt3 signaling with FL mimetic ligands. Such strategies would lead to deactivation or activation, respectively, of downstream pathways affecting hematopoietic cell proliferation and DC homeostasis/activity.

Accordingly, in an aspect, the invention relates to a method for identifying or designing a ligand which modulates Flt3 signaling, comprising the step of employing a three dimensional structure represented by a set of atomic coordinates presented in Table 3, or a subset thereof, or atomic coordinates which deviate from those in Table 3, or a subset thereof, by RMSD over protein backbone atoms by no more than 3 Å.

In an embodiment, said method further comprises the step of structure-based identification and/or design of a ligand based on the interaction of said ligand with the 3D structure represented by the atomic coordinates presented in Table 3, or a subset thereof, or atomic coordinates which deviate from those in Table 3, or a subset thereof, by RMSD over protein backbone atoms by no more than 3 Å.

In another embodiment, said method is a computer-implemented method, said computer comprising an inputting device, a processor, a user interface, and an outputting device, wherein said method comprises the steps of:

a) generating a three-dimensional structure of atomic coordinates presented in Table 3, or a subset thereof, or atomic coordinates which deviate from those in Table 3, or a subset thereof, by RMSD over protein backbone atoms by no more than 3 Å;
b) fitting the structure of step a) with the structure of a candidate ligand by computational modeling;
c) selecting a ligand that possesses energetically favorable interactions with the structure of step a).

In an embodiment, said fitting comprises superimposing the structure of step a) with the structure of said candidate ligand. In another embodiment, said modeling comprises docking modeling.

In a further embodiment, said ligand of step c) can bind to at least 1 amino acid residue of the structure of step a) without steric interference.

In another aspect, the invention relates to a method for identifying a ligand which modulates Flt3 signaling, comprising the steps of:

a) providing a candidate ligand;
b1) providing a polypeptide comprising a region of at least 5 consecutive amino acid residues of amino acid residues 245-345 of Flt3; or
b2) providing a polypeptide comprising a region of at least 5 consecutive amino acid residues of amino acid residues 5-20 of FL;
c) contacting said candidate ligand with said polypeptide of step b1) or step b2);
d) determining the binding of said candidate ligand with said region of step b1) or step b2); and
e) identifying said candidate ligand as a ligand which modulates Flt3 signaling if binding between said candidate ligand and said region of step b1) or step b2) is detected.

In a further aspect, the invention relates to an in vitro method for modulating Flt3 signaling, comprising the steps of:

a) providing a composition comprising an Flt3 polyprotein; and
b) contacting said composition with a ligand as identified or designed according to the methods as described herein.

In yet another aspect, the invention relates to the use of a polypeptide comprising a region of at least 5 consecutive amino acid residues of amino acid residues 245-345 of Flt3, a polypeptide comprising a region of at least 5 consecutive amino acid residues of amino acid residues 5-20 of FL, and/or the atomic coordinates presented in Table 3, or a subset thereof, or atomic coordinates which deviate from those in Table 3, or a subset thereof, by RMSD over protein backbone atoms by no more than 3 Å for designing and/or identifying a ligand which modulates Flt3 signaling.

In an embodiment, the ligand which is designed and/or identified according to the methods as described herein is an antagonist, which is preferably selected from the group consisting of an Alphabody™, a Nanobody®, an antibody, or a small molecule.

In an aspect, the invention also relates to an Alphabody™, a Nanobody®, an antibody, or a small molecule which binds to the region comprised within amino acid residues 245-345 of Flt3, or which binds to the region comprised within amino acid residues 5-20 of FL.

In a further aspect, the invention relates to a polypeptide comprising at most 200 consecutive amino acid residues of Flt3, wherein said polypeptide comprises at least 5 consecutive amino acid residues of amino acid residues 245-345 of FL.

In another aspect, the invention relates to a polypeptide comprising at most 50 consecutive amino acid residues of FL, wherein said polypeptide comprises at least 5 consecutive amino acid residues of amino acid residues 5-20 of FL.

In an aspect, the invention also relates to a ligand as designed and/or identified according to the methods as described herein, for use as a modulator of Flt3 signaling.

A further aspect of the invention relates to a computer system comprising:

a) a database containing the atomic coordinates presented in Table 3, or a subset thereof, or atomic coordinates which deviate from those in Table 3, or a subset thereof, by RMSD over protein backbone atoms by no more than 3 Å, stored on a computer readable storage medium; and
b) a user interface to view the information.

DESCRIPTION OF THE DRAWINGS

FIG. 1. High-Affinity Complex Formation Between FL and Flt3 Ectodomain Variants.

(A-B) Isolation of Flt3D1-D5:FL and Flt3D1-D4:FL by size-exclusion chromatography (SEC). Also shown are Coomassie-stained SDS-PAGE strips corresponding to the peak fraction of the isolated complexes. The elution profiles of the complexes are characterized by large shifts to a single, faster migrating peak corresponding to the respective complex. (C) Size-exclusion chromatography on the Flt3D1-D3:FL mixture at the end of an ITC experiment, showing that a large amount of Flt3D1-D3 remains in the unbound form. Identical elution profiles were obtained in standard SEC experiments as well, in the presence of a large molar excess of FL. (D-F) Binding isotherms and thermodynamic parameters of FL binding to Flt3 ectodomains obtained by Isothermal Titration calorimetry (ITC). All experiments were performed by titrating recombinant Flt3 extracellular domains with FL.

FIG. 2 Crystal Structure of the Flt3D1-D4:FL Complex.

(A) Domain organization of the Flt3 extracellular segment. The five Ig-like domains of Flt3 (D1: residues 79-161, D2: residues 167-244, D3: residues 245-345, D4: residues 348-434 and D5: residues 435-533) are shown as colored boxes: D1 is colored in yellow, D2 in blue, D3 in green, D4 in orange and D5 in gray. N-linked glycosylation sites are indicated by blue diamonds. Partially occupied glycosylation sites are indicated with an asterisk. Also shown is the disulfide bond network in Flt3D1-D4 as determined by mass-spectrometry. The putative disulfide bridges in Flt3D5 are shown as dashed lines, based on homology with Flt3D2 and KITD5. (B) Overall structure of the Flt3D1-D4:FL complex. The crystal structure of the Flt3D14:FL complex is shown in ribbon representation with the twofold symmetry axis of FL oriented along the vertical axis of the plane. FL is colored in magenta, while the different domains of Flt3D1-4 follow the same coloring scheme as in panel A. Disulfide bridges are shown as yellow spheres and N-linked glycans as green sticks. The structural panels to the right show FL in ribbon representation and the receptor in surface representation. A 90° rotation of the main figure along the horizontal axis of the plane allows a clear view on the symmetry of the FL-Flt3D2-D3 subcomplex, whereas a 90° rotation along the vertical axis of the plane shows how FL is bound by the membrane-distal tip of D3. This view also clearly shows the asymmetric projection of the two Flt3D1 away from the core of the complex.

FIG. 3 The Flt3-FL Binding Interface.

(A) Close-up view of the Flt3-FL binding interface. FL is colored in green, Flt3D3 in grey and Flt3D2 in orange. Residues that constitute the cytokine-receptor interface are labeled and shown as sticks protruding from spheres centered at their C-alpha positions. FL residues are colored in yellow and Flt3 residues are colored in green. The receptor-binding epitope on FL is almost entirely contained in the N-terminal loop (8-13) preceding helix A (see also the inset). At the receptor site, the residues involved in ligand binding are located in the BC loop and strands D and E, and in the DE loop (see also panel B). Residue D180, in the AB loop of Flt3D2 might interact with S13 of FL, but is the only residue from Flt3D2 that could possibly contact FL. (B) The unusual Flt3D2-Flt3D3 interface. Flt3D2-D4 is shown as a C trace coloured in red. The ligand is shown in ribbon representation and is coloured green. Flt3D2 is tightly packed against Flt3D3 burying ˜1000 Å2. The residues that participate in the hydrophobic interface are labeled and their sidechains are shown as black sticks. Disulfide bonds in the two receptor domains are labeled and shown as ball and sticks (yellow). (C) Structure-based alignment of diverse FL sequences. A comparison of the FL sequences from a wide variety of species shows that the PISSXF-segment (residues 10-15) within the N-terminal loop is strictly conserved (coloured in red). A complete alignment can be found in Supplementary FIG. 3. (D) Structural comparison of bound versus the unbound FL. FL undergoes a domain tilt by 6° about its dimer interface upon receptor binding, while the receptor-binding epitope remains virtually unchanged upon receptor binding (shown in red).

FIG. 4 The Flt3D3-Flt3D4 Elbow and the Absence of Receptor Homotypic Contacts in the Flt3:FL Complex.

(A) The Flt3D3-Flt3D4 elbow. Flt3D3 (partially shown) and Flt3D4 are shown in ribbon representations. The -strands of Flt3D4 are labelled as A-G. The locations of the atypical disulfide bridges in Flt3D4 (Cys368-Cys407 and Cys381-Cys391) are indicated. Residues mediating hydrophobic interactions between Flt3D3 and Flt3D4 are shown as green sticks (F261, V345, F349 and Y376). Residues in the Flt3D3-Flt3D4 linker are shown as yellow spheres centered at their C-positions (E346-G348). The side-chains of residues that mediate the contacts between the AA′ loop of Flt3D3 and the C′E loop of Flt3D4 could not be modelled due to the low resolution of our analysis. The EF-loop of Flt3D4 which constitutes the ‘tyrosine corner’ around Y416 (green sticks) is shown in orange. (B) KITD3-KITD4 orientation in the KIT:SCF complex. Homotypic contacts between tandem ectodomain 4 modules in the KIT-SCF complex are mediated by salt bridges, formed by R381 and E386 (green sticks), which reside on the EF loops (orange) of the interacting domains (PDB entry 2E9W). The residues that make up the hydrophobic KITD3-KITD4 interface (L222, V308, F312 and F340) are shown as green sticks. Residues in the KITD3-KITD4 linker region (D309-G311) are shown as yellow spheres. (C) Flt3D4 displays an atypical EF-loop within the RTKIII/V family. A sequence comparison shows that the pair of residues mediating the homotypic contacts in KITD4 and VEGFR-2D7 is well conserved in the corresponding domains of all RTKIII/V members but not in Flt3D4. (D) Sequence conservation of residues involved at the D3-D4 interface in KIT and Flt3. A sequence comparison between human and murine Flt3 and KIT sequences reveals that the residues in the Flt3D3-Flt3D4 linker region and those participating in the hydrophobic Flt3D3-Flt3D4 interface are strongly conserved in the homologous KIT receptor.

FIG. 5 Architecture of the Complete Flt3 Ectodomain Complex.

Surface representations of the full length Flt3 ectodomain complex. FL is coloured in magenta, D2 in blue, D3 in green, D4 in orange and D5. The central view shows the complex with the two-fold axis of FL oriented vertically in the plane of the paper. The left panel shows a view corresponding to a 45° rotation along the vertical axis, while the right panel shows a view at a 90° rotation along the horizontal axis. Whereas domains D2, D3 and D4 essentially follow the P2-symmetry of FL, domains 5 and 1 display varying degrees of plasticity. Like the Flt3D1-D4:FL complex, the Flt3D1-D5:FL complex is devoid of homotypic interactions as the tandem membrane-proximal modules Flt3D4-D5 remain separated by 20 Å.

FIG. 6 Comparison of Representative Extracellular Complexes for all Members of the RTKIII/V Family.

The structures shown represent the architecture of receptor-cytokine complexes for the different members of the RTKIII/V family: From left to right: human Flt3:FL (this study), human KIT:SCF (PDB 2E9W), murine CS-1R:CSF-1 (PDB 3EJJ), hPDGFR:PDGF (PDB 3MJG) and human VEGFR2:VEGF (PDB 2X1X). The dimeric ligands are colored in magenta. Receptor ectodomains are coloured as follows: D1 in pale yellow, D2 in blue, D3 in green, D4 in orange and D5 in grey.

FIG. 7 Asymmetric Unit of the D1-4 and D1-5 Complex.

(A) The asymmetric unit of Flt3D1-D4:FL complex crystals. The Flt3D1-D4:FL complex crystallized in spacegroup P21 with two complexes in the asymmetric unit (asu). The two helical ligands in the different complexes (chains A-B and chains C-D) make extensive interactions in the asu. The receptor chains are labeled E, F, H and G. No density was visible for domains D1 of receptor chains G and H. D4 of chain G was also not modelled because of its weak density. (B) The asymmetric unit of Flt3D1-D5:FL complex crystals. Like the Flt3D1-D4:FL complex, the Flt3D1-D5:FL complex crystallized in spacegroup P21 with two complexes in the assymetric unit (asu). The contacts between the two complexes are entirely mediated by the two ligands (chains A-B and chains C-D). The Flt3 receptor chains are labeled E, F, H and G. The structure was refined by rigid-body refinement in autoBuster 2.8 using the FL promoters (residues 3-132), Flt3D1 (residues 79-161), Flt3D2-D3 (residues 167-345), Flt3D4 (residues 348-434) and Flt3D5 (residues 437-529) as rigid bodies. D1 of chain F was not modelled because of its weak density.

FIG. 8 Final Quality of the Density Map for the D14 Complex.

(A) Stereo diagram illustrating the quality of the final 2Fo-Fc electron density map to 4.2 Å resolution (contoured at 1) for the Flt3D1-D4:FL complex. The figure is centered on the Flt3D2-D3 interface and junction, with the final model for Flt3D2 (left) and Flt3D3 (right) displayed in ribbon representation (blue). The N-linked NAG glycan residue modeled at Asn306 is shown in sticks (magenta). (B) Phase improvement by density modification based on a partial model of the Flt3D1-D4:FL complex consisting of only FL and Flt3D3. The electron density is contoured at 1. The final model for domains 3 and 4 in one of the receptor chains in the Flt3D1-D4-FL complex structure is shown in ribbon representation. N-linked glycans are shown in stick representation (magenta). This electron density map was obtained by applying NCS-averaging and solvent flattering protocols as implemented in PARROT1, and proved to be crucial early in the structure determination process providing the complete electron density trace for domain 4.

FIG. 9 Interspecies Comparison of the Flt3 Ligand (FL) Sequence.

Sequence numbering and secondary structure assignment are according to the determined structure of human Flt3 ligand (pdb 1ETE). Strictly conserved residues in the included FL sequences are shaded. Residues shown to interact with the receptor (according to the present invention) are marked with an asterix. The sequences were retrieved from the NCBI and Ensembl databases: Homo sapiens (NP_—001450.2), Mus musculus (NP_—038548.3), Rattus norvegicus (XP_—002725623.1), Papio cynocephalus (AAO72538.1), Felis catus (NP_—001009842.1), Ailuropoda melanoleuca (XP_—002917887.1), Canis lupus familiaris (NP_—001003350.1), Pteropus vampyrus (ENSPVAT00000010957), Ovis aries (NP_—001072128.1), Bos taurus (NP_—851373.1), Sus scrofa (ACZ63257.1), Sorex araneus (ENSSARP00000002887), Cavia porcellus (ENSCPOP00000020385), Monodelphis domestica (XP_—001379894), Xenopus tropicalis (XP_—002938571.1)

FIG. 10 Structural Characterization of the Flt3D1-5:FL Complex by Negative-Staining Electron Microscopy and SAXS Analysis of the Flt3D1-D5:FL Complex.

(A) The displayed gallery of 100 class averages of the Flt3D1-D5:FL complex allows to recognise features corresponding to projections of the crystal structure at different orientations, notably the slightly open horseshoe ring structure with well defined individual IgG domains. (B) The crystal structure of the Flt3D1-5-FL complex was refined as a rigid-body model against the experimental scattering curve obtained by SAXS. Fitting of the theoretical scattering curve calculated from the refined model (inset) to the experimental scattering curve shows a good agreement (X²=2.5).

FIG. 11 Mapping of Non-Synonymous Sequence Variants Identified in the Flt3 Ectodomain of AML Patients.

While the majority of oncogenic alterations in the Flt3 gene are located in the JM and TKD regions, several mutations in the extracellular domains have recently been identified in AML patients2, 3. Expression of Flt3 carrying a mutation at position 451 (S451F) in BaF3 cells resulted in cytokine-independent proliferation and constitutive Flt3 autophosphorylation, demonstrating the oncogenic potential of this sequence variant. S451 is located at the solvent exposed site of strand B in the membrane proximal domain 5. Although the D324N variant did not result in ligand independent activation it is associated with a higher risk of myeloid leukemias3. D324 is located in the EF-loop of domain 3. The possible role for all other sequence variants (T167A, V194M, Y364H) in leukemogenesis has not yet been demonstrated.

FIG. 12 Sequence Listing.

DETAILED DESCRIPTION

Before the present method and products of the invention are described, it is to be understood that this invention is not limited to particular methods, components, products or combinations described, as such methods, components, products and combinations may, of course, vary. It is also to be understood that the terminology used herein is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. It will be appreciated that the terms “comprising”, “comprises” and “comprised of” as used herein comprise the terms “consisting of”, “consists” and “consists of”.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

The term “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/−10% or less, preferably +/−5% or less, more preferably +/−1% or less, and still more preferably +/−0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

Whereas the terms “one or more” or “at least one”, such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any ≧3, ≧4, ≧5, ≧6 or ≧7 etc. of said members, and up to all said members.

All references cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings of all references herein specifically referred to are incorporated by reference.

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

In the following detailed description of the invention, reference is made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration only of specific embodiments in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural or logical changes may be made without departing from the scope of the present invention. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the present invention is defined by the appended claims.

Standard techniques commonly used in molecular biology are well known in the art, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989); Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988); Watson et al., Recombinant DNA, Scientific American Books, New York; Birren et al (eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York (1998).

As used herein, “Flt3” refers to fms-like tyrosine kinase receptor-3 (Entrez Gene ID of the human orthologue: 2322; NCBI reference mRNA sequence: NM_—004119.2 (SEQ ID NO: 1); NCBI reference protein sequence: NP_—004110.2 (SEQ ID NO: 2)). Unless explicitly indicated otherwise, all Flt3 amino acid residue positions referred to herein correspond to the amino acid residue positions as indicated in SEQ ID NO: 2. SEQ ID NO: 6 is a polypeptide consisting of a subset of contiguous amino acid residues of SEQ ID NO: 2, corresponding to the extracellular domain of Flt3, in particular Ig-like domains D1 to D5 (amino acid residues 27-541 of SEQ ID NO: 2). According to the invention, the Flt3 nucleotide and protein sequences referred to herein relate to Flt3 sequences originating from any organism, i.e. all orthologues of Flt3. Preferably, the Flt3 nucleotide and protein sequences referred to herein are from mammalian origin. Particularly preferred Flt3 sequences are human.

As used herein, “FL” refers to fms-like tyrosine kinase receptor-3 ligand (Entrez Gene ID of the human orthologue: 2323; NCBI reference mRNA sequence: NM_—001459.2 (SEQ ID NO: 3); NCBI reference protein sequence: NP_—001450.2 (SEQ ID NO: 4)). Amino acid residue positions 1 to 26 correspond to the signal peptide of FL. SEQ ID NO: 5 represents human mature FL in which the signal peptide is removed. Unless explicitly indicated otherwise, all FL amino acid residue positions referred to herein correspond to the amino acid residue positions as indicated in SEQ ID NO: 5. According to the invention, the FL nucleotide and protein sequences referred to herein relate to FL sequences originating from any organism, i.e. all orthologues of FL. Preferably, the FL nucleotide and protein sequences referred to herein are from mammalian origin. Particularly preferred FL sequences are human.

As used herein, the term “ligand” refers to a substance that is able to bind to and form a complex with a biomolecule to serve a biological purpose. The binding occurs by intermolecular forces, such as ionic bonds, hydrogen bonds and van der Waals forces. The docking (association) is usually, and preferably, reversible (dissociation). According to the invention, the ligand referred to herein is a ligand of Flt3 or a ligand of FL. As the ligands according to the present invention are able to modulate Flt3 signaling, the term “ligand” can be used interchangeably with the term “modulator”. In an embodiment, the ligand according to the invention are characterized by a dissociation constant (K_d) for its substrate (Flt3 or FL) of at most 10⁻⁵M, preferably at most 10⁻⁶M, at most 10⁻⁷M, at most 10⁻⁸M, at most 10⁻⁹M, or at most 10⁻¹⁰M.

As used herein, the term “binding site” or “binding interface” relates to the respective regions on either of two components where binding takes place. This region typically includes amino acid residues which are directly involved in binding and participate in non-covalent intermolecular interactions. This region may also include amino acid residues which are not directly involved in binding or participate in non-covalent intermolecular interactions, but which are merely interspersed between interacting amino acid residues, and/or provide a structural, special, energetic or other function. The term “binding site” or “binding interface” also refers to an area which determines an exclusion zone or competition zone of a component for two ligands with the same binding site. According to the present invention, the Flt3/FL binding interface or Flt3 and FL binding sites comprises or consists of amino acid residues 240-350, in particular D3, more in particular amino acid residues 245-345, even more in particular amino acid residues 279-311 of Flt3 and amino acid residues 5-20, in particular 5-18, 8-18, 5-15, or 8-15 of FL, preferably 5-15.

As used herein, the term “ligand which modulates Flt3 signaling” or “modulator of Flt3 signaling” refers to a ligand or modulator which is capable of influencing, regulating and/or otherwise altering Flt3 signaling. As such, contacting the ligand or modulator according to the present invention with its substrate results in a measurable effect on Flt3 signaling. Such effects can be for instance partial or full activation of Flt3 signaling, enhancement of Flt3 signaling, reduction of Flt3 signaling or partial or full inhibition of Flt3 signaling. Flt3 signaling is well documented in the art. Flt3 is a class III receptor tyrosine kinase, which activation resides in activation of the intracellular kinase domains by phosphorylation upon ligand binding. These phosphorylation events initiate downstream signaling via the PI3K/AKT and the RAS/RAF/MEK/ERK pathways. Modulation of Flt3 signaling can be easily and routinely evaluated for instance by measurement of a change in intracellular Flt3 phosphorylation or any of the downstream components. By means of example, and without limitation, Flt3 activation can be evaluated by measurement of tyrosine phosphorylation status (such as Y958 or Y969) by means of phospho-specific Flt3 antibodies, which are known in the art. By extension, modulation of Flt3 signaling can also be evaluated based on a specific biological event or outcome. It is known that Flt3 signaling is a potent regulator mechanism of for instance dendritic cell (DC) development and homeostasis and DC-mediated natural killer cell (NKC) activation. Therefore, a modulator of Flt3 signaling may also be evaluated or identified based on for instance measurement of DC proliferation, development, homeostasis or NKC activation.

The ligand according to the present invention can be of any chemical class of molecules, such as, without limitation, a naturally occurring or non-natural occurring protein, nucleic acid, hapten, lipid, carbohydrate, as well as chimeras and/or derivatives thereof, in monomeric, polymeric or conjugated forms. In a preferred embodiment, the ligand is an Alphabody™ (Complix, Belgium) a Nanobody® (Ablynx, Belgium), an antibody, or a small molecule, preferably an Alphabody™.

Antibodies, methods for obtaining antibodies, methods for screening antibodies are known in the art, and will not be detailed further. By means of further guidance, and without limitation, full length antibodies as well as functional fragments thereof, such as Fab, Fab′, (Fab′)₂, or Fv fragments, can be used as ligands to be identified or designed according to the invention. Also single chain antibodies (SCA) can be used.

Nanobodies® are antibody fragments consisting of a single monomeric variable antibody domain. These antibody-derived proteins contain the unique structural and functional properties of naturally-occurring heavy-chain antibodies. Originally derived from camelidae, these heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). The VHH domain is a perfectly stable polypeptide harbouring the full antigen-binding capacity of the original heavy-chain antibody. The isolated VHH domain is called a Nanobody®, and is described for instance in WO 94/04678, which is incorporated herein in its entirety by reference. In addition to sharing various common structural and functional features with conventional antibodies, in particular high target specificity, high affinity for their target, and low inherent toxicity, Nanobodies® offer several additional advantages. Due to their small size (about 1/10^thof conventional antibodies), like small molecule drugs they have the opportunity to inhibit enzymes and readily access receptor clefts. Furthermore, Nanobodies® are extremely stable, have the potential to be administered by means other than injection, and are easy to manufacture. These characteristics make Nanobodies® a versatile tool for drug development.

Accordingly, the invention also relates to a Nanobody® as identified or designed according to the methods as described herein.

Alphabodies™ are single-chain, triple-stranded coiled coil proteins with a molecular weight of between 10 and 14 kDa (10 to 15 times smaller than antibodies). Alphabodies™ are described in EP 2 188 303, EP 2 161 278 and WO 2010/066740 which are incorporated herein in their entirety by reference. Alphabodies™ can bind with high affinity to a wide range of molecular targets and display various beneficial characteristics as therapeutic drugs. Due to their unique structural properties, Alphabodies™ can bind to certain types of targets that are not easily accessible to antibodies or other types of protein scaffolds. Because of their small size, Alphabodies™ have a superior tissue penetration potential as compared to larger protein therapeutics, such as conventional antibodies. Despite their small size however, and simple structure, Alphabodies™ can display more than one antigen binding site on their surface; this means that a single Alphabody™ domain can display multi-specific target binding, a feature hardly achievable with antibodies or other known protein scaffolds. Furthermore, Alphabodies™ are extremely stable (melting temperature of >120° C.), can be autoclaved, can be lyophilized, and are highly resistant to various proteases. These properties allow the development of different formulations and alternative modes of administration (such as topical or pulmonary). Additional advantages of Alphabodies™ include the ease with which the in vivo half-life can be modulated (e.g. by standard techniques such as PEGylation) as well as the ease of production (e.g. by E. coli fermentation). Like Nanobodies®, these characteristics make Alphabodies™ a versatile tool for drug development. A particularly advantageous property of Alphabodies™ is their structural similarity with the cognate ligand of Flt3, FL (helical-shaped protein scaffolds), which makes this type of moieties excellent candidates for the design of non-naturally occurring ligands for Flt3. Accordingly, the invention also relates to a Alphabody™ as identified or designed according to the methods as described herein.

As used herein, the term “small molecule” refers to a low molecular weight organic, inorganic, or organometallic compound typically having a molecular weight of less than about 1000, as is generally known in the art. Small molecules can occur naturally (such as neurotransmitters, (steroid) hormones, etc.) or can be chemically synthesized. Most conventional pharmaceuticals, such as for instance aspirin, are small molecules. By means of example, small molecules include, but are not limited to, mono- or oligo-saccharides, -peptides, peptidomimetics, primary or secondary metabolites, etc. Small molecules can be of any chemical class, such as, without limitation, alcohols, ethers, esters, aldehydes, ketons, acids, amines, amides, etc. and can be chemically modified. Small molecule libraries offer a good source of small molecules for use in screening for particular activity. Methods for generating small molecule libraries are for instance disclosed in WO9424314. Various types of small molecule libraries can be obtained from commercial sources, such as, for instance, from ChemBridge (San Diego, Calif., USA).

As used herein, the term “crystal” refers to an ordered state of matter, in particular 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. The term “crystal” refers in particular to a solid physical crystal form such as an experimentally prepared crystal.

Proteins, by their nature are difficult to purify to homogeneity. Even highly purified proteins may be chronically heterogeneous due to modifications, the binding of ligands or a host of other effects. In addition, proteins are crystallized from generally complex solutions that may include not only the target molecule but also buffers, salts, precipitating agents, water and any number of small binding proteins. It is important to note that protein crystals are composed not only of protein, but also of a large percentage of solvents molecules, in particular water. These may vary from 30 to even 90%. Protein crystals may accumulate greater quantities and a diverse range of impurities which cannot be listed here or anticipated in detail. Frequently, heterogeneous masses serve as nucleation centers and the crystals simply grow around them. The skilled person knows that some crystals diffract better than others. Crystals vary in size from a barely observable 20 μm to 1 or more mm. Crystals useful for X-ray analysis are typically single, 0.05 mm or larger, and free of cracks and defects.

As used herein, the term “atomic coordinates” refers to a set of values which define the position of one or more atoms with reference to a system of axes. This term refers to the information of the three dimensional organization of the atoms contributing to a protein structure. The final map containing the atomic coordinates of the constituents of the crystal may be stored on a data carrier; typically the data is stored in PDB format or in x-plor format, both of which are known to the person skilled in the art. However, crystal coordinates may as well be stored in simple tables or text formats. The PDB format is organized according to the instructions and guidelines given by the Research Collaboratory for structural Bioinformatics. It will be understood by those skilled in the art that atomic coordinates may be varied, without affecting significantly the accuracy of models derived therefrom. Thus, although the invention provides a very accurate definition of a preferred atomic structure, it will be understood that minor variations are envisaged and the claims are intended to encompass such variations. The invention also relates to subsets of atomic coordinates as described herein, as well as the use of subsets in the methods as described herein. In a preferred embodiment, said subsets comprise or consist of the Flt3/FL binding interface, the FL binding site on Flt3, or the Flt3 binding site on FL. Particularly preferred subsets of the atomic coordinates as described herein are subsets comprising or consisting of atomic coordinates of atoms 1 to 681 of Table 3 or atoms 1 to 687 of Table 3 for atomic coordinates corresponding to Flt3; or atoms 688 to 1698 of Table 3 for atomic coordinates corresponding to FL. In other preferred embodiments, a subset of atomic coordinates may comprise or consist of atomic coordinates of atoms 227 to 456 of Table 3 for atomic coordinates corresponding to Flt3; or atoms 709 to 818 of Table 3 for atomic coordinates corresponding to FL; or a combination of both. In yet other preferred embodiments, the subsets of atomic coordinates may comprise or consist of atomic coordinates of atoms of Table 3, corresponding to any of the amino acid regions (of Flt3 and/or FL) as disclosed herein.

The term “root mean square deviation” (rmsd) is used as a means of comparing two closely related structures and relates to a deviation in the distance between related atoms of the two structures after structurally minimizing this distance in a superposition. Related proteins with closely related structures will be characterized by relatively low RMSD values whereas larger differences will result in an increase of the RMSD value.

As used herein, the terms “% identical” and “% homologous” in the context of polynucleic acid sequences or polypeptide sequences refer to the similarity between two sequences, preferably expressed as a percentage of identical nucleic acids or amino acids between two sequences after alignment of these sequences. Alignments and percentages of identity can be performed and calculated with various different programs and algorithms known in the art. Preferred alignment algorithms include BLAST (Altschul, 1990; available for instance at the NCBI website) and Clustal (reviewed in Chema, 2003; available for instance at the EBI website). Preferably, BLAST is used to calculate the percentage of identity between two sequences.

In an aspect, the invention relates to a crystal comprising Flt3, in particular the extracellular domain of Flt3. In an embodiment, said extracellular domain is at least 80%, preferably at least 85%, 90%, or 95% identical or homologous to SEQ ID NO: 6. In another embodiment, said extracellular domain has the sequence of SEQ ID NO: 6. The invention further relates to a crystal comprising Flt3, in particular the extracellular domain of Flt3, and a ligand. Preferably, said ligand is FL. In an embodiment, said ligand is at least 80%, preferably at least 85%, 90%, or 95% identical or homologous to SEQ ID NO: 5. In yet another embodiment, said ligand has the sequence of SEQ ID NO: 5.

The invention also relates to a crystal comprising a fragment of the extracellular domain of Flt3. The invention further relates to a crystal comprising a fragment of the extracellular domain of Flt3, and a ligand, preferably FL. Said fragment of the extracellular domain of Flt3 is extracellular domain (D) D1, D2, D3, D4, or D5, preferably D3. In an embodiment, said fragment of the extracellular domain of Flt3 is amino acid residues 79-161, 167-244, 245-345, 348-434, or 435-533, preferably 245-345. In another embodiment, said fragment of the extracellular domain of Flt3 is at least 80%, preferably at least 85%, 90%, or 95% identical or homologous to amino acid residues 79-161, 167-244, 245-345, 348-434, or 435-533 of SEQ ID NO 2, preferably 245-345.

The crystal of the invention preferably effectively diffracts x-rays for the determination of the atomic coordinates of the protein to a resolution better than 6 Å. More preferably the three dimensional structure determinations can be determined with a resolution of more than 5 Å, such as more than 4 Å or most preferably about 3.5 Å using the crystals according to the invention.

In a further embodiment, said crystal comprises a three-dimensional (3D) crystal structure characterized by the atomic coordinates in Table 3, or a subset thereof. Preferred subsets define one or more of the extracellular domains D1, D2, D3, D4, and/or D5 of Flt3. It will be understood that any reference herein, as well as in other aspects and embodiments of the invention as disclosed herein, to the atomic coordinates or subset of the atomic coordinates shown in Table 3 shall include, unless specified otherwise, atomic coordinates having a root mean square deviation of backbone atoms of not more than 3 Å, preferably not more than 2.5 Å, preferably not more than 1.5 Å, even more preferably not more than 1 Å, when superimposed on the corresponding backbone atoms described by the atomic coordinates shown in Table 3. Preferred variants are those in which the root mean square deviation (RMSD) of the x, y and z co-ordinates for all backbone atoms other than hydrogen is less than 1.5 Å (preferably less than 1 Å, 0.7 Å or less than 0.3 Å) compared with the coordinates given in Table 3. It will be readily appreciated by those skilled in the art that a 3D rigid body rotation and/or translation of the atomic coordinates does not alter the structure of the molecule concerned. In a highly preferred embodiment, the crystal has the atomic coordinates as shown in Table 3.

A person skilled in the art will appreciate that a set of atomic coordinates determined by X-ray crystallography is not without standard error. Accordingly, any set of structure coordinates for a crystal as described herein that has a root mean square deviation of protein backbone atoms of less than 0.75 Å when superimposed (using backbone atoms) on the atomic coordinates listed in Table 3 shall be considered identical.

The present invention also relates to the atomic coordinates of a crystal as described herein that substantially conforms to the atomic coordinates listed in Table 3. Accordingly, in an aspect, the invention relates to a set of atomic coordinates as shown in Table 3, or a subset thereof of both or either, in which the coordinates define a three dimensional structure of (the extracellular domain of) Flt3 and/or FL. The invention also relates to atomic coordinates which deviate from those in Table 3, or a subset thereof, by RMSD over protein backbone atoms by no more than 3 Å.

A structure that “substantially conforms” to a given set of atomic coordinates is a structure wherein at least about 50% of such structure has an RMSD of less than about 1.5 Å for the backbone atoms in secondary structure elements in each domain, and more preferably, less than about 1.3 Å for the backbone atoms in secondary structure elements in each domain, and, in increasing preference, less than about 1.0 Å, less than about 0.7 Å, less than about 0.5 Å, and most preferably, less than about 0.3 Å for the backbone atoms in secondary structure elements in each domain.

In a more preferred embodiment, a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 75% of such structure has the recited RMSD value, and more preferably, at least about 90% of such structure has the recited RMSD value, and most preferably, about 100% of such structure has the recited RMSD value. In an even more preferred embodiment, the above definition of “substantially conforms” can be extended to include atoms of amino acid side chains. As used herein, the phrase “common amino acid side chains” refers to amino acid side chains that are common to both the structure which substantially conforms to a given set of atomic coordinates and the structure that is actually represented by such atomic coordinates.

Those of skill in the art will understand that a set of structure coordinates for a protein or protein complex or a portion thereof, is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. The variations in coordinates may be generated by mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth in Table 3 could be manipulated by crystallographic permutations of the structure coordinates, fractionalization or matrix operations to sets of the structure coordinates or any combination of the above.

Various computational analyses are used to determine whether a molecular complex or a portion thereof is sufficiently similar to all or parts of the structure of the extracellular domain of IR described above. Such analyses may be carried out in current software applications, such as the Molecular Similarity program of QUANTA (Molecular Simulations Inc., San Diego, Calif.) version 4.1.

The Molecular Similarity program permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure. Comparisons typically involve calculation of the optimum translations and rotations required such that the root mean square difference of the fit over the specified pairs of equivalent atoms is an absolute minimum. This number is given in angstroms (Å).

Accordingly, structural coordinates of an (extracellular domain of) Flt3, or fragments thereof and/or FL within the scope of the present invention include structural coordinates related to the atomic coordinates listed in Table 3 by whole body translations and/or rotations. Accordingly, RMSD values listed herein assume that at least the backbone atoms of the structures are optimally superimposed which may require translation and/or rotation to achieve the required optimal fit from which to calculate the RMSD value. A three dimensional structure of an Flt3 and/or FL polypeptide or region thereof which substantially conforms to a specified set of atomic coordinates can be modeled by a suitable modeling computer program such as MODELER (Sali & Blundell, 1993), as implemented in the Insight II Homology software package (Insight II (97.0), MSI, San Diego), using information, for example, derived from the following data: (1) the amino acid sequence of the human Flt3 (extracellular domain) and/or FL; (2) the amino acid sequence of the related portion(s) of the protein represented by the specified set of atomic coordinates having a three dimensional configuration; and, (3) the atomic coordinates of the specified three dimensional configuration. A 3D structure of such polypeptides which substantially conforms to a specified set of atomic coordinates can also be calculated by a method such as molecular replacement, which is described in detail below.

In another aspect, the invention relates to the use of a crystal as defined herein for determining the 3D structure of (the extracellular domain) of Flt3, or fragments thereof, and/or FL, or fragments thereof, as well as a method for determining the 3D structure of (the extracellular domain) of Flt3, or fragments thereof, and/or FL, or fragments thereof, by means of said crystal.

In a further aspect, the invention relates to a three-dimensional structure obtained by or obtainable by the crystal as described herein.

In a further aspect, the invention relates to the use of the atomic coordinates as described in Table 3, or a subset thereof, or atomic coordinates which deviate from those in Table 3, or a subset thereof, by RMSD over protein backbone atoms by no more than 3 Å, for identifying and/or designing a modulator of Flt3 signaling, for identifying and/or designing a ligand of Flt3 or for identifying and/or designing a ligand of FL.

In a further aspect, the invention relates to a method for identifying and/or designing a modulator of Flt3 signaling, for identifying and/or designing a ligand of Flt3 or for identifying and/or designing a ligand of FL, comprising structure-based identification and/or design of a ligand based on the interaction of said ligand with the 3D structure represented by the atomic coordinates of Table 3, or a subset thereof, or atomic coordinates which deviate from those in Table 3, or a subset thereof, by RMSD over protein backbone atoms by no more than 3 Å. Said subset preferably comprises or consists of the Flt3/FL binding interface, the FL binding site of Flt3 and/or the Flt3 binding site of FL, as described herein.

Structure coordinates/atomic coordinates are typically loaded onto a machine readable-medium for subsequent computational manipulation. Thus models and/or atomic coordinates are advantageously stored on machine-readable media, such as magnetic or optical media and random-access or read-only memory, including tapes, diskettes, hard disks, CD-ROMs and DVDs, flash memory cards or chips, servers and the internet. The machine is typically a computer. Accordingly, in an aspect, the invention relates to a machine- or computer-readable data storage medium comprising a data storage material encoded with the structure coordinates, or at least a portion of the structure coordinates set forth in Table 3. Thus, in accordance with the present invention, the structure coordinates of (the extracellular domain of) Flt3, or fragments thereof and/or FL, or fragments thereof, can be stored in a machine- or computer-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery and X-ray crystallographic analysis of protein crystal. Accordingly, the invention also relates to a computer-readable media comprising the three-dimensional structure of the crystal as described herein. The invention further relates to a computer-readable media comprising the atomic coordinates of Table 3, or a subset thereof, or atomic coordinates which deviate from those in Table 3, or a subset thereof, by RMSD over protein backbone atoms by no more than 3 Å.

The storage medium may be local to a computer as described above, or the storage medium may be located in a net-worked storage medium including the internet, to which remote accessibility is possible.

The structure coordinates/atomic coordinates may be used in a computer to generate a representation, e.g. an image, of the three-dimensional structure of the IR ectodomain crystal which can be displayed by the computer and/or represented in an electronic file.

The structure coordinates/atomic coordinates and models derived therefrom may also be used for a variety of purposes such as drug discovery, biological reagent (binding protein) selection and X-ray crystallographic analysis of other protein crystals. Accordingly, in an aspect, the invention relates to the use of the crystal, the atomic coordinates or the computer-readable media as described herein for the identification and the design of ligands of Flt3 and/or FL. In another aspect, the invention relates to methods for identifying or designing ligands of Flt3 and/or FL by means of the crystal, the atomic coordinates or the computer-readable media as described herein. Alternatively, the invention also relates to the use of the crystal, the atomic coordinates or the computer-readable media as described herein for the identification of the binding-site for a ligand on Flt3 and/or FL. In another aspect, the invention relates to methods for identifying the binding-site for a ligand on Flt3 and/or FL by means of the crystal, the atomic coordinates or the computer-readable media as described herein.

Modulators of Flt3 signaling can be identified or designed with various computer-implemented modeling algorithms known in the art. As used herein, the term “modeling” includes the quantitative and qualitative analysis of molecular structure and/or function based on atomic structural information and interaction models. The term “modeling” 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. Molecular modeling techniques can be applied to the atomic coordinates as described herein or a subset thereof to derive a range of 3D models and to investigate the structure of binding sites, such as the binding sites of potential ligands. Such modeling methods are developed to design or select chemical entities that possess stereochemical complementary to particular target regions. By “stereochemical complementarity” is meant that the compound or a portion thereof makes a sufficient number of energetically favourable contacts with the target region as to have a net reduction of free energy on binding to the receptor. It is preferred that the stereochemical complementarity is such that the compound has a dissociation constant (K_d) for its substrate (Flt3 or FL) of at most 10⁻⁵M, preferably at most 10⁻⁶M, at most 10⁻⁷M, at most 10⁻⁸M, at most 10⁻⁹M, or at most 10⁻¹⁰M. It will be appreciated that it is not necessary that the complementarity between chemical entities and the receptor site extend over all residues of the target site in order to modulate Flt3 signaling.

Modeling and docking software that can be used for the identification or design of ligands is well known in the art and includes, without limitation DOCK, FLEXR, GOLD, FLO, FRED, GLIDE, LIGFIT, MOE, MVP, QUANTA, INSIGHT, SYBYL, AMBER, CHARMM, GRID, MCSS, AUTODOCK, CAVEAT, MACCS-3D, HOOK. By means of example, and without limitation, the following approach may be used to identify and/or design ligands. Ligands are in silico directly docked from a three-dimensional structural database, to the target site, using mostly, but not exclusively, geometric criteria to assess the goodness-of-fit of a particular molecule to the site. This approach is illustrated by Kuntz et al. (1982) and Ewing et al. (2001), the contents of which are hereby incorporated by reference, whose algorithm for ligand design is implemented in a commercial software package, DOCK version 4.0, distributed by the Regents of the University of California and further described in a document, provided by the distributor, which is entitled “Overview of the DOCK program suite” the contents of which are hereby incorporated by reference. Ligands identified on the basis of geometric parameters, can then be modified to satisfy criteria associated with chemical complementarity, such as hydrogen bonding, ionic interactions and Van der Waals interactions. The scoring functions may include, but are not limited to force-field scoring functions (affinities estimated by summing Van der Waals and electrostatic interactions of all atoms in the complex between the target site and the ligand), empirical scoring functions (counting the number of various interactions, for instance number of hydrogen bonds, hydrophobic-hydrophobic contacts and hydrophilic-hydrophobic contacts, between the target site and the ligand), and knowledge based scoring functions (with basis on statistical findings of intermolecular contacts involving certain types of atoms or functional groups). Scoring functions involving terms from any of the two of the mentioned scoring functions may also be combined into a single function used in database virtual screening of chemical libraries. Different scoring functions can be employed to rank and select the best molecule from a database. See for example Bohm & Stahl (1999). The software package FlexX, marketed by Tripos Associates, Inc. (St. Louis, Mo.) is another program that can be used in this direct docking approach (see Rarey et al., 1996).

Once a ligand has been designed or identified, the efficiency with which the ligand may bind to the target site can be tested and optimized by computational evaluation. An effective ligand must preferably demonstrate a relatively small difference in energy between its bound and free states (i.e., a small deformation energy of binding). Thus, the most efficient ligand should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole, preferably, not greater than 7 kcal/mole.

A compound designed or identified as binding to a target site may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target protein. Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions. Specifically, the sum of all electrostatic interactions between ligand and the target site, preferably make a neutral or favorable contribution to the enthalpy of binding. Once an Flt3- or FL-binding ligand has been optimally selected or designed, as described above, substitutions may then be made in some of its atoms or side groups to improve or modify its binding properties. Generally, initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided. Such substituted chemical compounds may then be analyzed for efficiency of fit to the target site by the same computer methods described above.

The identification and/or design methods may be implemented in hardware or software, or a combination of both. However, preferably, the methods are implemented in computer programs executing on programmable computers each comprising a processor, a data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device. Program code is applied to input data to perform the functions described above and generate output information. The output information is applied to one or more output devices, in known fashion. The computer may be, for example, a personal computer, microcomputer, or workstation of conventional design. Each program is preferably implemented in a high level procedural or object-oriented programming language to communicate with a computer system. However, the programs can be implemented in assembly or machine language, if desired. In any case, the language may be compiled or interpreted language. Accordingly, the invention relates to a computer system comprising:

a) a database containing information on the three dimensional structure of the crystal as described herein, stored on a computer readable storage medium; and
b) a user interface to view the information.

In an embodiment, said database contains the atomic coordinates presented in Table 3, or a subset thereof, or atomic coordinates which deviate from those in Table 3, or a subset thereof, by RMSD over protein backbone atoms by no more than 3 Å, stored on a computer readable storage medium. Said subset preferably comprises or consists of the Flt3/FL binding interface, the FL binding site of Flt3 and/or the Flt3 binding site of FL, as described herein.

In an aspect, the invention relates to a method of identifying or designing a ligand which modulates Flt3 signaling, a ligand of (the region comprised within amino acid residues 240-350, preferably 245-345 of) Flt3 or a ligand of (the region comprised within amino acid residues 5-20 of) FL, comprising the step of employing a three dimensional structure of the crystal as described herein or the atomic coordinates as described herein, or a subset thereof or atomic coordinates which deviate from those in Table 3, or a subset thereof, by RMSD over protein backbone atoms by no more than 3 Å.

In an embodiment, said method further comprises the step of structure-based identification and/or design of a ligand based on the interaction of said ligand with the 3D structure represented by the atomic coordinates of Table 3, or a subset thereof, or atomic coordinates which deviate from those in Table 3, or a subset thereof, by RMSD over protein backbone atoms by no more than 3 Å.

In an embodiment, said method is a computer-implemented method, said computer preferably comprising an inputting device, a processor, a user interface, and/or an outputting device. Said inputting device may comprise for instance a CD-rom driver, a USB-port, a keyboard. Said processor may comprise hardware and software (such as the modeling algorithms and programs as described herein). Said user interface may comprise a computer screen. Said outputting device may comprise a printer.

In an embodiment, said method, comprises the steps of:

a) generating a three-dimensional structure of atomic coordinates presented in Table 3, or a subset thereof, or atomic coordinates which deviate from those in Table 3, or a subset thereof, by RMSD over protein backbone atoms by no more than 3 Å;
b) fitting the structure of step a) with the structure of a candidate ligand by computational modeling;
c) selecting a ligand that possesses energetically favorable interactions with the structure of step a).

In an embodiment, said fitting comprises superimposing the structure of step a) with the structure of said candidate ligand. In another embodiment, said modeling comprises docking modeling. In a further embodiment, said ligand of step c) can bind to at least 1 amino acid residue, such as at least 2, 3, 4, 5, 6, 7, or 8 amino acid residues of the structure of step a) without steric interference.

It will be understood by the skilled person that generating the structures, as well as the modeling and fitting operations as described above may be performed with the algorithms, programs and platforms as disclosed in the present specification.

In an aspect, the invention also relates to a method for identifying modulators of Flt3 signaling. In particular, the invention relates to a method for identifying a ligand which modulates Flt3 signaling, comprising the steps of:

a) providing a candidate ligand;
b1) providing a polypeptide comprising or consisting of a region of at least 5 consecutive amino acid residues of amino acid residues 240-350, preferably 245-345 of Flt3; or
b2) providing a polypeptide comprising or consisting of a region of at least 5 consecutive amino acid residues of amino acid residues 5-20 of FL;
c) contacting said candidate ligand with said polypeptide of step b1) or step b2);
d) determining the binding of said candidate ligand with said region of step b1) or step b2); and
e) identifying said candidate ligand as a ligand which modulates Flt3 signaling if binding between said candidate ligand and said region of step b1) or step b2) is detected.

The invention also relates to a method for identifying a ligand of Flt3, comprising the steps of:

a) providing a candidate ligand;
b) providing a polypeptide comprising or consisting of a region of at least 5 consecutive amino acid residues of amino acid residues 240-350, preferably 245-345 of Flt3;
c) contacting said candidate ligand with said polypeptide of step b);
d) determining the binding of said candidate ligand with said region of step b); and
e) identifying said candidate ligand as a ligand which modulates Flt3 signaling if binding between said candidate ligand and said region of step b) detected.

The invention also relates to a method for identifying a ligand of Flt3 which binds to the FL binding site, in particular the region of Flt3 comprised within of consisting of amino acid residues 240-350, preferably 245-345, the method comprising the steps of:

a) providing a candidate ligand;
b) providing a polypeptide comprising or consisting of said FL binding site or said region,
c) contacting said candidate ligand with said polypeptide;
d) determining the binding of said candidate ligand with said region;
e) identifying said candidate ligand as a ligand of Flt3 if binding is detected.

The invention further relates to a method for identifying a ligand of FL, comprising the steps of:

a) providing a candidate ligand;
b) providing a polypeptide comprising or consisting of a region of at least 5 consecutive amino acid residues of amino acid residues 5-20 of FL;
c) contacting said candidate ligand with said polypeptide of step b);
d) determining the binding of said candidate ligand with said region of step b); and
e) identifying said candidate ligand as a ligand which modulates Flt3 signaling if binding between said candidate ligand and said region of step b) detected.

The invention also relates to a method for identifying a ligand of FL that binds to the Flt3 binding site, in particular the region of FL comprised within or consisting of amino acid residues 5-20, the method comprising the steps of:

a) providing a candidate ligand;
b) providing a polypeptide comprising or consisting of said Flt3 binding site or said region,
c) contacting said candidate ligand with said polypeptide;
d) determining the binding of said candidate ligand with said region;
e) identifying said candidate ligand as a ligand of FL if binding is detected.

According to an aspect of the invention, a candidate ligand is brought into contact with any one of the above indicated polypeptides or fragments of Flt3 or FL, after which binding between said candidate ligand and said polypeptides or fragments of Flt3 or FL is evaluated. In a particularly preferred embodiment, the binding between said candidate ligand and the respective region of Flt3 or FL which constitutes the Flt3/FL binding interface is determined. Methods for identifying interactions between compounds, such as interactions between proteins, are well known in the art, and will not be detailed further. By means of example, and without limitation, interactions can be evaluated by techniques such as pull-down, co-immunoprecipitation, yeast two-hybrid, bimolecular fluorescence complementation (BiFC), affinity electrophoresis, label transfer, phage display, ELISA, RIA, in-vivo crosslinking, tandem affinity purification (TAP), chemical crosslinking, dual polarisation interferometry (DPI), surface plasmon resonance (SPR), static light scattering (SLS), dynamic light scattering (DLS or QELS), fluorescence polarization/anisotropy, fluorescence correlation spectroscopy, fluorescence resonance energy transfer (FRET), EMSA, NMR, isothermal titration calorimetry (ITC). Particularly preferred techniques include competition or displacement assays, which are well known in the art. Briefly, a known ligand (such as (a fragment of) FL or Flt3) competes with the candidate ligand for binding. Either one or both of the known or candidate ligand can be labeled for ease of (differential) detection. Different types of labels are well known in the art, such as labels which allow fluorescent detection or affinity purification. Typically, a dilution series of candidate or known ligand is incubated with the binding partner and with fixed concentration of known or candidate ligand. Concentration-dependent changes in the detection of binding of the known or candidate ligand identifies candidate ligands as effective ligands. An alternative technique to validate candidate ligands comprises on the one hand incubating the candidate ligand with a wild type binding partner or fragment thereof (Flt3 or FL) and on the other hand incubating the candidate ligand with a mutated binding partner or fragment thereof (Flt3 or FL), wherein the mutated Flt3 or FL comprises at least one mutation in the respective binding domain of Flt3 or FL which constitutes the Flt3/FL binding interface. It will be understood by a person skilled in the art that preferred mutations constitute non-conservative mutations.

Accordingly, in an aspect, the invention relates to a method for identifying a ligand of Flt3, comprising the steps of

a) providing a candidate ligand;
b1) providing a first polypeptide comprising or consisting of a region of at least 5 consecutive amino acid residues of amino acid residues 240-350, preferably 245-345 of Flt3;
b2) providing a second polypeptide comprising or consisting of said region, wherein at least one amino acid residue of amino acid residues 240-350, preferably 245-345 is mutated;
c) contacting said candidate ligand with said polypeptide of step b1) or step b2);
d) determining the binding of said candidate ligand with said region of step b1) and step b2); and
e) identifying said candidate ligand as a ligand of Flt3 if binding between said candidate ligand and said region of step b1) is detected and if no binding between said candidate ligand and said region (or polypeptide) of step b2) is detected.

Particularly preferred amino acid residues to be mutated on Flt3 comprise one or more of amino acid residues at position 279, 281, 301, 302, 303, 307, 309, and 311. Accordingly, in an embodiment, the invention relates to a method as describes above, wherein said at least 5 consecutive amino acid residues comprise one or more of amino acid residues at position 279, 281, 301, 302, 303, 307, 309, and 311 of which one or more is mutated. In a further aspect, the invention relates to an Flt3 (isolated) polypeptide or a fragment thereof (such as D3, or a fragment corresponding to amino acid residues 245-345 of SEQ ID NO: 2), as well as the polynucleic acid sequences encoding these polypeptides, wherein at least one of the amino acid residues, or the corresponding nucleotide(s) in the polynucleic acid sequence encoding said polypeptide, comprised within the FL binding domain is mutated. In an embodiment, one or more amino acid residue, or the corresponding nucleotide(s), comprises within amino acid residues 240-350, preferably 245-345, more preferably 279-311 is mutated. In a preferred embodiment, one or more of amino acids 279, 280, 281, 301, 302, 303, 307, 309, or 311 is mutated.

In a further aspect, the invention relates to a method for identifying a ligand of FL, comprising the steps of

a) providing a candidate ligand;
b1) providing a first polypeptide comprising or consisting of a region of at least 5 consecutive amino acid residues of amino acid residues 5-20 of FL;
b2) providing a second polypeptide comprising or consisting of said region wherein at least one amino acid residue of amino acid residues 5-20 is mutated;
c) contacting said candidate ligand with said polypeptide of step b1) or step b2);
d) determining the binding of said candidate ligand with said region of step b1) and step b2); and
e) identifying said candidate ligand as a ligand of FL if binding between said candidate ligand and said region of step b1) is detected and if no binding between said candidate ligand and said region (or polypeptide) of step b2) is detected.

Particularly preferred amino acid residues to be mutated on FL comprise one or more of amino acid residues at position 8, 9, 10, 11, 12, 13, 14, and 15. Accordingly, in an embodiment, the invention relates to a method as describes above, wherein said at least 5 consecutive amino acid residues comprise one or more of amino acid residues at position 8, 9, 10, 11, 12, 13, 14, and 15 of which one or more is mutated.

Underlying the present invention is the surprising finding that the binding interface of Flt3 and its cognate ligand FL comprises a subset of extracellular domain 3 (D3) of Flt3 (comprised within amino acid residues 240-350, preferably 245-345 of Flt3) and an N-terminal part of FL (comprised within amino acid residues 5-20 of FL). Accordingly, in an aspect, the present invention relates to a method for the identification of ligands which modulate (or modulators) of Flt3 signaling, wherein said ligands or modulators are capable of binding to the respective binding site of Flt3 or FL which contribute to the Flt3/FL binding interface. In another aspect, the invention relates to a method for the identification of ligands of Flt3, wherein said ligands are capable of binding to the binding site of Flt3 which contributes to the Flt3/FL binding interface. In a further aspect, the invention relates to a method for the identification of ligands of FL, wherein said ligands are capable of binding to the binding site of FL which contributes to the Flt3/FL binding interface.

According to an aspect of the invention, the methods as described herein for identifying a ligand of Flt3 or a ligand which modulates Flt3 signaling comprise a step of providing a polypeptide or the atomic coordinates of Table 3, or a subset thereof, or atomic coordinates which deviate from those in Table 3, or a subset thereof, by RMSD over protein backbone atoms by no more than 3 Å comprising a region of at least 5 consecutive amino acid residues of amino acid residues 240-350, preferably 245-345, more preferably 279-311 of Flt3. In an embodiment, said polypeptide or atomic coordinates comprises a region of at least 5 consecutive amino acid residues of amino acid residues 240-350, preferably 245-345, more preferably 279-311 of a protein which is at least 80%, preferably at least 85%, 90%, or 95% identical or homologous to SEQ ID NO: 2. In another embodiment, said polypeptide or atomic coordinates comprises a region of at least 5 consecutive amino acid residues of amino acid residues 240-350, preferably 245-345, more preferably 279-311 of SEQ ID NO: 2. In an embodiment, said polypeptide or atomic coordinates comprises at least 5 consecutive amino acid residues of amino acid residues 250-350, 250-340, 260-350, 260-340, 270-340, 270-330, 270-320, 275-320, 275-315, or 279-311 of Flt3, of SEQ ID NO: 2, or of a protein which is at least 80%, preferably at least 85%, 90%, or 95% identical or homologous to SEQ ID NO: 2. In another embodiment, said polypeptide or atomic coordinates comprises 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or at least the recited number of consecutive amino acid residues of the region comprised within any of the amino acid residues 250-350, 260-350, 250-340, 260-340, 270-340, 270-330, 270-320, 275-320, 275-315, or 279-311 of Flt3, of SEQ ID NO: 2, or of a protein which is at least 80%, preferably at least 85%, 90%, or 95% identical or homologous to SEQ ID NO: 2. In a further embodiment, said polypeptide or atomic coordinates consists of extracellular domain 3 (D3) of Flt3. In another embodiment, said polypeptide or atomic coordinates consists of a fragment of D3 of Flt3, wherein said fragment of D3 comprises at least 5 consecutive amino acid residues of amino acid residues 240-350, preferably 245-345, more preferably 279-311 of Flt3, of SEQ ID NO: 2, or of a protein which is at least 80%, preferably at least 85%, 90%, or 95% identical or homologous to SEQ ID NO: 2. In an embodiment, said fragment of D3 comprises at least 5 consecutive amino acid residues of amino acid residues 260-350, 250-340, 260-340, 270-340, 270-330, 270-320, 275-320, 275-315, or 279-311 of Flt3, of SEQ ID NO: 2, or of a protein which is at least 80%, preferably at least 85%, 90%, or 95% identical or homologous to SEQ ID NO: 2. In another embodiment, said fragment of D3 comprises 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or at least the recited number of consecutive amino acid residues of the region comprised within amino acid residues 250-350, 250-340, 260-350, 260-340, 270-340, 270-330, 270-320, 275-320, 275-315, or 279-311 of Flt3, of SEQ ID NO: 2, or of a protein which is at least 80%, preferably at least 85%, 90%, or 95% identical or homologous to SEQ ID NO: 2. In yet another embodiment, said polypeptide or atomic coordinates consists of amino acid residues 245-345 of Flt3, more preferably 279-311 of SEQ ID NO: 2, or of a protein which is at least 80%, preferably at least 85%, 90%, or 95% identical or homologous to SEQ ID NO: 2.

In an aspect, the invention also specifically relates to the (isolated) Flt3 polypeptide sequences as well as the as the (isolated) polynucleic acid sequences encoding said polypeptide sequences as described herein. In a preferred embodiment, said Flt3 polypeptide sequence comprises at most 200 amino acid residues, preferably at most 175, 150, 125, or 100 amino acid residues. A particularly preferred Flt3 polypeptide according to an embodiment of the invention comprises D3 as the sole Flt3-derived polypeptide fragment or is at least 80%, preferably at least 85%, 90%, or 95% identical or homologous to amino acid residues 245-345 of SEQ ID NO: 2.

In an aspect, the invention relates to the use of the polypeptides and/or fragments thereof or atomic coordinates as described herein for designing and/or identifying a ligand which modulates Flt3 signaling.

In a further aspect, the invention also relates to the use of the polypeptides and/or fragments thereof or atomic coordinates as described herein for designing and/or identifying a ligand of Flt3, in particular, the FL-binding region of Flt3. As these polypeptide fragments or atomic coordinates of Flt3 comprise the FL binding site, these fragments may be used to inhibit Flt3 signaling. Accordingly, in an aspect, the invention relates to the use of said fragment as an antagonist of Flt3 signaling, as well as a method for antagonizing Flt3 signaling by using said fragments.

According to another aspect of the invention, the methods as described herein for identifying a ligand of FL or a ligand which modulates Flt3 signaling comprises a step of providing a polypeptide or atomic coordinates comprising a region of at least 5 consecutive amino acid residues of amino acid residues 5-20 of FL. In an embodiment, said polypeptide comprises a region of at least 5 consecutive amino acid residues of amino acid residues 5-20 of a protein which is at least 80%, preferably at least 85%, 90%, or 95% identical or homologous to SEQ ID NO: 5. In another embodiment, said polypeptide or atomic coordinates comprises a region of at least 5 consecutive amino acid residues of amino acid residues 5-20 of SEQ ID NO: 5. In an embodiment, said polypeptide or atomic coordinates comprises at least 5 consecutive amino acid residues of amino acid residues 5-19, 5-18, 5-17, 5-16, 5-15, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15 of FL, of SEQ ID NO: 5, preferably 8-15 or 5-15 of FL or SEQ ID NO: 5, or of a protein which is at least 80%, preferably at least 85%, 90%, or 95% identical or homologous to SEQ ID NO: 5. In another embodiment, said polypeptide or atomic coordinates consists of a fragment of at most 50 consecutive amino acid residues of FL, wherein said fragment comprises at least 5 consecutive amino acid residues of amino acid residues 5-19, 5-18, 5-17, 5-16, 5-15, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15 of FL, of SEQ ID NO: 5, preferably 8-15 or 5-15 of FL or SEQ ID NO: 5, or of a protein which is at least 80%, preferably at least 85%, 90%, or 95% identical or homologous to SEQ ID NO: 5. In another embodiment, said polypeptide or atomic coordinates comprises 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 or at least the recited number of consecutive amino acid residues of the region comprised within any of the amino acid residues 5-19, 5-18, 5-17, 5-16, 5-15, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15 of FL, of SEQ ID NO: 5, preferably 8-15 or 5-15 of FL or SEQ ID NO: 5, or of a protein which is at least 80%, preferably at least 85%, 90%, or 95% identical or homologous to SEQ ID NO: 5. In a further embodiment, said polypeptide or atomic coordinates consists of amino acid residues 8-15 of FL, of SEQ ID NO: 5, or of a protein which is at least 80%, preferably at least 85%, 90%, or 95% identical or homologous to SEQ ID NO: 5.

In an aspect, the invention also specifically relates to the FL (isolated) polypeptide sequences as well as the as the (isolated) polynucleic acid sequences encoding said polypeptide sequences as described herein. In a preferred embodiment, said FL polypeptide sequence comprises at most 50 amino acid residues, preferably at most 40, 30, 20, or 10 amino acid residues. A particularly preferred FL polypeptide according to an embodiment of the is at least 80%, preferably at least 85%, 90%, or 95% identical or homologous to amino acid residues 5-20, more preferably 8-18 of SEQ ID NO: 5.

In an aspect, the invention also relates to the use of the polypeptides and/or fragments thereof or atomic coordinates as described herein for designing and/or identifying a ligand of FL, in particular, the Flt3-binding region of FL. As these polypeptide fragments of FL comprise the Flt3 binding site, these fragments may be used to inhibit Flt3 signaling. Accordingly, in an aspect, the invention relates to the use of said fragment as an antagonist of Flt3 signaling, as well as a method for antagonizing Flt3 signaling by using said fragments.

It will be appreciated by a skilled person that the Flt3 and/or FL polypeptides and polynucleotides as described herein can be fused to heterologous polypeptide or polynucleotide sequences. As used herein, the term “heterologous polypeptide” and “heterologous polynucleotide” relate to polypeptides or polynucleotides which are not derived from or originate from Flt3 or FL. Examples of such heterologous sequences include for instance tags, such as tags for detection and/or isolation and/or immobilization and/or reporter tags, etc.

The invention also relates to polypeptide and polynucleic acid sequences comprising or encoding the herein described respective region of Flt3 or FL which constitutes the Flt3/FL binding interface, as well as the full length Flt3 or FL or fragments thereof wherein one or more of the amino acid residues of the respective region of Flt3 or FL which constitutes the Flt3/FL binding interface, or the corresponding nucleotide(s) in the polynucleic acid encoding the polypeptides, are mutated.

A person skilled in the art will appreciate that the polynucleic acids disclosed herein can be cloned in a vector, with techniques which are well known in the art (such as PCR amplification or restriction digests). Accordingly, in an aspect, the invention relates to vector comprising a polynucleic acid as described herein. In an embodiment, said vector is an expression vector, such as a eukaryotic or prokaryotic expression vector. Vectors in general and eukaryotic or prokaryotic expression vectors in particular are well known in the art and hence will not be detailed further. In an aspect, the invention also relates to a host cell comprising a polynucleic acid or a vector as described herein. Suitable host cells include prokaryotic and eukaryotic host cells, such as bacteria, yeast, insect cells and mammalian cells. Methods for transiently or stably introducing polynucleic acids in these host cells (such as transformation, infection, electroporation, transfection), as well as methods for expressing polypeptides encoded by these polynucleic acids (inducible or constitutive) are well known in the art. The invention in an aspect also relates to the use of these host cells for the expression of the polypeptides as disclosed herein, as well as methods for expressing the polypeptides as disclosed herein by use of these host cells.

The invention also relates to ligands of Flt3 or FL, preferably ligands which bind to the respective domains of Flt3 or FL constituting the Flt3/FL binding interface. As Flt3 and FL are known binding partners and hence per se ligands of each other, the full length FL and Flt3 polypeptides are hereby explicitly disclaimed as ligands.

In an aspect, the invention relates to a ligand which is identified by the methods as described herein. In an embodiment, said ligand is an Alphabody™, a Nanobody®, an antibody, or a small molecule, preferably an Alphabody™.

In another aspect, the invention relates to a ligand which binds to the FL binding site of Flt3. In an embodiment, said ligand is an Alphabody™, a Nanobody®, an antibody, or a small molecule, preferably an Alphabody™.

In another aspect, the invention relates to a ligand which binds to the Flt3 binding site of FL. In an embodiment, said ligand is an Alphabody™, a Nanobody®, an antibody, or a small molecule, preferably an Alphabody™.

In a further aspect, the invention relates to the ligands as described herein for use in modulating Flt3 signaling or for use as a medicament. In a further aspect, the invention relates to the use of the ligands as described herein for the manufacture of a medicament. In a further aspect, the invention relates to a method for treating diseases or disorders characterized by abnormal Flt3 signaling with a ligand as described herein. Diseases or disorders characterized by abnormal Flt3 signaling can be caused by a lack of or insufficient Flt3 signaling or alternatively can be caused by inappropriate or increased Flt3 signaling.

In an embodiment, the ligand as described herein may be coupled to a therapeutic compound or drug, and hence may function as a drug-delivery vehicle. Accordingly, in an aspect, the invention relates to a ligand as described herein for use in drug delivery, wherein said ligand is coupled to said drug.

In an embodiment, the ligand according to the present invention is a ligand which modulates or interferes with Flt3 dimerization. The ligand according to this embodiment is a monovalent ligand which completely or partially prevents ligand-mediated dimerization of Flt3 receptors. In an embodiment, the ligand according to the present invention is a ligand which modulates or interferes with Flt3/FL binding. The ligand according to this embodiment completely or partially prevents binding of the cognate ligand FL to Flt3. In another embodiment, the ligand according to the present invention is a ligand which modulates Flt3 (kinase) activation. The ligand according to this embodiment completely or partially alters Flt3 phosphorylation. In a further embodiment, the ligand according to the present invention is a therapeutical agent. The ligand according to this embodiment modulates Flt3 signaling such that a biological effect results in a therapeutic application. In another embodiment, the ligand according to the present invention is an agonist or an antagonist of Flt3 signaling. An agonist according to this embodiment completely or partially activates or enhances Flt3 signaling. An antagonist according to this embodiment completely or partially inhibits or reduces Flt3 signaling. It is to be understood that the ligand according to the invention exerts its function either on Flt3 (if it is a ligand of Flt3) or on FL (if it is a ligand of FL).

In an embodiment, the invention relates to a ligand as described herein for use in modulating Flt3 signaling. Preferred indications which benefit from Flt3 modulation include cancer, precancerous state, autoimmune diseases (such as rheumatoid arthritis), transplantation or grafting, inflammation, immunomodulation, musculo-skeletal disorders (in particular bone disorders such as characterized by abnormal bone resorption), angiogenesis, ophthalmological disorders (such as diabetic macular edema and macular degeneration), apoptosis, cell cycle regulation, dermatological abnormalities (such as dermal fibrosis, mastocytis and psoriasis), CNS disorders (such as multiple sclerosis). In a preferred embodiment, the invention relates to a ligand as described herein for use in treating cancer. In another preferred embodiment, the invention relates to a ligand as described herein for use in treating autoimmune diseases, preferably rheumatoid arthritis, psoriasis or multiple sclerosis. In yet another preferred embodiment, the invention relates to a ligand as described herein for use in cell or organ transplantation. Said ligand is preferably administered prior to, during and/or after transplantation.

In another embodiment, the invention relates to a ligand as described herein for use in any of:

a) treating cancer, said cancer not being characterized by increased Flt3 signaling, if said ligand is an Flt3 agonist;
b) treating cancer, said cancer being characterized by increased Flt3 signaling, if said ligand is an Flt3 antagonist;
c) treating autoimmune diseases if said ligand is an Flt3 antagonist;
d) stimulating an immune response if said ligand is an Flt3 agonist;
e) suppressing an immune response if said ligand is an Flt3 antagonist, such as in the case of transplantation;
f) expansion of dendritic cells if said ligand is an Flt3 agonist;
g) activating Flt3 signaling if said ligand is an agonist;
h) suppressing Flt3 signaling if said ligand is an antagonist.

Cancer treatment which benefits from Flt3 antagonists relates to cancers characterized by an inappropriately increased Flt3 signaling, such as acute myeloid leukemia (AML), bile duct cancer, bladder cancer, brain tumors (in particular (anaplastic) astrocytoma or glioblastoma), breast cancer, uterine cancer, leukemia (in particular (chronic) lymphocytic or myelogenous leukemia, colon cancer, colorectal cancer, stomach cancer, head and neck cancer (in particular squamous cell carcinoma), hematological malignancies (in particular (systemic) mastocytosis or myoproliferative diseases), kidney cancer (in particular urothelial or renal cell carcinoma), liver cancer (in particular hepatocellular carcinoma), lymphoma, melanoma, mesothelioma, multiple myeloma, neoplasia, neuroendocrine tumors (in particular advanced pancreatic neuroendocrine tumors), lung cancer (in particular non-small cell lung cancer), ovarial cancer, pancreatic cancer, prostate cancer, sarcoma or thyroid cancer. In a preferred embodiment, the invention relates to a ligand which is an antagonist designed and/or identified as described herein, for use in treating acute myeloid leukemia. On the other hand, cancer treatment which benefits from Flt3 agonists relates to cancers which are not characterized by an inappropriately increased Flt3 signaling. Particularly beneficial applications of Flt3 agonists relate to immunotherapy in such cancers. In particular, Flt3 signaling is involved in DC homeostasis and DC-mediated activation of NK cells. Hence, activation of Flt3 signaling by Flt3 agonists in DC cells leads to DC proliferation and expansion in aiding immunotherapy. It will be appreciated by the skilled person that, FL as the cognate ligand of Flt3, dimerizes and as a consequence thereof brings Flt3 individual receptors in close proximity upon interaction of an FL dimer with two Flt3 receptors. Such association of Flt3 receptors will lead to intermolecular Flt3 phosphorylation and downstream signaling. Accordingly, a ligand which functions as an agonist preferably is bivalent with respect to Flt3 binding. Alternatively, two monovalent ligands may be coupled to each other to mimic a bivalent ligand. Such ligands may be coupled covalently, for instance by linker or hinge regions, or non-covalently, for instance by self-association or dimerization.

The invention also relates to medicaments or compositions comprising or consisting of the ligands as described herein. In a preferred embodiment, the invention relates to such medicaments or compositions, wherein said ligand is identified according to the methods as described herein. In an embodiment, said compositions are pharmaceutical compositions comprising a ligand as described herein and one or more pharmaceutically acceptable excipients, such as without limitation buffers (such as for instance isotonic saline solutions or PBS), salts, stabilizers, solubilizers, coating agents, emulgators, etc. Pharmaceutical compositions or medicaments containing a compound of the present invention may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pa. The compositions may appear in conventional forms, for example capsules, tablets, aerosols, solutions, suspensions or topical applications. Routes of administration include topical, parenteral, intramuscular, oral, intravenous, intra-peritoneal, intranasal inhalation, lung inhalation, intradermal or intra-articular. Due to the high stability of Nanobodies® and Alphabodies™, oral administration of medicaments comprising Nanobodies® and Alphabodies™ as described herein is preferred.

The invention further relates to such compositions for use as a medicament. The invention further relates to the use of such compositions for the manufacture of a medicament. The invention further relates to a method of treatment by using such compositions.

The invention also relates to a method for modulating Flt3 signaling, comprising the steps of:

a) providing (a composition comprising) an Flt3 polyprotein; and
b) contacting said (composition comprising an) Flt3 polyprotein with a ligand as described herein.

In an embodiment, said method is an in vitro method, wherein said Flt3 polypeptide is provided in an isolated form from an individual, such as an isolated cancer cell, DC, etc.

The invention also relates to a method for determining a mutation in the ligand-binding region of Flt3, comprising the step of determining one or more mutation in the region corresponding to amino acid residues 240-350, preferably 245-345 of Flt3 and/or the polynucleic acid encoding said region. Preferably, said region comprises or consists of amino acid residues 240-350, in particular D3, more in particular amino acid residues 245-345, even more in particular amino acid residues 279-311 of Flt3. Particularly preferred Flt3 mutations comprise mutations of amino acid residues at positions 279, 280, 281, 301, 302, 303, 307, 309, or 311.

In another aspect, the invention relates to a method for diagnosing a disorder which is characterized by aberrant Flt3 signaling, the method comprising the step of determining one or more mutation in the region corresponding to amino acid residues 240-350, preferably 245-345 of Flt3 and/or the polynucleic acid encoding said region. Preferably said method is an in vitro method. Accordingly, said mutation(s) is (are) detected in a sample isolated from an individual.

In a further aspect, the invention relates to the use of a ligand as designed or identified according to any one of the methods defined herein, as a modulator of Flt3 signaling.

The invention will now be illustrated by means of the following examples, which do not limit the scope of the invention in any way.

EXAMPLES
Example 1
Preparation of Recombinant Human Flt3 and Flt3-FL Complexes

cDNA encoding human Flt3 ectodomain variants, Flt3D1(27-161), Flt3D12 (27-244), Flt3D13(27-346), Flt3D14(27-434) and Flt3D15(27-541) were cloned in the mammalian expression vectors, pHLsec (Aricescu) and pcDNA4/TO (Invitrogen), which contained a p-phosphatase secretion signal and a C-terminal hexahistine tag.

Transient protein expression in HEK293T was carried out as previously described (Aricescu, 2006). Briefly, confluent cells, grown in tissue culture flasks or roller bottles (Greiner Bio-One) were transfected with purified plasmid DNA (Plasmid Mega Kit, Qiagen) mixed with 25 kDa branched polyethylenimine (Aldrich) and allowed to secrete the recombinant protein for 5-7 days in serum free medium, in the presence of kifunensine.

The pcDNA4/TO constructs were used to establish stable secreting cell lines in HEK293S GnTI−/− cells, as follows. 70% confluent cells were transfected with the plasmid-DNA according the calcium phosphate precipitation method. Stably transfected clones were selected using Zeocine (Invitrogen) at a concentration of 200 μg/mL, and allowed to grow for 3 weeks. Individual colonies were picked up with trypsin-soaked pieces of filter paper, expanded and subsequently tested for their protein expression. The presence of the recombinant protein in the medium was detected by Western blot analysis using a anti-His(C-term)-antibody coupled to horseradish peroxidase (Invitrogen). For large scale expression experiments, the medium of 50 confluent 175 cm2 tissue culture flasks was replaced with serum-free DMEM-F12 medium containing 5 mM sodium butyrate (Sigma) and 2 μg/mL tetracycline to induce protein expression.

The receptor variants were purified using IMAC: conditioned medium (1-3 liter) was applied to a Talon column (Clontech) with a bed volume of 20 mL, washed and eluted using imidazole. The proteins were further purified by gel-filtration chromatography on a Superdex 200 column (GE Healthcare). Ligand-receptor complexes were formed by adding excess molar amounts of recombinant FL (Verstraete, 2009) to purified receptor ectodomains, followed by purification by gel filtration chromatography.

Example 2
Mapping of Disulfide Bridges and Glycosylation Sites by Mass Spectrometry

Gel slices containing recombinant Flt3D1-D5 obtained from Coomassie-stained polyacrylamide gels were digested with trypsin (Promega) as previously described (Vanrobaeys, 2003). After digestion overnight at 37° C., the digestion mixture were dried and redissolved in 20 ml 0.1% formic acid. One microliter of the digestion mixture was mixed with an equal volume of 3 mg/ml a-cyano hydroxycinnamic acid (Sigma) in 50% acetronitrile/0.1% TFA and was subsequently subjected to mass spectrometric analyses on a 4800 plus TOF/TOF analyzer (Applied Biosystems).

About 75 pmoles of purified recombinant Flt3D1-D5 were dissolved in 20 mM Tris-HCl, pH 8.0, and digested with trypsin (Promega), Glu-C and Asp-N endoproteinases (Sigma) at E/S= 1/35. After incubation at 37° C. overnight, 1 ml of the digestion mixture were mixed with 5 ml of 3 mg/ml α-cyano hydroxycinnamic acid (Sigma) in 50% acetronitrile/0.1% TFA prior to mass spectrometric analyses as described earlier. The remaining volume of the digestion mixture was applied on a Spheri-5 PTC-C18 column (220×2.1 mm, Higgins Analytical) at a flow rate of 100 ml/min. Reversed phase chromatography of peptide mixture was performed on an Ettan LC (Amersham Biosciences) with on-line 96-well plate Frac-950 fractionator set at 20 ml/min. One microliter of the collected fractions was mixed with an equal volume of 3 mg/ml a-cyano hydroxycinnamic acid as described earlier. The results are depicted in Table 1.

TABLE 1

Mapping of disulfide bridges and N-linked glycosylation

sites in the Flt3 ectodomain by mass-spectrometry.

measured
calculated
sequence

mass
mass
positions
remarks

Asp-N peptides

2013.3
2013.09
248-265*
No glycosylation at #250

2081.56
2081.28
96-118
Glycosylation at #100, disulfide

bridge between #103 and #114

3486.61
3486.78
29-43
Glycosylation at #43, disulfide bridge

between #35 and #65

62-76*

4156.83
4156.11
29-43
Glycosylation at #43, disulfide bridge

between #35 and #65

62-83

Glu-C peptides

1499.56
1499.61
196-204
Disulfide bridge between #199

and #206

205-208

1628.6
1628.65
196-204
Disulfide bridge between #199

and #206

205-209*

1573.53
1573.72
347-357
Glycosylation at #351 and #354

1777.52
1777.78
347-360*
Glycosylation at #351 or #354

1980.58
1980.86
347-360*
Glycosylation at #351 and #354

4018.67
4018.32**
25-44*
Glycosylation at #43, disulfide bridge

between #35 and #65

63-77*

(4134.96)
4133.35**
25-44*
Glycosylation at #43, disulfide bridge

between #35 and #65

62-77

(4263.21)
4262.39**
24-44*
Glycosylation at #43, disulfide bridge

between #35 and #65

62-77

5559.91
5560.18**
25-58
Glycosylation at #43, disulfide bridge

between #35 and #65

62-77

Tryptic peptides

1735.62
1735.76
381-387
Disulfide bridge between #381

and #392

389-395

1775.74
1775.71
323-334
Glycosylation at #323, disulfide

bridge between #330 and #272

272-273

2187.03
1983.98
491-508
Glycosylation at #502

2214.78
2214.95
317-307
Glycosylation at #306***

2485.03
2281.98
467-485
Glycosylation at #473

3542.46
3542.78
133-161
Glycosylation at #151

3811.3
3811.62
348-372
Glycosylation at #351 or #354,

disulfide bridge between #368

and #407

406-410

4014.43
4014.7
348-372
Glycosylation at #351 and #354,

disulfide bridge between #368

and #407

406-410

(5332.29)
5333.06**
176-215*
Disulfide bridges between #184-#231

and #232-#241 (from X-ray data)

231-234

240-243

6614.06
6614.55**
176-215*
Disulfide bridges between #184-#231

and #232-#241 (from X-ray data)

220-234*

240-243

8998.44
8998.13**
35-41
Glycosylation at 100***, disulfide

bridges between #35-#65 and

#103-#114

50-108

109-123

*Peptide containing in-complete or on-specific cleavage

**Averaged values

***contains also very small amount of fucose (+146 Da)

Example 3
Crystallography of Flt3:FL Complexes

Purified recombinant Flt3D1-D4: FL (5 mg/mL in 10 mM Hepes pH 7.4, 100 mM NaCl) and Flt3D1-D5: FL (8 mg/mL in 10 mM Hepes pH 7.4, 100 mM NaCl) complexes were used to carry out an extensive crystallization screen based on 1 μL crystallization droplets (0.5 μL protein sample and 0.5 μL crystallization condition) equilibrated in sitting- and hanging-drop geometry over 250 μL reservoirs containing a given crystallization condition. This led to the identification of multiple lead conditions that typically combined 0.1-0.2 M monovalent or divalent salts, pH 6-7.5, and 10-20% PEG of various molecular weights.

Diffraction quality crystals of Flt3_D1-D4:FL and Flt3_D1-D5:FL could be grown over the course of several days as rectangular rods measuring 0.1×0.1×0.3 mm from both lead conditions using the vapor-diffusion method based on the ‘sitting drop’ geometry as follows: for each complex, crystallization droplets consisting of 1 μL protein sample (Flt3_D1-D4:FL at 5 mg/mL in 10 mM Hepes pH 7.4, 150 mM NaCl; Flt3_D1-D5:FL at 5 mg/mL in 10 mM Hepes pH 7.4, 150 mM NaCl) were mixed with 1 μL reservoir solution (Flt3_D1-D4:FL: 100 mM MgCl₂, 50 mM MES pH 6.5, 11-13% w/v PEG 4000; Flt3_D1-D5:FL: 200 mM lithium citrate, 100 mM Tris pH 7.0, 12-14% w/v PEG 3350) and were equilibrated against 0.5 mL reservoir solution. For data collection under cryogenic conditions (100 K), single crystals were flash cooled—with the help of a nylon loop—in liquid nitrogen after brief serial incubations (typically 1-2 minutes per step) in mother liquor containing a gradually higher percentage of cryoprotectant (PEG 400 for Flt3_D1-D4:FL and glycerol for Flt3_D1-D5:FL). The optimal concentration of the cryoprotectant was 20% v/v for both crystal types.

Diffraction experiments were conducted on the X06SA (PXI) and X06DA (PXIII) beamlines at the Swiss Light Source (Paul Scherrer Institute, Villigen, Switzerland) and the 1D23-1 beamline at the ESRF (Grenoble, France). All data were integrated and scaled using the XDS suite (Kabsch, 2010).

The structure of Flt3D1-D4: FL was determined by maximum-likelihood molecular replacement as implemented in the program suite PHASER (McCoy et al., 2007), using the structure of human FL as search model (PDB entry 1ETE, Savvides 2000). Following density modification employing solvent flattening and 4-fold ncs-averaging via the program PARROT (Cowtan, 2010), the electron density maps revealed contiguous density for domains 2 and 3 of the Flt3 ectodomain. Model (re)building was carried out manually in electron density maps after density modification, using the program COOT (Emsley, 2010), and in the later stages via a combination of automated methods as implemented in the program BUCCANEER (Cowtan 2006). Chain tracing was facilitated by mapping of the disulfide bridges and glycosylation sites in Flt3 by mass-spectrometry. Crystallographic refinement and structure validation was carried out using PHENIX (Adams, 2010) and Buster-TNT (Blanc, 2004). The structure of Flt3D1-D5: FL was determined by maximum-likelihood molecular replacement as implemented in the program suite PHASER (McCoy et al., 2007), using the structure of the Flt3D2-D3: FL subcomplex as determined in the Flt3D1-D4: FL complex. The remaining domains were placed via additional rounds of molecular replacement and manual placement in electron density maps after density modification. Due to the low resolution of the analysis we only applied rigid-body refinement to optimize model placement.

Example 4
Small-Angle X-Ray Scattering

Data were collected at beamline X33 at DESY, Hamburg. The measurements were carried out at 298 K, within a momentum transfer range of 0.01 Å-1<s<0.45 Å-1 where s=4π sin(θ)/λ and 2θ is the scattering angle. All samples were measured at several solute concentrations ranging from 0.5 to 6 mg/ml in 50 mM NaPO4 pH 7.40, 100 mM NaCl, with intermittent buffer solution (50 mM NaPO4, pH 7.40, 100 mM NaCl). To monitor radiation damage, consecutive 30 sec. exposures at the highest protein concentration were compared. The data were processed using standard procedures, corrected for buffer contribution and extrapolated to infinite dilution using the program PRIMUS20. The radius of gyration Rg and forward scattering I(O), the maximum particle dimension Dmax and the distance distribution function p(r) were evaluated using the program GNOM21. The molecular masses of the different constructs were calculated by comparison with the reference bovine serum albumin (BSA) samples. The scattering patterns from the high-resolution models were calculated using the program CRYSOL22. Constrained rigid-body refinement runs were carried out in SASREF723. Rigid-body refinement of the unliganded receptors was carried out under P1 symmetry; refinement convergence was optimal with specified ambiguous distance contacts at the D3-D3* and D4-D4* interfaces. Rigid-body refinement of the hCSF-1L:hCSF-1RD1-D3 complex was carried out with twofold symmetry imposed.

Example 5
Electron Microscopy

For preparation of negatively stained Flt3D1-D5/FL complex, purified complex at ˜0.05 mg/mL in PBS buffer was applied to the clear side of carbon on a carbon-mica interface and stained with 2% (w/v) uranyl acetate. Images were recorded under low-dose conditions with a JEOL 1200 EX II microscope at 100 kV and at nominal 40000× magnification. Selected negatives were then digitized on a Zeiss scanner (Photoscan TD) at a step size of 14 micrometer giving a pixel size of 3.5 Å at the specimen level. Using the boxer routine of the EMAN image processing software (Ludtke, 1999), 25134 subframes of 96×96 pixels containing individual Flt3D1-D5/FL complex particles were selected interactively, CTF-corrected with CTFFIND3 (Mindell and Grigorieff, 2003) and bsoft (Heymann et al., 2008), and low-path-filtered at 15 Å with Imagic-5. Subsequent data processing was performed with Imagic-5 software package (van Heel et al., 1996) The translationally centered data set was subjected to multivariate statistical analysis and classification that provided a set of references for multireference alignment. Class averages obtained after several cycles of multireference alignment, multivariate statistical analysis and classification were compared to projections of the SAXS model of the Flt3D1-D5/FL complex (FIG. 10).

Example 6
Isothermal Titration Calorimetry

Experiments were carried out using a VP-ITC MicroCalorimeter (MicroCal, MA) at 37° C., and data were analyzed using the Origin ITC analysis software package supplied by MicroCal. Purified recombinant Flt3 ectodomain constructs and FL were dialyzed overnight against 10 mM Tris-HCl, pH 7.4, at 4° C. Protein concentrations were measured spectrophotometrically at 280 nm using calculated theoretical extinction coefficients and all solutions were extensively degassed prior to use. The sample was stirred at a speed of 400 rpm throughout. The thermal titration data were fit to the “one binding site model”, and apparent molar reaction enthalpy (ΔH°), apparent entropy (ΔS°), association constant (Ka) and stoichiometry of binding (N) were determined. Several titrations were performed to evaluate reproducibility.

Example 7
Isolation of Recombinant Flt3 Ectodomain Complexes and Thermodynamic Binding Profile of Complex Formation

A series of constructed recombinant Flt3 ectodomains was constructed (Flt3D1-D5, Flt3D1-D4, Flt3D1-D3, Flt3D1-D2, and Flt3D1) based on intron/exon boundaries and sequence alignments with homologous receptors. The constructs were produced via transient protein expression in human embryonic kidney 293T cells. Faced with prohibitively poor protein yields (100-200 μg per liter of media) tetracycline-inducible cell lines were established in HEK293S cells deficient in N-acetylglucosaminyltransferase I (HEK293S GnTI−/−) (Reeves 2006) that could secrete the target ectodomain variants with limited and homogeneous glycosylation to mg amounts. The yields and expression levels for both Flt3D1-D3 and Flt3D1-D2 were much lower than for all other constructs, and the two constructs suffered from significant solubility and stability problems, especially Flt3D1-D2.

High-affinity stoichiometric complexes of purified glycosylated Flt3D1-D5, Flt3D1-D4, and Flt3D1-D3 with recombinant human FL produced in E. coli (Verstraete 2009) consistent with bivalent binding of FL to each of the ectodomain constructs were initially established by analytical size-exclusion chromatography, and subsequent batches for structural studies were obtained via preparative size-exclusion chromatography in the presence of excess molar amounts of purified FL (FIG. 1A-C). The observed elution profiles for all three ectodomain complexes were indicative of ligand-induced receptor dimerization. In contrast to our preparations of Flt3D1-D5 and Flt3D1-D4, preparations of recombinant Flt3D1-D3 consistently contained a significant portion of receptor that was incapable of binding the ligand (FIG. 10). On the other hand, we were not able to observe complex formation for Flt3D1, and Flt3D1-D2, providing the first direct evidence that these ectodomain constructs do not carry a high-affinity ligand binding site.

To quantify the thermodynamics, stoichiometry and affinity of extracellular complexes and to dissect the contribution of individual ectodomain modules to complex formation, isothermal titration calorimetry (ITC) was employed. This led to a number of consensus observations (FIG. 1D-F). A first consensus is that all three characterized complexes exhibit high-affinity binding characterized by a strongly exothermic enthalpic term coupled to an entropic penalty (FIG. 1D-F). Secondly, FL exhibits bivalent binding to both Flt3D1-D5 and Flt3D1-D4 (N=0.5, 2 molecules of Flt3 to 1 FL). Furthermore, the sequential exclusion of the membrane-proximal domains Flt3D4 and Flt3D5 leads to a very modest decrease in affinity (Kd [Flt3D1-D5:FL]=8.7 nM; Kd [Flt3D1-D4:FL]=40 nM; Kd [Flt3D1-D3:FL]=70 nM) (FIG. 1D-F). Taken together, the data indicate that the membrane-proximal module Flt3D4-D5 does not contribute to the overall stability of the complex. It is noted that the inherent instability of recombinant Flt3D1-D3 (FIG. 10) is the likely reason for the observed deviation in stoichiometry of binding for the Flt3D1-D3:FL complex in the ITC measurements. Nonetheless, the data clearly show that Flt3D1-D3 is capable of engaging in a high-affinity interaction just like the larger ectodomain constructs. Upon adjusting the Flt3 concentration to the approximate active receptor fraction (˜50%) estimated via the chromatographic elution profile of complex formation, a stoichiometry and thermodynamic signature consistent with that of the other two complexes is detected (FIG. 1F).

Example 8
Crystal Structure of the Flt3D1-D4:FL Complex

Highly pure and monodisperse preparations of Flt3D1-D4:FL complex yielded crystals of appreciable size (typically 0.2×0.1×0.25 mm), which diffracted synchrotron X-rays anisotropically to 4.5-5.5 Å resolution. The crystals exhibited great variation in diffraction quality even within the same crystal. Trimming of the N-glycosylation via treatment with endoglycosidase H, in an effort to the improve crystal quality resulted in receptor-ligand preparations with drastically reduced solubility and stability, which proved inadequate for crystal optimization. Optimization of the crystal cryoprotection protocol and a large scale screening of Flt3D1-D4:FL complex crystals resulted in a dataset to 4.3 Å resolution (Table 2).

TABLE 2

X-ray data collection and refinement statistics

(the values in parentheses refer to the highest resolution shell)

Flt3_D1-D4:FL
Flt3_D1-D5:FL

Data collection

Source
ESRF ID23-1
ESRF ID23-1

Wavelength
0.9714
1.0762

(Å)

Detector
ADSC-Q315R
ADSC-Q315R

Resolution (Å)
40.00-4.3 (4.45-4.30)
40.00-7.8 (8-7.8)

Space group
P2₁
P2₁

Unit cell
a = 103.89
a = 124.74

parameters
b = 146.26
b = 153.51

c = 105.95
c = 133.85

α = γ = 90°, β = 109.7°
α = γ = 90°, β = 94.6°

Wilson B (Å²)
135
436

Unique
20184 (1942)
5575 (374)

reflections

Redundancy
3.8 (3.8)
3.1 (3.0)

Completeness
98.8 (98.9)
95.3 (90.8)

(%)

R_meas(%)^a
10.8 (75.9)
12.5 (75.9)

Average //σ(/)
12.04 (2.08)
8.8 (1.8)

Refinement

Resolution (Å)
40.00-4.3 (4.53-4.30)
35-7.8 (8.7-7.8)

Reflections
19172/1010 (2622/141)
5090/565 (1437/159)

(working set/

test set)

R_work, R_free
0.258/0.286 (0.270/0.268)
0.367/0.350 (0.336/0.334)

R.m.s.

deviations

Bonds (Å)
0.010
n.a.

Angles (°)
1.16
n.a.

Average
174
370

ADP (Å²)

Ramachandran

analysis (%)

Favorable
97.7
n.a.

Outliers
2.3
n.a.

Protein Data

Bank access

code

^aR_meas= Σ_h√n_h/(n_h− 1) Σ_hΣ_i|/(h, i) − </(h)>|/Σ_hΣ_i/(h, i), where n_his the multiplicity, /(h, i) is the intensity of the i^thmeasurement of reflection h, and </(h)> is the average value over multiple measurements.

In the first phase of the structure determination process, molecular replacement solutions for FL and Flt3D3 were found using the crystal structure of FL (Savvides et al., 2000) and a homology model for Flt3D3 based on the third extracellular domain of KIT (Yuzawa et al., 2007). This showed the presence of two Flt3D1-D4:FL complexes in the crystal asymmetric unit (FIG. 7). Electron density modification exploiting the presence of improper 4-fold non-crystallographic symmetry and the high solvent content of the crystals, allowed us to select the correct MR solution for a homology model of Flt3D4 based on domain 4 of KIT, and to manually place a model for Flt3D2 based on domain 5 of KIT. In the later stages of model building and refinement, the core structure of Flt3D1 was modeled for one of the two receptor complexes. To facilitate chain tracing we determined the atypical disulfide-bond network of Flt3 as well as the actual number of N-linked glycosylation sites in extracellular Flt3 by mass-spectrometry. It was confirmed that all nine N-linked glycosylation sites are at least partially occupied and that all cysteines present in Flt3D1-D4 are engaged in disulfide-bond formation (FIG. 2A).

Example 9
Overall Structure of the Flt3 D1-D4:FL Complex

The structure of the Flt3D1-D4:FL complex was found to be unlike any of the structurally characterized RTKIII/V complexes to date and was found to be characterized by a number of surprising features (FIG. 2B). The FL-Flt3D1-D4 extracellular complex can be described as a moderately open horseshoe ring structure measuring 100 Å×75 Å×110 Å, comprising FL, Flt3D2, Flt3D3 and Flt3D4. FL is bound bivalently by two receptor molecules and is accommodated by a binding epitope contributed exclusively by Flt3D3. Flt3D2 leans against the concave side of Flt3D3 and is stowed underneath FL in the ring opening without engaging in interactions with the cytokine ligand. Intriguingly, the apparent two-fold symmetry of the complex about the FL dimer interface only holds for the FL:Flt3D2-D3 subcomplex, as both Flt3D1 and Flt3D4 adopt asymmetric orientations compared to their tandem modules in the complex (FIG. 2B). Remarkably, Flt3D4 does not engage in any obvious homotypic interactions as seen in the KIT structure (Yuzawa 2007). The N-terminal Flt3D1 exhibits significant disorder and domain plasticity manifested by at least two different orientations about the D1-D2 linker region (residues 162-166), and protrudes perpendicularly away from the plane of the ring assembly at the level of Flt3D2 without making any interactions with the rest of the complex. Our electron density maps (FIG. 8) allowed us to model only the core of the Flt3D1 structure (residues 79-161), but residual positive difference electron density extending away from the N-terminus of the model suggested that the atypical 50 amino acid module of Flt3D1 is likely associated with the core domain structure.

Example 10
Flt3 Employs a Remarkably Compact Cytokine-Binding Epitope

Perhaps the most unanticipated feature of the Flt3D1-D4:FL complex is that the ligand-binding epitope is exclusively contributed by Flt3D3 (FIG. 3A). This module is a member of the “I-set” Ig domains and is structurally homologous to extracellular domain 3 of KIT (Liu 2007; Yuzawa 2007) and FMS (Chen 2008), featuring 8 β-strands making up the ABED and A′FGC β-sheets.

However, the topology of Flt3D3 is unusual such that the polypeptide chain extending from Flt3D2 forms the N-terminal A strand in Flt3D3 (residues 246-249) by complementing strand B in a parallel fashion, while the AA′ loop of Flt3D3 (residues 250-258) adopts an extended conformation. Flt3D2, which in all other RTKIII/V complexes contributes roughly half of the ligand-binding epitope, packs against the hydrophobic patch projected by the ABED-face of Flt3D3 centered around Trp269 burying ˜1000 Å2 (FIG. 3B). Flt3D2 is homologous to KITD5 and is a member of the C2 subset of IgSF (ABED/CFG topology), but contains an additional solvent-exposed disulfide bridging strands F and G. Although the AB and EF loops of Flt3D2 point in the direction of the ligand they generally remain too far to engage in any interactions. The FL binding epitope on Flt3D3 engages in extensive interactions with the N-terminal loop of FL leading to αA (residues 8-13) and Lys18 on αA, and is mainly contributed by the BC loop of Flt3D3 (residues 279-280) and strands D (residues 301-303). Additional interactions are mediated by the DE loop of Flt3D3 (residues 307) which contacts a small patch on the C-terminal region of helix αC of FL defined by residues 73 and 78. Therefore, the Flt3 ligand-receptor interaction results in a single, highly polar contact site covering merely ˜900 Å2 of buried surface area.

Example 11
FL Plasticity Upon Receptor Binding

Comparison of FL in its unbound (Savvides 2000) and now in its receptor-bound form revealed that the cytokine ligand does not undergo any significant local structural changes at its receptor binding epitope (FIG. 3D). This is contrary to what has been observed in Stem Cell Factor in complex with KIT, whereby the cytokine ligand undergoes a cascade of structural rearrangements (Liu 2007, Yuzawa 2007). However, the two FL subunits display a hinge-like rigid-body rearrangement about the dimer interface, resulting in an increase in the tilt angle between the two promoters by 5-6° (FIG. 3D). A similar motion was previously observed for human Stem Cell Factor (SCF) binding to KIT (Yuzawa 2007), although SCF already appears to have significant variability in the receptor-free form as shown by the range of intersubunit tilt angles (2° to 6°) in two independent crystal structures of SCF (1EXZ and 1SCF).

Example 12
The Flt3D3-Flt3D4 Domain Elbow and the Absence of Homotypic Receptor Interactions

A second striking feature of the Flt3D1-D4:FL complex is the absence of any obvious specific homotypic receptor interactions. Based on the current paradigm of RTKIII activation, such interactions are mediated by Ig-like domain 4. While Flt3D4 points to its tandem Flt3D4′ in the complex, the two receptor domains stay clearly away from each other and deviate from the two-fold symmetry of the complex. The inability of Flt3D4 to engage in homotypic interactions may also explain the observed disorder for this part of the structure, as a only a complete Flt3D4-Flt3D4′ tandem could reliably be modeled and refined in only one of the two complexes in the asymmetric unit of the crystal, whereas the second could only place one of the two domains.

Closer inspection of Flt3D4 topology and sequence revealed that Flt3D4 does not possess the conserved structure-sequence fingerprints seen in all other RTKIII/V homologues for this domain. For instance, Flt3D4 has two additional disulfide bridges, a solvent exposed cross-strand disulfide bridge connecting strands B and E, and a second connecting its unusual C′E loop with strand C. Most importantly, Flt3D4 displays an EF-loop which drastically differs both in structure and sequence from all homologues (FIG. 4). The EF-loop constitutes the conserved ‘tyrosine corner’ motif in I-set Ig-domains (Harpaz and Chothia), and has been shown to mediate homotypic interactions in the case of KITD4 and VEGFRD7.

Structural comparisons of the two independent Flt3D1-D4:FL complexes in the crystal asymmetric unit revealed slight orientational plasticity of Flt3D4 about the Flt3D3-Flt3D4 linker region. This stretch of residues and the A strand of Flt3D4 are strongly conserved in Flt3 and KIT and other RTKIIIs, suggesting a common functional role. Indeed, a comparison of KIT in the bound and unbound form showed that the KITD3-KITD4 linker region acts as a hinge to reorient KITD4 for homotypic interactions upon ligand binding. However, the orientational flexibility of Flt3D4 appears to be restricted by a core of hydrophobic interactions mediated by Phe261 (A′ strand of Flt3D3), Val345 (Flt3D3-Flt3D4 linker), Phe349 (A strand of Flt3D4) and Tyr376 (BC loop of Flt3D4), as well as additional interactions between the AA′ loop of Flt3D3 and the C′E loop of Flt3D4 (FIG. 4). It thus appears that the domain elbow defined by Flt3D3 and Flt3D4 in cytokine-bound Flt3 is preserved in the ligand-free receptor.

Example 13
Architecture of the Complete Flt3 Extracellular Signaling Complex

Structural studies of the complete extracellular complex of Flt3 (Flt3D1-D5:FL) were pursued via a combined approach involving X-ray crystallography, negative-stain electron microscopy (EM), and Small-angle X-ray Scattering (SAXS).

Crystals of Flt3D1-D5:FL grew reproducibly from a number of crystallization conditions but proved to be of low diffraction quality. Despite repeated efforts to improve diffraction quality via reduction of glycosylation and by applying several crystal manipulation techniques, only a complete data set to 7.8 Å resolution was obtained (Table 3). Nonetheless, this data set proved sufficient to elucidate the architecture of the complete extracellular Flt3 signaling complex by molecular replacement based on the Flt3D2-D3: FL subcomplex as refined in the Flt3D1-D4:FL crystal structure. All remaining receptor domains in the two complexes in the crystal asymmetric unit, including a conservative homology model of Flt3D5 derived from the structure of human KIT, were subsequently placed into electron density and optimized by rigid-body refinement protocols (Yuzawa 2007) (Table 3).

In the full-length ectodomain complex the core structure observed in Flt3D1-D4:FL is mounted onto two membrane-proximal Flt3D5 facing each other to form an assembly resembling a hollow tennis racket (140×75×110 Å) (FIG. 5, 6). Remarkably, the asymmetry exhibited by the tandem Flt3D4 modules in Flt3D1-D4:FL is not present in the complete extracellular complex. Instead, the two Flt3D4 segments face each other symmetrically according to the 2-fold symmetry of the Flt3D2-D3:FL core structure and approach to about 20 Å from each other. While this inter-receptor separation is maintained at the ensuing Flt3D5 modules, the apparent two-fold symmetry breaks down. Furthermore, the asymmetric projection of the N-terminal Flt3D1 domains perpendicularly our of the plane of the racket head occurs in a manner analogous to what was observed in the Flt3D1-D4:FL complex.

Complementary studies of the full-length signaling complex by negative-stain EM and by SAXS in solution corroborated the overall structural features revealed by the crystal structure (FIG. 10). In retrospect, the inherent flexibility and asymmetry of the Flt3 complexes, coupled to the extensive receptor glycosylation might explain why structural studies of extracellular complexes of Flt3 have proved so challenging.

Example 14
Flt3 Agonist Ligand Identification

cDNA encoding full length human Flt3 is cloned in the mammalian expression vectors, pcDNA4/TO (Invitrogen).

Transient protein expression in HEK293T is carried out as previously described (Aricescu, 2006). Briefly, confluent cells, grown in tissue culture flasks or roller bottles (Greiner Bio-One) are transfected with purified plasmid DNA (Plasmid Mega Kit, Qiagen) by means of Ca-phosphate transfection method, essentially as described in Kingston et al. (2003). Flt3 expression is induced according to the manufacturer's instructions.

A dilution series of candidate ligand is added to the culture medium in a concentration ranging between 0.01 and 1000 ng/ml for 15 minutes.

Cells are lysed in Laemmli lysis buffer and subjected to Western blot analysis. Flt3 phosphorylation is evaluated with Phospho-FLT3 (Tyr591) Antibody #3461 (Cell Signaling). Data are normalized for total Flt3 expression levels with FLT3 (8F2) Rabbit mAb #3462 (Cell Signaling).

Candidate ligands are identified as Flt3 agonists if capable to induce Flt3 phosphorylation. EC50 values give information about the strength of the agonist.

Example 15
Flt3 Antagonist Ligand Identification

cDNA encoding full length human Flt3 is cloned in the mammalian expression vectors, pcDNA4/TO (Invitrogen).

Human recombinant FL (hFLT3L #8924, Cell Signaling) is added to the culture medium in a concentration ranging between 0.1 and 100 ng/ml for 15 minutes. For each FL concentration, a dilution series of candidate ligand (0.01-1000 ng/ml) is concomitantly added for the same time.

Candidate ligands are identified as Flt3 antagonists if capable to decrease FL-induced Flt3 phosphorylation relative to the Flt3 phosphorylation which is induced by FL. EC50 values give information about the strength of the antagonist.

Example 16
Expansion of Dendritic Cells

Cells having the CD34+ phenotype are isolated with a CD34 specific monoclonal antibody.

The CD34+ cells which are selected then are cultured in McCoy's enhanced media with 20 ng/ml each of GM-CSF, 1L-4, TNF-a (negative control); 20 ng/ml each of GM-CSF, 1L-4, TNF-a, and 100 ng/ml FL (positive control); and 20 ng/ml each of GM-CSF, 1L-4, TNF-a, and 0.01-1000 ng/ml candidate Flt3 ligand (experimental setup). The culture is continued for approximately two weeks at 37° C. in 10% C02 in humid air. Cells then are sorted by flow cytometry for CDIa+ and HLA-DR+ expression.

Candidate ligands are identified as Flt3 agonists if capable to expand dendritic cells. EC50 values give information about the strength of the agonist.

Example 17
Cell Proliferation Assay

Monocytic human leukemic OCI-AML3 and THP-1 cell lines, which express the wild type Flt3 receptor and proliferate in response to FL, are purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ) (Braunschweig, Germany). OCI-AML3 cells are cultured in alpha-MEM with nucleosides (Gibco, Karlsruhe, Germany) and THP-1 cells are cultured in RPMI1640 (Gibco, Karlsruhe, Germany), with both media supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS) (Gibco, Karlsruhe, Germany) and 1% (v/v) Penicillin/Streptomycin (PAA Laboratories, Pasching, Austria) at 37° C. and 5% CO2, in a humidified atmosphere. Recombinant human FL (rhFL) produced in insect cells is used as a positive control (from R&D Systems; Minneapolis, Minn., USA).

The proliferation behavior of cells is assessed using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit from Promega (Madison, Wis., USA) according to manufacturer's recommendations. In brief, 5,000 cells are seeded per well of a 96-well-plate in medium with 1% FCS (starvation medium) with or without the addition of a candidate modulator of Flt3 signaling or rhFL and cultured for 70 h at 37° C. and 5% CO2, in a humidified atmosphere. After adding the CellTiter 96® aqueous one solution reagent, cells are incubated for further 2 h at 37° C. and 5% CO2. Absorbance is recorded at 450 nm in an Anthos htII spectrometer (Anthos Labtec Instruments, Wals, Austria). Each assay is performed in triplicate in at least three independent experiments.

Flt3 modulators are identified by their propensity to stimulate cell proliferation.

Modulation of Flt3 signaling is verified by Flt3 modulator-dependent phosphorylation of the Flt3 receptor and the downstream signaling molecule MEK via Western blot analysis, using mouse monoclonal anti-phospho FLT3 antibody, rabbit polyclonal anti phospho-MEK1/2 antibody, and mouse monoclonal anti-MEK1/2 antibody (Cell Signaling Technology, Danvers, Mass., USA); mouse monoclonal anti-human FLT3 antibody (R&D Systems); and mouse monoclonal anti-GAPDH antibody (Abcam, Cambridge, UK).

Example 18
Screening for Ligands

Screening for ligands of Flt3 or FL is carried out by phage display, essentially as described in Clackson & Lowman (2004). DNA encoding candidate ligands, preferably Alphabodies™ or Nanobodies®, is cloned into the pIII or pVIII gene of bacteriophage M13 in a phagmid vector, and transformed into E. coli. Viral production initiates upon coinfection of E. coli with helper phages. In this way, a phage library is established.

Full length Flt3 or FL protein is immobilized on a solid substrate and incubated with the phage library, preferably via avidin/biotin coupling. The substrate is washed by which non-bound phages are removed. Retained phages are eluted and used to infect E. coli. After amplification, the phagmid containing the DNA sequence of the candidate ligand is extracted and the DNA sequence of the candidate ligand is determined. It will be clear to the person skilled in the art that multiple consecutive cycles of infection may be performed after each elution step in order to gradually enrich the final population of phages containing strongly binding candidate ligands.

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TABLE 3

Atomic coordinates

ATOM
1
N
PHE
B
245
−16.843
30.204
−81.964
1.00
161.52
N

ATOM
2
CA
PHE
B
245
−16.008
31.221
−82.589
1.00
161.52
C

ATOM
3
C
PHE
B
245
−15.016
31.792
−81.597
1.00
161.52
C

ATOM
4
O
PHE
B
245
−15.390
32.078
−80.462
1.00
161.52
O

ATOM
5
CB
PHE
B
245
−16.837
32.332
−83.225
1.00
161.52
C

ATOM
6
CG
PHE
B
245
−16.014
33.145
−84.190
1.00
161.52
C

ATOM
7
CD1
PHE
B
245
−15.862
32.740
−85.510
1.00
161.52
C

ATOM
8
CD2
PHE
B
245
−15.357
34.294
−83.771
1.00
161.52
C

ATOM
9
CE1
PHE
B
245
−15.080
33.484
−86.401
1.00
161.52
C

ATOM
10
CE2
PHE
B
245
−14.581
35.040
−84.659
1.00
161.52
C

ATOM
11
CZ
PHE
B
245
−14.447
34.632
−85.969
1.00
161.52
C

ATOM
12
N
THR
B
246
−13.746
31.924
−82.017
1.00
159.24
N

ATOM
13
CA
THR
B
246
−12.687
32.414
−81.145
1.00
159.24
C

ATOM
14
C
THR
B
246
−11.776
33.427
−81.844
1.00
159.24
C

ATOM
15
O
THR
B
246
−11.549
33.330
−83.046
1.00
159.24
O

ATOM
16
CB
THR
B
246
−11.875
31.234
−80.609
1.00
159.24
C

ATOM
17
N
ILE
B
247
−11.270
34.404
−81.063
1.00
158.96
N

ATOM
18
CA
ILE
B
247
−10.340
35.475
−81.440
1.00
158.96
C

ATOM
19
C
ILE
B
247
−9.153
35.378
−80.483
1.00
158.96
C

ATOM
20
O
ILE
B
247
−9.288
35.730
−79.304
1.00
158.96
O

ATOM
21
CB
ILE
B
247
−11.025
36.879
−81.400
1.00
158.96
C

ATOM
22
CG1
ILE
B
247
−12.145
36.998
−82.450
1.00
158.96
C

ATOM
23
CG2
ILE
B
247
−10.001
38.022
−81.541
1.00
158.96
C

ATOM
24
CD1
ILE
B
247
−12.974
38.289
−82.374
1.00
158.96
C

ATOM
25
N
ASP
B
248
−8.012
34.863
−80.964
1.00
161.81
N

ATOM
26
CA
ASP
B
248
−6.845
34.693
−80.105
1.00
161.81
C

ATOM
27
C
ASP
B
248
−5.915
35.888
−80.180
1.00
161.81
C

ATOM
28
O
ASP
B
248
−5.301
36.142
−81.218
1.00
161.81
O

ATOM
29
CB
ASP
B
248
−6.080
33.401
−80.429
1.00
161.81
C

ATOM
30
N
LEU
B
249
−5.821
36.629
−79.068
1.00
166.16
N

ATOM
31
CA
LEU
B
249
−4.897
37.751
−78.935
1.00
166.16
C

ATOM
32
C
LEU
B
249
−3.627
37.157
−78.324
1.00
166.16
C

ATOM
33
O
LEU
B
249
−3.396
37.134
−77.098
1.00
166.16
O

ATOM
34
CB
LEU
B
249
−5.489
38.928
−78.145
1.00
166.16
C

ATOM
35
CG
LEU
B
249
−6.842
39.445
−78.639
1.00
166.16
C

ATOM
36
CD1
LEU
B
249
−7.336
40.567
−77.769
1.00
166.16
C

ATOM
37
CD2
LEU
B
249
−6.778
39.890
−80.086
1.00
166.16
C

ATOM
38
N
ASN
B
250
−2.913
36.504
−79.237
1.00
170.32
N

ATOM
39
CA
ASN
B
250
−1.670
35.759
−79.150
1.00
170.32
C

ATOM
40
C
ASN
B
250
−1.326
35.382
−80.602
1.00
170.32
C

ATOM
41
O
ASN
B
250
−0.226
35.692
−81.068
1.00
170.32
O

ATOM
42
CB
ASN
B
250
−1.808
34.531
−78.228
1.00
170.32
C

ATOM
43
N
GLN
B
251
−2.327
34.822
−81.350
1.00
174.18
N

ATOM
44
CA
GLN
B
251
−2.235
34.375
−82.754
1.00
174.18
C

ATOM
45
C
GLN
B
251
−1.929
35.517
−83.737
1.00
174.18
C

ATOM
46
O
GLN
B
251
−2.239
36.685
−83.469
1.00
174.18
O

ATOM
47
CB
GLN
B
251
−3.533
33.663
−83.193
1.00
174.18
C

ATOM
48
N
THR
B
252
−1.313
35.149
−84.882
1.00
176.76
N

ATOM
49
CA
THR
B
252
−0.954
36.050
−85.978
1.00
176.76
C

ATOM
50
C
THR
B
252
−2.227
36.490
−86.711
1.00
176.76
C

ATOM
51
O
THR
B
252
−3.092
35.647
−86.977
1.00
176.76
O

ATOM
52
CB
THR
B
252
0.042
35.373
−86.927
1.00
176.76
C

ATOM
53
N
PRO
B
253
−2.369
37.801
−87.025
1.00
178.90
N

ATOM
54
CA
PRO
B
253
−3.594
38.274
−87.694
1.00
178.90
C

ATOM
55
C
PRO
B
253
−3.889
37.571
−89.023
1.00
178.90
C

ATOM
56
O
PRO
B
253
−2.975
37.261
−89.794
1.00
178.90
O

ATOM
57
CB
PRO
B
253
−3.327
39.766
−87.911
1.00
178.90
C

ATOM
58
CG
PRO
B
253
−2.317
40.129
−86.878
1.00
178.90
C

ATOM
59
CD
PRO
B
253
−1.445
38.921
−86.747
1.00
178.90
C

ATOM
60
N
GLN
B
254
−5.188
37.295
−89.259
1.00
181.38
N

ATOM
61
CA
GLN
B
254
−5.712
36.639
−90.459
1.00
181.38
C

ATOM
62
C
GLN
B
254
−5.953
37.674
−91.546
1.00
181.38
C

ATOM
63
O
GLN
B
254
−6.766
38.585
−91.357
1.00
181.38
O

ATOM
64
CB
GLN
B
254
−7.016
35.867
−90.146
1.00
181.38
C

ATOM
65
N
THR
B
255
−5.250
37.538
−92.683
1.00
182.34
N

ATOM
66
CA
THR
B
255
−5.407
38.437
−93.822
1.00
182.34
C

ATOM
67
C
THR
B
255
−6.691
38.043
−94.572
1.00
182.34
C

ATOM
68
O
THR
B
255
−6.651
37.592
−95.720
1.00
182.34
O

ATOM
69
CB
THR
B
255
−4.152
38.410
−94.688
1.00
182.34
C

ATOM
70
N
THR
B
256
−7.834
38.190
−93.868
1.00
184.42
N

ATOM
71
CA
THR
B
256
−9.206
37.894
−94.300
1.00
184.42
C

ATOM
72
C
THR
B
256
−10.237
38.692
−93.453
1.00
184.42
C

ATOM
73
O
THR
B
256
−11.316
39.021
−93.971
1.00
184.42
O

ATOM
74
CB
THR
B
256
−9.484
36.382
−94.215
1.00
184.42
C

ATOM
75
N
LEU
B
257
−9.875
39.024
−92.165
1.00
184.95
N

ATOM
76
CA
LEU
B
257
−10.694
39.717
−91.149
1.00
184.95
C

ATOM
77
C
LEU
B
257
−12.019
38.954
−91.013
1.00
184.95
C

ATOM
78
O
LEU
B
257
−13.043
39.423
−91.518
1.00
184.95
O

ATOM
79
CB
LEU
B
257
−10.912
41.203
−91.489
1.00
184.95
C

ATOM
80
N
PRO
B
258
−11.951
37.714
−90.448
1.00
185.16
N

ATOM
81
CA
PRO
B
258
−13.126
36.809
−90.385
1.00
185.16
C

ATOM
82
C
PRO
B
258
−14.563
37.396
−90.425
1.00
185.16
C

ATOM
83
O
PRO
B
258
−14.914
38.304
−89.668
1.00
185.16
O

ATOM
84
CB
PRO
B
258
−12.890
36.061
−89.077
1.00
185.16
C

ATOM
85
CG
PRO
B
258
−11.353
36.030
−88.933
1.00
185.16
C

ATOM
86
CD
PRO
B
258
−10.761
37.038
−89.883
1.00
185.16
C

ATOM
87
N
GLN
B
259
−15.397
36.833
−91.335
1.00
185.06
N

ATOM
88
CA
GLN
B
259
−16.799
37.206
−91.538
1.00
185.06
C

ATOM
89
C
GLN
B
259
−17.700
36.134
−90.934
1.00
185.06
C

ATOM
90
O
GLN
B
259
−17.851
35.060
−91.519
1.00
185.06
O

ATOM
91
CB
GLN
B
259
−17.112
37.413
−93.034
1.00
185.06
C

ATOM
92
N
LEU
B
260
−18.254
36.407
−89.735
1.00
185.40
N

ATOM
93
CA
LEU
B
260
−19.160
35.495
−89.017
1.00
185.40
C

ATOM
94
C
LEU
B
260
−20.556
35.544
−89.645
1.00
185.40
C

ATOM
95
O
LEU
B
260
−20.988
36.618
−90.070
1.00
185.40
O

ATOM
96
CB
LEU
B
260
−19.236
35.867
−87.523
1.00
185.40
C

ATOM
97
CG
LEU
B
260
−18.869
34.778
−86.509
1.00
185.40
C

ATOM
98
CD1
LEU
B
260
−18.618
35.382
−85.139
1.00
185.40
C

ATOM
99
CD2
LEU
B
260
−19.957
33.711
−86.410
1.00
185.40
C

ATOM
100
N
PHE
B
261
−21.257
34.396
−89.721
1.00
183.44
N

ATOM
101
CA
PHE
B
261
−22.579
34.364
−90.348
1.00
183.44
C

ATOM
102
C
PHE
B
261
−23.600
33.597
−89.535
1.00
183.44
C

ATOM
103
O
PHE
B
261
−23.268
32.538
−88.992
1.00
183.44
O

ATOM
104
CB
PHE
B
261
−22.480
33.743
−91.749
1.00
183.44
C

ATOM
105
CG
PHE
B
261
−22.169
34.723
−92.856
1.00
183.44
C

ATOM
106
CD1
PHE
B
261
−23.186
35.269
−93.628
1.00
183.44
C

ATOM
107
CD2
PHE
B
261
−20.854
35.089
−93.139
1.00
183.44
C

ATOM
108
CE1
PHE
B
261
−22.896
36.158
−94.669
1.00
183.44
C

ATOM
109
CE2
PHE
B
261
−20.565
35.997
−94.165
1.00
183.44
C

ATOM
110
CZ
PHE
B
261
−21.587
36.522
−94.927
1.00
183.44
C

ATOM
111
N
LEU
B
262
−24.850
34.123
−89.457
1.00
178.10
N

ATOM
112
CA
LEU
B
262
−25.926
33.414
−88.743
1.00
178.10
C

ATOM
113
C
LEU
B
262
−27.323
33.949
−89.107
1.00
178.10
C

ATOM
114
O
LEU
B
262
−27.464
35.134
−89.419
1.00
178.10
O

ATOM
115
CB
LEU
B
262
−25.726
33.364
−87.203
1.00
178.10
C

ATOM
116
CG
LEU
B
262
−25.617
34.646
−86.359
1.00
178.10
C

ATOM
117
CD1
LEU
B
262
−25.841
34.322
−84.898
1.00
178.10
C

ATOM
118
CD2
LEU
B
262
−24.246
35.286
−86.467
1.00
178.10
C

ATOM
119
N
LYS
B
263
−28.342
33.051
−89.113
1.00
171.62
N

ATOM
120
CA
LYS
B
263
−29.725
33.393
−89.453
1.00
171.62
C

ATOM
121
C
LYS
B
263
−30.414
34.127
−88.307
1.00
171.62
C

ATOM
122
O
LYS
B
263
−30.245
33.729
−87.147
1.00
171.62
O

ATOM
123
CB
LYS
B
263
−30.508
32.141
−89.830
1.00
171.62
C

ATOM
124
N
VAL
B
264
−31.192
35.203
−88.632
1.00
168.89
N

ATOM
125
CA
VAL
B
264
−31.897
36.018
−87.609
1.00
168.89
C

ATOM
126
C
VAL
B
264
−32.683
35.108
−86.636
1.00
168.89
C

ATOM
127
O
VAL
B
264
−33.477
34.274
−87.077
1.00
168.89
O

ATOM
128
CB
VAL
B
264
−32.806
37.124
−88.226
1.00
168.89
C

ATOM
129
CG1
VAL
B
264
−33.831
37.668
−87.226
1.00
168.89
C

ATOM
130
CG2
VAL
B
264
−31.971
38.266
−88.778
1.00
168.89
C

ATOM
131
N
GLY
B
265
−32.417
35.278
−85.335
1.00
167.11
N

ATOM
132
CA
GLY
B
265
−33.026
34.509
−84.250
1.00
167.11
C

ATOM
133
C
GLY
B
265
−32.031
33.612
−83.537
1.00
167.11
C

ATOM
134
O
GLY
B
265
−32.213
33.326
−82.347
1.00
167.11
O

ATOM
135
N
GLU
B
266
−30.960
33.163
−84.272
1.00
162.99
N

ATOM
136
CA
GLU
B
266
−29.887
32.293
−83.765
1.00
162.99
C

ATOM
137
C
GLU
B
266
−29.001
33.033
−82.716
1.00
162.99
C

ATOM
138
O
GLU
B
266
−28.962
34.268
−82.715
1.00
162.99
O

ATOM
139
CB
GLU
B
266
−29.032
31.754
−84.929
1.00
162.99
C

ATOM
140
N
PRO
B
267
−28.299
32.324
−81.802
1.00
159.20
N

ATOM
141
CA
PRO
B
267
−27.500
33.037
−80.793
1.00
159.20
C

ATOM
142
C
PRO
B
267
−26.048
33.295
−81.214
1.00
159.20
C

ATOM
143
O
PRO
B
267
−25.320
32.371
−81.581
1.00
159.20
O

ATOM
144
CB
PRO
B
267
−27.572
32.118
−79.571
1.00
159.20
C

ATOM
145
CG
PRO
B
267
−27.950
30.755
−80.117
1.00
159.20
C

ATOM
146
CD
PRO
B
267
−28.240
30.867
−81.587
1.00
159.20
C

ATOM
147
N
LEU
B
268
−25.621
34.559
−81.130
1.00
156.12
N

ATOM
148
CA
LEU
B
268
−24.258
34.932
−81.484
1.00
156.12
C

ATOM
149
C
LEU
B
268
−23.305
34.584
−80.357
1.00
156.12
C

ATOM
150
O
LEU
B
268
−23.648
34.786
−79.198
1.00
156.12
O

ATOM
151
CB
LEU
B
268
−24.195
36.423
−81.799
1.00
156.12
C

ATOM
152
CG
LEU
B
268
−22.825
37.011
−82.115
1.00
156.12
C

ATOM
153
CD1
LEU
B
268
−22.205
36.363
−83.351
1.00
156.12
C

ATOM
154
CD2
LEU
B
268
−22.910
38.517
−82.283
1.00
156.12
C

ATOM
155
N
TRP
B
269
−22.111
34.073
−80.694
1.00
154.61
N

ATOM
156
CA
TRP
B
269
−21.097
33.679
−79.713
1.00
154.61
C

ATOM
157
C
TRP
B
269
−19.679
33.957
−80.203
1.00
154.61
C

ATOM
158
O
TRP
B
269
−19.236
33.344
−81.178
1.00
154.61
O

ATOM
159
CB
TRP
B
269
−21.240
32.192
−79.379
1.00
154.61
C

ATOM
160
CG
TRP
B
269
−22.337
31.905
−78.407
1.00
154.61
C

ATOM
161
CD1
TRP
B
269
−23.600
31.482
−78.696
1.00
154.61
C

ATOM
162
CD2
TRP
B
269
−22.271
32.033
−76.983
1.00
154.61
C

ATOM
163
CE2
TRP
B
269
−23.527
31.648
−76.469
1.00
154.61
C

ATOM
164
CE3
TRP
B
269
−21.259
32.410
−76.088
1.00
154.61
C

ATOM
165
NE1
TRP
B
269
−24.328
31.341
−77.538
1.00
154.61
N

ATOM
166
CZ2
TRP
B
269
−23.797
31.619
−75.102
1.00
154.61
C

ATOM
167
CZ3
TRP
B
269
−21.541
32.416
−74.738
1.00
154.61
C

ATOM
168
CH2
TRP
B
269
−22.796
32.026
−74.256
1.00
154.61
C

ATOM
169
N
ILE
B
270
−18.958
34.869
−79.518
1.00
152.78
N

ATOM
170
CA
ILE
B
270
−17.587
35.230
−79.898
1.00
152.78
C

ATOM
171
C
ILE
B
270
−16.665
35.244
−78.671
1.00
152.78
C

ATOM
172
O
ILE
B
270
−16.753
36.159
−77.858
1.00
152.78
O

ATOM
173
CB
ILE
B
270
−17.514
36.590
−80.647
1.00
152.78
C

ATOM
174
CG1
ILE
B
270
−18.825
36.995
−81.330
1.00
152.78
C

ATOM
175
CG2
ILE
B
270
−16.365
36.616
−81.615
1.00
152.78
C

ATOM
176
CD1
ILE
B
270
−19.492
38.096
−80.641
1.00
152.78
C

ATOM
177
N
ARG
B
271
−15.779
34.244
−78.550
1.00
154.11
N

ATOM
178
CA
ARG
B
271
−14.831
34.155
−77.438
1.00
154.11
C

ATOM
179
C
ARG
B
271
−13.596
34.942
−77.748
1.00
154.11
C

ATOM
180
O
ARG
B
271
−13.067
34.838
−78.844
1.00
154.11
O

ATOM
181
CB
ARG
B
271
−14.448
32.699
−77.135
1.00
154.11
C

ATOM
182
N
CYS
B
272
−13.119
35.718
−76.798
1.00
157.73
N

ATOM
183
CA
CYS
B
272
−11.899
36.459
−77.018
1.00
157.73
C

ATOM
184
C
CYS
B
272
−10.874
36.045
−75.991
1.00
157.73
C

ATOM
185
O
CYS
B
272
−10.980
36.438
−74.826
1.00
157.73
O

ATOM
186
CB
CYS
B
272
−12.152
37.954
−76.988
1.00
157.73
C

ATOM
187
SG
CYS
B
272
−10.724
38.929
−77.497
1.00
157.73
S

ATOM
188
N
LYS
B
273
−9.897
35.218
−76.415
1.00
153.79
N

ATOM
189
CA
LYS
B
273
−8.863
34.668
−75.529
1.00
153.79
C

ATOM
190
C
LYS
B
273
−7.558
35.410
−75.659
1.00
153.79
C

ATOM
191
O
LYS
B
273
−6.873
35.293
−76.675
1.00
153.79
O

ATOM
192
CB
LYS
B
273
−8.632
33.166
−75.785
1.00
153.79
C

ATOM
193
N
ALA
B
274
−7.208
36.168
−74.630
1.00
150.31
N

ATOM
194
CA
ALA
B
274
−5.960
36.908
−74.637
1.00
150.31
C

ATOM
195
C
ALA
B
274
−4.994
36.257
−73.694
1.00
150.31
C

ATOM
196
O
ALA
B
274
−5.395
35.865
−72.595
1.00
150.31
O

ATOM
197
CB
ALA
B
274
−6.200
38.350
−74.249
1.00
150.31
C

ATOM
198
N
VAL
B
275
−3.733
36.091
−74.120
1.00
150.55
N

ATOM
199
CA
VAL
B
275
−2.768
35.439
−73.228
1.00
150.55
C

ATOM
200
C
VAL
B
275
−1.668
36.402
−72.855
1.00
150.55
C

ATOM
201
O
VAL
B
275
−1.009
36.931
−73.744
1.00
150.55
O

ATOM
202
CB
VAL
B
275
−2.163
34.124
−73.776
1.00
150.55
C

ATOM
203
CG1
VAL
B
275
−1.945
33.124
−72.643
1.00
150.55
C

ATOM
204
CG2
VAL
B
275
−3.011
33.517
−74.896
1.00
150.55
C

ATOM
205
N
HIS
B
276
−1.436
36.607
−71.555
1.00
151.82
N

ATOM
206
CA
HIS
B
276
−0.391
37.529
−71.118
1.00
151.82
C

ATOM
207
C
HIS
B
276
0.424
36.934
−69.966
1.00
151.82
C

ATOM
208
O
HIS
B
276
−0.028
35.971
−69.350
1.00
151.82
O

ATOM
209
CB
HIS
B
276
−1.020
38.871
−70.726
1.00
151.82
C

ATOM
210
CG
HIS
B
276
−0.044
39.953
−70.365
1.00
151.82
C

ATOM
211
CD2
HIS
B
276
0.936
40.522
−71.106
1.00
151.82
C

ATOM
212
ND1
HIS
B
276
−0.047
40.538
−69.107
1.00
151.82
N

ATOM
213
CE1
HIS
B
276
0.918
41.442
−69.125
1.00
151.82
C

ATOM
214
NE2
HIS
B
276
1.539
41.468
−70.305
1.00
151.82
N

ATOM
215
N
VAL
B
277
1.631
37.509
−69.693
1.00
153.56
N

ATOM
216
CA
VAL
B
277
2.595
37.118
−68.643
1.00
153.56
C

ATOM
217
C
VAL
B
277
2.040
37.425
−67.227
1.00
153.56
C

ATOM
218
O
VAL
B
277
1.822
36.493
−66.447
1.00
153.56
O

ATOM
219
CB
VAL
B
277
3.969
37.798
−68.852
1.00
153.56
C

ATOM
220
CG1
VAL
B
277
5.001
37.253
−67.872
1.00
153.56
C

ATOM
221
CG2
VAL
B
277
4.449
37.633
−70.288
1.00
153.56
C

ATOM
222
N
ASN
B
278
1.864
38.722
−66.886
1.00
153.90
N

ATOM
223
CA
ASN
B
278
1.299
39.153
−65.604
1.00
153.90
C

ATOM
224
C
ASN
B
278
−0.215
39.186
−65.730
1.00
153.90
C

ATOM
225
O
ASN
B
278
−0.724
39.187
−66.845
1.00
153.90
O

ATOM
226
CB
ASN
B
278
1.845
40.525
−65.214
1.00
153.90
C

ATOM
227
N
HIS
B
279
−0.943
39.220
−64.615
1.00
155.49
N

ATOM
228
CA
HIS
B
279
−2.406
39.284
−64.676
1.00
155.49
C

ATOM
229
C
HIS
B
279
−2.883
40.689
−65.076
1.00
155.49
C

ATOM
230
O
HIS
B
279
−4.072
40.897
−65.331
1.00
155.49
O

ATOM
231
CB
HIS
B
279
−3.014
38.898
−63.325
1.00
155.49
C

ATOM
232
CG
HIS
B
279
−2.900
39.982
−62.304
1.00
155.49
C

ATOM
233
CD2
HIS
B
279
−3.685
41.069
−62.108
1.00
155.49
C

ATOM
234
ND1
HIS
B
279
−1.854
40.013
−61.399
1.00
155.49
N

ATOM
235
CE1
HIS
B
279
−2.045
41.100
−60.672
1.00
155.49
C

ATOM
236
NE2
HIS
B
279
−3.128
41.775
−61.072
1.00
155.49
N

ATOM
237
N
GLY
B
280
−1.959
41.642
−65.037
1.00
157.26
N

ATOM
238
CA
GLY
B
280
−2.209
43.043
−65.338
1.00
157.26
C

ATOM
239
C
GLY
B
280
−2.559
43.329
−66.781
1.00
157.26
C

ATOM
240
O
GLY
B
280
−1.692
43.702
−67.577
1.00
157.26
O

ATOM
241
N
PHE
B
281
−3.843
43.144
−67.110
1.00
158.55
N

ATOM
242
CA
PHE
B
281
−4.461
43.393
−68.408
1.00
158.55
C

ATOM
243
C
PHE
B
281
−5.933
43.050
−68.338
1.00
158.55
C

ATOM
244
O
PHE
B
281
−6.337
42.180
−67.564
1.00
158.55
O

ATOM
245
CB
PHE
B
281
−3.777
42.619
−69.555
1.00
158.55
C

ATOM
246
CG
PHE
B
281
−4.106
41.149
−69.693
1.00
158.55
C

ATOM
247
CD1
PHE
B
281
−4.795
40.675
−70.802
1.00
158.55
C

ATOM
248
CD2
PHE
B
281
−3.687
40.232
−68.735
1.00
158.55
C

ATOM
249
CE1
PHE
B
281
−5.069
39.312
−70.944
1.00
158.55
C

ATOM
250
CE2
PHE
B
281
−3.972
38.868
−68.875
1.00
158.55
C

ATOM
251
CZ
PHE
B
281
−4.650
38.418
−69.985
1.00
158.55
C

ATOM
252
N
GLY
B
282
−6.716
43.763
−69.129
1.00
161.67
N

ATOM
253
CA
GLY
B
282
−8.154
43.579
−69.261
1.00
161.67
C

ATOM
254
C
GLY
B
282
−8.512
43.370
−70.716
1.00
161.67
C

ATOM
255
O
GLY
B
282
−7.623
43.316
−71.570
1.00
161.67
O

ATOM
256
N
LEU
B
283
−9.804
43.227
−71.010
1.00
164.54
N

ATOM
257
CA
LEU
B
283
−10.302
43.036
−72.374
1.00
164.54
C

ATOM
258
C
LEU
B
283
−11.608
43.801
−72.580
1.00
164.54
C

ATOM
259
O
LEU
B
283
−12.352
44.008
−71.613
1.00
164.54
O

ATOM
260
CB
LEU
B
283
−10.516
41.548
−72.657
1.00
164.54
C

ATOM
261
CG
LEU
B
283
−9.332
40.766
−73.162
1.00
164.54
C

ATOM
262
CD1
LEU
B
283
−8.565
40.167
−72.022
1.00
164.54
C

ATOM
263
CD2
LEU
B
283
−9.794
39.647
−74.038
1.00
164.54
C

ATOM
264
N
THR
B
284
−11.900
44.216
−73.828
1.00
167.63
N

ATOM
265
CA
THR
B
284
−13.121
44.959
−74.120
1.00
167.63
C

ATOM
266
C
THR
B
284
−13.636
44.649
−75.491
1.00
167.63
C

ATOM
267
O
THR
B
284
−12.857
44.428
−76.418
1.00
167.63
O

ATOM
268
CB
THR
B
284
−12.889
46.459
−73.980
1.00
167.63
C

ATOM
269
N
TRP
B
285
−14.966
44.665
−75.615
1.00
174.19
N

ATOM
270
CA
TRP
B
285
−15.708
44.420
−76.851
1.00
174.19
C

ATOM
271
C
TRP
B
285
−16.307
45.717
−77.417
1.00
174.19
C

ATOM
272
O
TRP
B
285
−16.684
46.590
−76.630
1.00
174.19
O

ATOM
273
CB
TRP
B
285
−16.839
43.410
−76.591
1.00
174.19
C

ATOM
274
CG
TRP
B
285
−16.436
41.963
−76.675
1.00
174.19
C

ATOM
275
CD1
TRP
B
285
−16.478
41.040
−75.670
1.00
174.19
C

ATOM
276
CD2
TRP
B
285
−16.004
41.258
−77.849
1.00
174.19
C

ATOM
277
CE2
TRP
B
285
−15.763
39.919
−77.468
1.00
174.19
C

ATOM
278
CE3
TRP
B
285
−15.778
41.634
−79.187
1.00
174.19
C

ATOM
279
NE1
TRP
B
285
−16.070
39.812
−76.136
1.00
174.19
N

ATOM
280
CZ2
TRP
B
285
−15.305
38.957
−78.372
1.00
174.19
C

ATOM
281
CZ3
TRP
B
285
−15.321
40.680
−80.080
1.00
174.19
C

ATOM
282
CH2
TRP
B
285
−15.079
39.363
−79.668
1.00
174.19
C

ATOM
283
N
GLU
B
286
−16.445
45.824
−78.770
1.00
177.27
N

ATOM
284
CA
GLU
B
286
−17.026
47.009
−79.435
1.00
177.27
C

ATOM
285
C
GLU
B
286
−17.712
46.688
−80.788
1.00
177.27
C

ATOM
286
O
GLU
B
286
−17.348
45.687
−81.412
1.00
177.27
O

ATOM
287
CB
GLU
B
286
−15.929
48.062
−79.675
1.00
177.27
C

ATOM
288
N
LEU
B
287
−18.675
47.567
−81.261
1.00
180.30
N

ATOM
289
CA
LEU
B
287
−19.319
47.440
−82.593
1.00
180.30
C

ATOM
290
C
LEU
B
287
−18.998
48.702
−83.437
1.00
180.30
C

ATOM
291
O
LEU
B
287
−18.682
49.752
−82.859
1.00
180.30
O

ATOM
292
CB
LEU
B
287
−20.853
47.210
−82.508
1.00
180.30
C

ATOM
293
CG
LEU
B
287
−21.549
46.424
−83.654
1.00
180.30
C

ATOM
294
CD1
LEU
B
287
−22.777
45.730
−83.148
1.00
180.30
C

ATOM
295
CD2
LEU
B
287
−22.005
47.330
−84.790
1.00
180.30
C

ATOM
296
N
GLU
B
288
−19.056
48.586
−84.795
1.00
182.72
N

ATOM
297
CA
GLU
B
288
−18.803
49.668
−85.766
1.00
182.72
C

ATOM
298
C
GLU
B
288
−19.762
50.845
−85.542
1.00
182.72
C

ATOM
299
O
GLU
B
288
−20.946
50.759
−85.889
1.00
182.72
O

ATOM
300
CB
GLU
B
288
−18.921
49.155
−87.214
1.00
182.72
C

ATOM
301
N
ASN
B
289
−19.243
51.924
−84.909
1.00
183.79
N

ATOM
302
CA
ASN
B
289
−19.955
53.152
−84.534
1.00
183.79
C

ATOM
303
C
ASN
B
289
−21.165
52.855
−83.600
1.00
183.79
C

ATOM
304
O
ASN
B
289
−21.973
53.751
−83.358
1.00
183.79
O

ATOM
305
CB
ASN
B
289
−20.386
53.952
−85.774
1.00
183.79
C

ATOM
306
N
LYS
B
290
−21.264
51.617
−83.050
1.00
185.08
N

ATOM
307
CA
LYS
B
290
−22.341
51.213
−82.143
1.00
185.08
C

ATOM
308
C
LYS
B
290
−21.786
50.680
−80.825
1.00
185.08
C

ATOM
309
O
LYS
B
290
−20.818
49.890
−80.805
1.00
185.08
O

ATOM
310
CB
LYS
B
290
−23.266
50.173
−82.786
1.00
185.08
C

ATOM
311
N
ALA
B
291
−22.409
51.149
−79.716
1.00
187.65
N

ATOM
312
CA
ALA
B
291
−22.084
50.775
−78.335
1.00
187.65
C

ATOM
313
C
ALA
B
291
−22.750
49.446
−78.009
1.00
187.65
C

ATOM
314
O
ALA
B
291
−23.982
49.335
−78.047
1.00
187.65
O

ATOM
315
CB
ALA
B
291
−22.527
51.862
−77.359
1.00
187.65
C

ATOM
316
N
LEU
B
292
−21.919
48.432
−77.736
1.00
187.87
N

ATOM
317
CA
LEU
B
292
−22.353
47.070
−77.484
1.00
187.87
C

ATOM
318
C
LEU
B
292
−23.057
46.887
−76.151
1.00
187.87
C

ATOM
319
O
LEU
B
292
−22.657
47.483
−75.143
1.00
187.87
O

ATOM
320
CB
LEU
B
292
−21.152
46.127
−77.550
1.00
187.87
C

ATOM
321
CG
LEU
B
292
−21.255
45.013
−78.579
1.00
187.87
C

ATOM
322
CD1
LEU
B
292
−19.884
44.508
−78.959
1.00
187.87
C

ATOM
323
CD2
LEU
B
292
−22.140
43.867
−78.077
1.00
187.87
C

ATOM
324
N
GLU
B
293
−24.103
46.025
−76.159
1.00
188.78
N

ATOM
325
CA
GLU
B
293
−24.865
45.625
−74.980
1.00
188.78
C

ATOM
326
C
GLU
B
293
−23.971
44.672
−74.190
1.00
188.78
C

ATOM
327
O
GLU
B
293
−24.002
43.448
−74.397
1.00
188.78
O

ATOM
328
CB
GLU
B
293
−26.218
44.992
−75.364
1.00
188.78
C

ATOM
329
N
GLU
B
294
−23.101
45.278
−73.331
1.00
189.56
N

ATOM
330
CA
GLU
B
294
−22.117
44.629
−72.443
1.00
189.56
C

ATOM
331
C
GLU
B
294
−22.824
43.800
−71.342
1.00
189.56
C

ATOM
332
O
GLU
B
294
−22.150
43.160
−70.521
1.00
189.56
O

ATOM
333
CB
GLU
B
294
−21.161
45.672
−71.823
1.00
189.56
C

ATOM
334
N
GLY
B
295
−24.170
43.826
−71.369
1.00
186.66
N

ATOM
335
CA
GLY
B
295
−25.084
43.052
−70.532
1.00
186.66
C

ATOM
336
C
GLY
B
295
−25.528
41.813
−71.295
1.00
186.66
C

ATOM
337
O
GLY
B
295
−26.710
41.455
−71.314
1.00
186.66
O

ATOM
338
N
ASN
B
296
−24.543
41.182
−71.962
1.00
181.79
N

ATOM
339
CA
ASN
B
296
−24.590
39.991
−72.802
1.00
181.79
C

ATOM
340
C
ASN
B
296
−23.135
39.436
−72.946
1.00
181.79
C

ATOM
341
O
ASN
B
296
−22.923
38.373
−73.550
1.00
181.79
O

ATOM
342
CB
ASN
B
296
−25.217
40.357
−74.153
1.00
181.79
C

ATOM
343
N
TYR
B
297
−22.147
40.177
−72.350
1.00
175.21
N

ATOM
344
CA
TYR
B
297
−20.700
39.924
−72.307
1.00
175.21
C

ATOM
345
C
TYR
B
297
−20.254
39.395
−70.925
1.00
175.21
C

ATOM
346
O
TYR
B
297
−20.660
39.949
−69.900
1.00
175.21
O

ATOM
347
CB
TYR
B
297
−19.935
41.215
−72.645
1.00
175.21
C

ATOM
348
N
PHE
B
298
−19.397
38.335
−70.911
1.00
170.91
N

ATOM
349
CA
PHE
B
298
−18.893
37.678
−69.690
1.00
170.91
C

ATOM
350
C
PHE
B
298
−17.370
37.445
−69.690
1.00
170.91
C

ATOM
351
O
PHE
B
298
−16.779
37.101
−70.719
1.00
170.91
O

ATOM
352
CB
PHE
B
298
−19.607
36.337
−69.465
1.00
170.91
C

ATOM
353
CG
PHE
B
298
−19.245
35.641
−68.173
1.00
170.91
C

ATOM
354
CD1
PHE
B
298
−19.689
36.135
−66.948
1.00
170.91
C

ATOM
355
CD2
PHE
B
298
−18.448
34.505
−68.175
1.00
170.91
C

ATOM
356
CE1
PHE
B
298
−19.345
35.496
−65.744
1.00
170.91
C

ATOM
357
CE2
PHE
B
298
−18.126
33.857
−66.976
1.00
170.91
C

ATOM
358
CZ
PHE
B
298
−18.565
34.362
−65.767
1.00
170.91
C

ATOM
359
N
GLU
B
299
−16.767
37.570
−68.497
1.00
163.47
N

ATOM
360
CA
GLU
B
299
−15.335
37.446
−68.279
1.00
163.47
C

ATOM
361
C
GLU
B
299
−14.935
36.359
−67.282
1.00
163.47
C

ATOM
362
O
GLU
B
299
−15.356
36.372
−66.126
1.00
163.47
O

ATOM
363
CB
GLU
B
299
−14.770
38.782
−67.787
1.00
163.47
C

ATOM
364
N
MET
B
300
−14.049
35.462
−67.734
1.00
159.67
N

ATOM
365
CA
MET
B
300
−13.421
34.382
−66.956
1.00
159.67
C

ATOM
366
C
MET
B
300
−11.918
34.540
−67.008
1.00
159.67
C

ATOM
367
O
MET
B
300
−11.399
35.071
−67.991
1.00
159.67
O

ATOM
368
CB
MET
B
300
−13.787
32.999
−67.504
1.00
159.67
C

ATOM
369
CG
MET
B
300
−15.224
32.656
−67.377
1.00
159.67
C

ATOM
370
SD
MET
B
300
−15.589
30.997
−67.964
1.00
159.67
S

ATOM
371
CE
MET
B
300
−15.225
31.157
−69.714
1.00
159.67
C

ATOM
372
N
SER
B
301
−11.206
34.054
−65.999
1.00
155.29
N

ATOM
373
CA
SER
B
301
−9.753
34.162
−66.004
1.00
155.29
C

ATOM
374
C
SER
B
301
−9.134
33.024
−65.243
1.00
155.29
C

ATOM
375
O
SER
B
301
−9.706
32.618
−64.241
1.00
155.29
O

ATOM
376
CB
SER
B
301
−9.312
35.492
−65.399
1.00
155.29
C

ATOM
377
OG
SER
B
301
−9.744
35.631
−64.057
1.00
155.29
O

ATOM
378
N
THR
B
302
−7.988
32.480
−65.713
1.00
154.59
N

ATOM
379
CA
THR
B
302
−7.263
31.431
−64.967
1.00
154.59
C

ATOM
380
C
THR
B
302
−5.744
31.589
−65.215
1.00
154.59
C

ATOM
381
O
THR
B
302
−5.318
32.423
−66.029
1.00
154.59
O

ATOM
382
CB
THR
B
302
−7.807
29.990
−65.196
1.00
154.59
C

ATOM
383
CG2
THR
B
302
−7.310
29.345
−66.476
1.00
154.59
C

ATOM
384
OG1
THR
B
302
−7.451
29.177
−64.073
1.00
154.59
O

ATOM
385
N
TYR
B
303
−4.939
30.813
−64.492
1.00
154.51
N

ATOM
386
CA
TYR
B
303
−3.498
30.891
−64.618
1.00
154.51
C

ATOM
387
C
TYR
B
303
−2.900
29.746
−65.465
1.00
154.51
C

ATOM
388
O
TYR
B
303
−3.509
28.679
−65.613
1.00
154.51
O

ATOM
389
CB
TYR
B
303
−2.883
30.919
−63.234
1.00
154.51
C

ATOM
390
CG
TYR
B
303
−3.004
32.264
−62.561
1.00
154.51
C

ATOM
391
CD1
TYR
B
303
−1.911
33.111
−62.460
1.00
154.51
C

ATOM
392
CD2
TYR
B
303
−4.200
32.672
−61.984
1.00
154.51
C

ATOM
393
CE1
TYR
B
303
−2.003
34.334
−61.805
1.00
154.51
C

ATOM
394
CE2
TYR
B
303
−4.307
33.897
−61.334
1.00
154.51
C

ATOM
395
CZ
TYR
B
303
−3.202
34.723
−61.242
1.00
154.51
C

ATOM
396
OH
TYR
B
303
−3.291
35.928
−60.597
1.00
154.51
O

ATOM
397
N
SER
B
304
−1.703
29.997
−66.034
1.00
159.68
N

ATOM
398
CA
SER
B
304
−0.941
29.082
−66.896
1.00
159.68
C

ATOM
399
C
SER
B
304
0.554
29.023
−66.459
1.00
159.68
C

ATOM
400
O
SER
B
304
0.938
29.767
−65.551
1.00
159.68
O

ATOM
401
CB
SER
B
304
−1.068
29.531
−68.350
1.00
159.68
C

ATOM
402
OG
SER
B
304
−0.201
28.842
−69.235
1.00
159.68
O

ATOM
403
N
THR
B
305
1.377
28.133
−67.104
1.00
164.27
N

ATOM
404
CA
THR
B
305
2.824
27.856
−66.906
1.00
164.27
C

ATOM
405
C
THR
B
305
3.414
28.516
−65.639
1.00
164.27
C

ATOM
406
O
THR
B
305
3.108
28.056
−64.537
1.00
164.27
O

ATOM
407
CB
THR
B
305
3.651
28.223
−68.154
1.00
164.27
C

ATOM
408
CG2
THR
B
305
3.762
27.073
−69.126
1.00
164.27
C

ATOM
409
OG1
THR
B
305
3.093
29.367
−68.806
1.00
164.27
O

ATOM
410
N
ASN
B
306
4.237
29.575
−65.786
1.00
167.32
N

ATOM
411
CA
ASN
B
306
4.798
30.272
−64.635
1.00
167.32
C

ATOM
412
C
ASN
B
306
4.031
31.561
−64.390
1.00
167.32
C

ATOM
413
O
ASN
B
306
4.201
32.524
−65.140
1.00
167.32
O

ATOM
414
CB
ASN
B
306
6.294
30.533
−64.811
1.00
167.32
C

ATOM
415
CG
ASN
B
306
7.202
29.402
−64.374
1.00
167.32
C

ATOM
416
ND2
ASN
B
306
6.618
28.204
−64.045
1.00
167.32
N

ATOM
417
OD1
ASN
B
306
8.437
29.575
−64.351
1.00
167.32
O

ATOM
418
N
ARG
B
307
3.135
31.551
−63.372
1.00
162.29
N

ATOM
419
CA
ARG
B
307
2.278
32.669
−62.959
1.00
162.29
C

ATOM
420
C
ARG
B
307
1.769
33.506
−64.183
1.00
162.29
C

ATOM
421
O
ARG
B
307
1.696
34.735
−64.130
1.00
162.29
O

ATOM
422
CB
ARG
B
307
3.012
33.547
−61.930
1.00
162.29
C

ATOM
423
N
THR
B
308
1.423
32.798
−65.277
1.00
160.17
N

ATOM
424
CA
THR
B
308
0.907
33.314
−66.552
1.00
160.17
C

ATOM
425
C
THR
B
308
−0.611
33.484
−66.422
1.00
160.17
C

ATOM
426
O
THR
B
308
−1.225
32.808
−65.600
1.00
160.17
O

ATOM
427
CB
THR
B
308
1.290
32.341
−67.700
1.00
160.17
C

ATOM
428
CG2
THR
B
308
1.266
32.987
−69.084
1.00
160.17
C

ATOM
429
OG1
THR
B
308
2.575
31.760
−67.450
1.00
160.17
O

ATOM
430
N
MET
B
309
−1.221
34.373
−67.219
1.00
158.25
N

ATOM
431
CA
MET
B
309
−2.664
34.580
−67.130
1.00
158.25
C

ATOM
432
C
MET
B
309
−3.362
34.532
−68.484
1.00
158.25
C

ATOM
433
O
MET
B
309
−2.910
35.147
−69.461
1.00
158.25
O

ATOM
434
CB
MET
B
309
−2.981
35.901
−66.433
1.00
158.25
C

ATOM
435
CG
MET
B
309
−3.815
35.723
−65.197
1.00
158.25
C

ATOM
436
SD
MET
B
309
−5.488
36.353
−65.422
1.00
158.25
S

ATOM
437
CE
MET
B
309
−6.009
36.486
−63.724
1.00
158.25
C

ATOM
438
N
ILE
B
310
−4.471
33.780
−68.522
1.00
154.55
N

ATOM
439
CA
ILE
B
310
−5.329
33.634
−69.689
1.00
154.55
C

ATOM
440
C
ILE
B
310
−6.625
34.305
−69.337
1.00
154.55
C

ATOM
441
O
ILE
B
310
−7.145
34.065
−68.237
1.00
154.55
O

ATOM
442
CB
ILE
B
310
−5.550
32.151
−70.120
1.00
154.55
C

ATOM
443
CG1
ILE
B
310
−4.275
31.301
−70.013
1.00
154.55
C

ATOM
444
CG2
ILE
B
310
−6.172
32.056
−71.522
1.00
154.55
C

ATOM
445
CD1
ILE
B
310
−4.543
29.867
−69.601
1.00
154.55
C

ATOM
446
N
ARG
B
311
−7.151
35.147
−70.246
1.00
154.13
N

ATOM
447
CA
ARG
B
311
−8.437
35.804
−70.016
1.00
154.13
C

ATOM
448
C
ARG
B
311
−9.403
35.537
−71.166
1.00
154.13
C

ATOM
449
O
ARG
B
311
−9.041
35.687
−72.333
1.00
154.13
O

ATOM
450
CB
ARG
B
311
−8.282
37.311
−69.779
1.00
154.13
C

ATOM
451
CG
ARG
B
311
−7.849
37.678
−68.361
1.00
154.13
C

ATOM
452
CD
ARG
B
311
−8.413
39.026
−67.908
1.00
154.13
C

ATOM
453
NE
ARG
B
311
−7.549
39.711
−66.931
1.00
154.13
N

ATOM
454
CZ
ARG
B
311
−7.690
39.653
−65.606
1.00
154.13
C

ATOM
455
NH1
ARG
B
311
−8.666
38.931
−65.064
1.00
154.13
N + 1

ATOM
456
NH2
ARG
B
311
−6.851
40.308
−64.814
1.00
154.13
N

ATOM
457
N
ILE
B
312
−10.618
35.103
−70.822
1.00
154.45
N

ATOM
458
CA
ILE
B
312
−11.697
34.838
−71.764
1.00
154.45
C

ATOM
459
C
ILE
B
312
−12.723
35.931
−71.604
1.00
154.45
C

ATOM
460
O
ILE
B
312
−13.346
36.019
−70.547
1.00
154.45
O

ATOM
461
CB
ILE
B
312
−12.357
33.437
−71.573
1.00
154.45
C

ATOM
462
CG1
ILE
B
312
−11.356
32.281
−71.700
1.00
154.45
C

ATOM
463
CG2
ILE
B
312
−13.573
33.247
−72.516
1.00
154.45
C

ATOM
464
CD1
ILE
B
312
−11.974
30.898
−71.430
1.00
154.45
C

ATOM
465
N
LEU
B
313
−12.932
36.744
−72.637
1.00
158.55
N

ATOM
466
CA
LEU
B
313
−13.961
37.776
−72.586
1.00
158.55
C

ATOM
467
C
LEU
B
313
−14.915
37.534
−73.731
1.00
158.55
C

ATOM
468
O
LEU
B
313
−14.729
38.098
−74.809
1.00
158.55
O

ATOM
469
CB
LEU
B
313
−13.345
39.174
−72.646
1.00
158.55
C

ATOM
470
CG
LEU
B
313
−14.212
40.276
−72.049
1.00
158.55
C

ATOM
471
CD1
LEU
B
313
−13.637
40.772
−70.723
1.00
158.55
C

ATOM
472
CD2
LEU
B
313
−14.356
41.438
−73.007
1.00
158.55
C

ATOM
473
N
PHE
B
314
−15.885
36.626
−73.541
1.00
162.00
N

ATOM
474
CA
PHE
B
314
−16.769
36.316
−74.658
1.00
162.00
C

ATOM
475
C
PHE
B
314
−17.975
37.225
−74.690
1.00
162.00
C

ATOM
476
O
PHE
B
314
−18.410
37.727
−73.659
1.00
162.00
O

ATOM
477
CB
PHE
B
314
−17.196
34.837
−74.699
1.00
162.00
C

ATOM
478
CG
PHE
B
314
−17.853
34.281
−73.468
1.00
162.00
C

ATOM
479
CD1
PHE
B
314
−19.193
34.539
−73.198
1.00
162.00
C

ATOM
480
CD2
PHE
B
314
−17.159
33.436
−72.615
1.00
162.00
C

ATOM
481
CE1
PHE
B
314
−19.809
34.012
−72.058
1.00
162.00
C

ATOM
482
CE2
PHE
B
314
−17.774
32.909
−71.475
1.00
162.00
C

ATOM
483
CZ
PHE
B
314
−19.099
33.192
−71.208
1.00
162.00
C

ATOM
484
N
ALA
B
315
−18.485
37.460
−75.893
1.00
165.31
N

ATOM
485
CA
ALA
B
315
−19.677
38.256
−76.139
1.00
165.31
C

ATOM
486
C
ALA
B
315
−20.733
37.353
−76.703
1.00
165.31
C

ATOM
487
O
ALA
B
315
−20.407
36.457
−77.489
1.00
165.31
O

ATOM
488
CB
ALA
B
315
−19.369
39.383
−77.103
1.00
165.31
C

ATOM
489
N
PHE
B
316
−21.993
37.549
−76.300
1.00
170.05
N

ATOM
490
CA
PHE
B
316
−23.037
36.673
−76.805
1.00
170.05
C

ATOM
491
C
PHE
B
316
−24.387
37.395
−76.959
1.00
170.05
C

ATOM
492
O
PHE
B
316
−24.750
38.202
−76.109
1.00
170.05
O

ATOM
493
CB
PHE
B
316
−23.142
35.423
−75.900
1.00
170.05
C

ATOM
494
CG
PHE
B
316
−24.447
35.145
−75.196
1.00
170.05
C

ATOM
495
CD1
PHE
B
316
−25.460
34.421
−75.829
1.00
170.05
C

ATOM
496
CD2
PHE
B
316
−24.658
35.581
−73.889
1.00
170.05
C

ATOM
497
CE1
PHE
B
316
−26.665
34.139
−75.169
1.00
170.05
C

ATOM
498
CE2
PHE
B
316
−25.869
35.314
−73.230
1.00
170.05
C

ATOM
499
CZ
PHE
B
316
−26.865
34.591
−73.874
1.00
170.05
C

ATOM
500
N
VAL
B
317
−25.131
37.082
−78.042
1.00
169.64
N

ATOM
501
CA
VAL
B
317
−26.475
37.627
−78.302
1.00
169.64
C

ATOM
502
C
VAL
B
317
−27.478
36.488
−78.167
1.00
169.64
C

ATOM
503
O
VAL
B
317
−27.303
35.456
−78.816
1.00
169.64
O

ATOM
504
CB
VAL
B
317
−26.594
38.330
−79.674
1.00
169.64
C

ATOM
505
CG1
VAL
B
317
−28.008
38.852
−79.902
1.00
169.64
C

ATOM
506
CG2
VAL
B
317
−25.577
39.455
−79.799
1.00
169.64
C

ATOM
507
N
SER
B
318
−28.506
36.667
−77.317
1.00
170.99
N

ATOM
508
CA
SER
B
318
−29.536
35.665
−77.046
1.00
170.99
C

ATOM
509
C
SER
B
318
−30.198
35.224
−78.341
1.00
170.99
C

ATOM
510
O
SER
B
318
−30.098
34.056
−78.711
1.00
170.99
O

ATOM
511
CB
SER
B
318
−30.568
36.213
−76.065
1.00
170.99
C

ATOM
512
N
SER
B
319
−30.823
36.169
−79.046
1.00
173.70
N

ATOM
513
CA
SER
B
319
−31.464
35.957
−80.339
1.00
173.70
C

ATOM
514
C
SER
B
319
−31.083
37.128
−81.228
1.00
173.70
C

ATOM
515
O
SER
B
319
−31.377
38.273
−80.865
1.00
173.70
O

ATOM
516
CB
SER
B
319
−32.979
35.829
−80.187
1.00
173.70
C

ATOM
517
N
VAL
B
320
−30.361
36.862
−82.351
1.00
175.37
N

ATOM
518
CA
VAL
B
320
−29.890
37.923
−83.262
1.00
175.37
C

ATOM
519
C
VAL
B
320
−31.024
38.488
−84.135
1.00
175.37
C

ATOM
520
O
VAL
B
320
−32.059
37.846
−84.318
1.00
175.37
O

ATOM
521
CB
VAL
B
320
−28.678
37.533
−84.146
1.00
175.37
C

ATOM
522
CG1
VAL
B
320
−27.453
37.235
−83.300
1.00
175.37
C

ATOM
523
CG2
VAL
B
320
−29.002
36.383
−85.092
1.00
175.37
C

ATOM
524
N
ALA
B
321
−30.802
39.703
−84.671
1.00
178.14
N

ATOM
525
CA
ALA
B
321
−31.713
40.430
−85.565
1.00
178.14
C

ATOM
526
C
ALA
B
321
−30.905
41.352
−86.518
1.00
178.14
C

ATOM
527
O
ALA
B
321
−29.697
41.153
−86.666
1.00
178.14
O

ATOM
528
CB
ALA
B
321
−32.723
41.231
−84.750
1.00
178.14
C

ATOM
529
N
ARG
B
322
−31.563
42.335
−87.170
1.00
178.72
N

ATOM
530
CA
ARG
B
322
−30.913
43.280
−88.079
1.00
178.72
C

ATOM
531
C
ARG
B
322
−29.890
44.145
−87.344
1.00
178.72
C

ATOM
532
O
ARG
B
322
−28.777
44.316
−87.841
1.00
178.72
O

ATOM
533
CB
ARG
B
322
−31.961
44.172
−88.753
1.00
178.72
C

ATOM
534
N
ASN
B
323
−30.269
44.645
−86.141
1.00
181.16
N

ATOM
535
CA
ASN
B
323
−29.494
45.513
−85.237
1.00
181.16
C

ATOM
536
C
ASN
B
323
−28.150
44.907
−84.783
1.00
181.16
C

ATOM
537
O
ASN
B
323
−27.210
45.670
−84.527
1.00
181.16
O

ATOM
538
CB
ASN
B
323
−30.332
45.881
−83.987
1.00
181.16
C

ATOM
539
CG
ASN
B
323
−31.599
46.651
−84.304
1.00
181.16
C

ATOM
540
ND2
ASN
B
323
−32.422
46.993
−83.338
1.00
181.16
N

ATOM
541
OD1
ASN
B
323
−31.933
46.897
−85.460
1.00
181.16
O

ATOM
542
N
ASP
B
324
−28.061
43.554
−84.685
1.00
179.55
N

ATOM
543
CA
ASP
B
324
−26.871
42.812
−84.229
1.00
179.55
C

ATOM
544
C
ASP
B
324
−25.792
42.602
−85.330
1.00
179.55
C

ATOM
545
O
ASP
B
324
−24.635
42.334
−84.980
1.00
179.55
O

ATOM
546
CB
ASP
B
324
−27.275
41.463
−83.617
1.00
179.55
C

ATOM
547
CG
ASP
B
324
−28.237
41.604
−82.455
1.00
179.55
C

ATOM
548
OD1
ASP
B
324
−27.903
42.327
−81.488
1.00
179.55
O

ATOM
549
OD2
ASP
B
324
−29.331
41.025
−82.525
1.00
179.55
O + 1

ATOM
550
N
THR
B
325
−26.152
42.744
−86.635
1.00
175.46
N

ATOM
551
CA
THR
B
325
−25.201
42.640
−87.750
1.00
175.46
C

ATOM
552
C
THR
B
325
−24.241
43.864
−87.708
1.00
175.46
C

ATOM
553
O
THR
B
325
−24.717
45.002
−87.583
1.00
175.46
O

ATOM
554
CB
THR
B
325
−25.957
42.532
−89.079
1.00
175.46
C

ATOM
555
N
GLY
B
326
−22.918
43.614
−87.743
1.00
171.84
N

ATOM
556
CA
GLY
B
326
−21.892
44.663
−87.701
1.00
171.84
C

ATOM
557
C
GLY
B
326
−20.437
44.241
−87.539
1.00
171.84
C

ATOM
558
O
GLY
B
326
−20.130
43.053
−87.469
1.00
171.84
O

ATOM
559
N
TYR
B
327
−19.525
45.229
−87.479
1.00
168.24
N

ATOM
560
CA
TYR
B
327
−18.086
45.021
−87.300
1.00
168.24
C

ATOM
561
C
TYR
B
327
−17.723
44.987
−85.804
1.00
168.24
C

ATOM
562
O
TYR
B
327
−17.661
46.038
−85.157
1.00
168.24
O

ATOM
563
CB
TYR
B
327
−17.289
46.115
−88.028
1.00
168.24
C

ATOM
564
N
TYR
B
328
−17.492
43.777
−85.256
1.00
164.98
N

ATOM
565
CA
TYR
B
328
−17.159
43.565
−83.839
1.00
164.98
C

ATOM
566
C
TYR
B
328
−15.663
43.482
−83.623
1.00
164.98
C

ATOM
567
O
TYR
B
328
−14.964
42.824
−84.394
1.00
164.98
O

ATOM
568
CB
TYR
B
328
−17.816
42.283
−83.301
1.00
164.98
C

ATOM
569
CG
TYR
B
328
−19.318
42.351
−83.109
1.00
164.98
C

ATOM
570
CD1
TYR
B
328
−19.880
42.281
−81.840
1.00
164.98
C

ATOM
571
CD2
TYR
B
328
−20.181
42.398
−84.203
1.00
164.98
C

ATOM
572
CE1
TYR
B
328
−21.263
42.296
−81.660
1.00
164.98
C

ATOM
573
CE2
TYR
B
328
−21.564
42.417
−84.034
1.00
164.98
C

ATOM
574
CZ
TYR
B
328
−22.100
42.355
−82.762
1.00
164.98
C

ATOM
575
OH
TYR
B
328
−23.460
42.388
−82.594
1.00
164.98
O

ATOM
576
N
THR
B
329
−15.169
44.122
−82.562
1.00
162.52
N

ATOM
577
CA
THR
B
329
−13.740
44.089
−82.283
1.00
162.52
C

ATOM
578
C
THR
B
329
−13.439
43.917
−80.807
1.00
162.52
C

ATOM
579
O
THR
B
329
−14.062
44.547
−79.938
1.00
162.52
O

ATOM
580
CB
THR
B
329
−13.014
45.319
−82.840
1.00
162.52
C

ATOM
581
CG2
THR
B
329
−13.525
46.641
−82.259
1.00
162.52
C

ATOM
582
OG1
THR
B
329
−11.607
45.175
−82.601
1.00
162.52
O

ATOM
583
N
CYS
B
330
−12.426
43.085
−80.550
1.00
158.77
N

ATOM
584
CA
CYS
B
330
−11.939
42.796
−79.213
1.00
158.77
C

ATOM
585
C
CYS
B
330
−10.555
43.417
−79.027
1.00
158.77
C

ATOM
586
O
CYS
B
330
−9.658
43.178
−79.844
1.00
158.77
O

ATOM
587
CB
CYS
B
330
−11.919
41.292
−78.960
1.00
158.77
C

ATOM
588
SG
CYS
B
330
−11.389
40.836
−77.291
1.00
158.77
S

ATOM
589
N
SER
B
331
−10.386
44.217
−77.960
1.00
160.47
N

ATOM
590
CA
SER
B
331
−9.110
44.858
−77.660
1.00
160.47
C

ATOM
591
C
SER
B
331
−8.725
44.620
−76.212
1.00
160.47
C

ATOM
592
O
SER
B
331
−9.559
44.784
−75.320
1.00
160.47
O

ATOM
593
CB
SER
B
331
−9.174
46.351
−77.956
1.00
160.47
C

ATOM
594
N
SER
B
332
−7.465
44.213
−75.983
1.00
162.45
N

ATOM
595
CA
SER
B
332
−6.937
43.960
−74.643
1.00
162.45
C

ATOM
596
C
SER
B
332
−5.987
45.080
−74.205
1.00
162.45
C

ATOM
597
O
SER
B
332
−5.417
45.779
−75.049
1.00
162.45
O

ATOM
598
CB
SER
B
332
−6.243
42.605
−74.574
1.00
162.45
C

ATOM
599
N
SER
B
333
−5.851
45.265
−72.873
1.00
163.81
N

ATOM
600
CA
SER
B
333
−5.009
46.301
−72.269
1.00
163.81
C

ATOM
601
C
SER
B
333
−3.552
46.123
−72.679
1.00
163.81
C

ATOM
602
O
SER
B
333
−2.955
47.053
−73.222
1.00
163.81
O

ATOM
603
CB
SER
B
333
−5.140
46.296
−70.747
1.00
163.81
C

ATOM
604
N
LYS
B
334
−2.998
44.925
−72.473
1.00
164.27
N

ATOM
605
CA
LYS
B
334
−1.620
44.667
−72.849
1.00
164.27
C

ATOM
606
C
LYS
B
334
−1.572
43.831
−74.146
1.00
164.27
C

ATOM
607
O
LYS
B
334
−0.676
42.993
−74.286
1.00
164.27
O

ATOM
608
CB
LYS
B
334
−0.862
43.995
−71.687
1.00
164.27
C

ATOM
609
N
HIS
B
335
−2.515
44.076
−75.111
1.00
165.44
N

ATOM
610
CA
HIS
B
335
−2.561
43.346
−76.394
1.00
165.44
C

ATOM
611
C
HIS
B
335
−3.158
44.162
−77.573
1.00
165.44
C

ATOM
612
O
HIS
B
335
−3.831
45.167
−77.325
1.00
165.44
O

ATOM
613
CB
HIS
B
335
−3.327
42.021
−76.244
1.00
165.44
C

ATOM
614
CG
HIS
B
335
−2.530
40.991
−75.524
1.00
165.44
C

ATOM
615
CD2
HIS
B
335
−2.742
40.427
−74.314
1.00
165.44
C

ATOM
616
ND1
HIS
B
335
−1.315
40.547
−76.020
1.00
165.44
N

ATOM
617
CE1
HIS
B
335
−0.844
39.707
−75.115
1.00
165.44
C

ATOM
618
NE2
HIS
B
335
−1.673
39.594
−74.074
1.00
165.44
N

ATOM
619
N
PRO
B
336
−2.926
43.745
−78.859
1.00
165.68
N

ATOM
620
CA
PRO
B
336
−3.482
44.508
−79.998
1.00
165.68
C

ATOM
621
C
PRO
B
336
−4.900
44.085
−80.406
1.00
165.68
C

ATOM
622
O
PRO
B
336
−5.199
42.888
−80.453
1.00
165.68
O

ATOM
623
CB
PRO
B
336
−2.499
44.208
−81.127
1.00
165.68
C

ATOM
624
CG
PRO
B
336
−1.970
42.838
−80.815
1.00
165.68
C

ATOM
625
CD
PRO
B
336
−2.124
42.592
−79.334
1.00
165.68
C

ATOM
626
N
SER
B
337
−5.755
45.069
−80.748
1.00
165.23
N

ATOM
627
CA
SER
B
337
−7.141
44.836
−81.162
1.00
165.23
C

ATOM
628
C
SER
B
337
−7.230
43.949
−82.422
1.00
165.23
C

ATOM
629
O
SER
B
337
−6.367
44.054
−83.298
1.00
165.23
O

ATOM
630
CB
SER
B
337
−7.849
46.162
−81.417
1.00
165.23
C

ATOM
631
N
GLN
B
338
−8.252
43.051
−82.484
1.00
164.33
N

ATOM
632
CA
GLN
B
338
−8.537
42.151
−83.626
1.00
164.33
C

ATOM
633
C
GLN
B
338
−10.037
42.126
−83.876
1.00
164.33
C

ATOM
634
O
GLN
B
338
−10.828
42.212
−82.926
1.00
164.33
O

ATOM
635
CB
GLN
B
338
−7.986
40.736
−83.408
1.00
164.33
C

ATOM
636
N
SER
B
339
−10.439
42.040
−85.142
1.00
162.18
N

ATOM
637
CA
SER
B
339
−11.858
42.150
−85.424
1.00
162.18
C

ATOM
638
C
SER
B
339
−12.419
41.085
−86.341
1.00
162.18
C

ATOM
639
O
SER
B
339
−11.687
40.443
−87.090
1.00
162.18
O

ATOM
640
CB
SER
B
339
−12.147
43.512
−86.038
1.00
162.18
C

ATOM
641
OG
SER
B
339
−11.268
44.499
−85.524
1.00
162.18
O

ATOM
642
N
ALA
B
340
−13.757
40.941
−86.288
1.00
163.31
N

ATOM
643
CA
ALA
B
340
−14.585
40.029
−87.075
1.00
163.31
C

ATOM
644
C
ALA
B
340
−15.936
40.678
−87.355
1.00
163.31
C

ATOM
645
O
ALA
B
340
−16.484
41.354
−86.484
1.00
163.31
O

ATOM
646
CB
ALA
B
340
−14.772
38.715
−86.338
1.00
163.31
C

ATOM
647
N
LEU
B
341
−16.474
40.495
−88.560
1.00
165.75
N

ATOM
648
CA
LEU
B
341
−17.734
41.148
−88.908
1.00
165.75
C

ATOM
649
C
LEU
B
341
−18.880
40.163
−88.996
1.00
165.75
C

ATOM
650
O
LEU
B
341
−18.867
39.253
−89.827
1.00
165.75
O

ATOM
651
CB
LEU
B
341
−17.604
41.937
−90.217
1.00
165.75
C

ATOM
652
CG
LEU
B
341
−16.809
43.254
−90.143
1.00
165.75
C

ATOM
653
CD1
LEU
B
341
−15.323
43.055
−90.486
1.00
165.75
C

ATOM
654
CD2
LEU
B
341
−17.415
44.309
−91.052
1.00
165.75
C

ATOM
655
N
VAL
B
342
−19.879
40.368
−88.125
1.00
166.66
N

ATOM
656
CA
VAL
B
342
−21.097
39.569
−87.986
1.00
166.66
C

ATOM
657
C
VAL
B
342
−22.113
39.962
−89.078
1.00
166.66
C

ATOM
658
O
VAL
B
342
−22.572
41.106
−89.131
1.00
166.66
O

ATOM
659
CB
VAL
B
342
−21.692
39.698
−86.555
1.00
166.66
C

ATOM
660
CG1
VAL
B
342
−23.050
39.015
−86.444
1.00
166.66
C

ATOM
661
CG2
VAL
B
342
−20.734
39.133
−85.518
1.00
166.66
C

ATOM
662
N
THR
B
343
−22.452
38.991
−89.936
1.00
167.53
N

ATOM
663
CA
THR
B
343
−23.398
39.132
−91.025
1.00
167.53
C

ATOM
664
C
THR
B
343
−24.585
38.222
−90.784
1.00
167.53
C

ATOM
665
O
THR
B
343
−24.459
37.011
−90.546
1.00
167.53
O

ATOM
666
CB
THR
B
343
−22.728
38.864
−92.354
1.00
167.53
C

ATOM
667
N
ILE
B
344
−25.748
38.829
−90.837
1.00
171.98
N

ATOM
668
CA
ILE
B
344
−27.006
38.154
−90.615
1.00
171.98
C

ATOM
669
C
ILE
B
344
−27.527
37.638
−91.972
1.00
171.98
C

ATOM
670
O
ILE
B
344
−27.315
38.273
−93.006
1.00
171.98
O

ATOM
671
CB
ILE
B
344
−27.953
39.140
−89.854
1.00
171.98
C

ATOM
672
CG1
ILE
B
344
−28.166
38.722
−88.384
1.00
171.98
C

ATOM
673
CG2
ILE
B
344
−29.259
39.467
−90.569
1.00
171.98
C

ATOM
674
CD1
ILE
B
344
−27.075
39.243
−87.392
1.00
171.98
C

ATOM
675
N
VAL
B
345
−28.144
36.454
−91.966
1.00
176.21
N

ATOM
676
CA
VAL
B
345
−28.704
35.867
−93.185
1.00
176.21
C

ATOM
677
C
VAL
B
345
−30.200
35.563
−92.999
1.00
176.21
C

ATOM
678
O
VAL
B
345
−30.677
35.276
−91.871
1.00
176.21
O

ATOM
679
CB
VAL
B
345
−27.939
34.632
−93.748
1.00
176.21
C

ATOM
680
CG1
VAL
B
345
−26.675
35.055
−94.480
1.00
176.21
C

ATOM
681
CG2
VAL
B
345
−27.633
33.587
−92.674
1.00
176.21
C

ATOM
682
N
GLU
B
346
−30.925
35.677
−94.135
1.00
178.80
N

ATOM
683
CA
GLU
B
346
−32.356
35.436
−94.288
1.00
178.80
C

ATOM
684
C
GLU
B
346
−32.610
33.983
−94.695
1.00
178.80
C

ATOM
685
O
GLU
B
346
−33.632
33.409
−94.314
1.00
178.80
O

ATOM
686
CB
GLU
B
346
−32.953
36.401
−95.323
1.00
178.80
C

TER
687

GLU
B
346

ATOM
688
N
THR
A
1
5.321
54.347
−52.494
1.00
185.01
N

ATOM
689
CA
THR
A
1
5.559
52.904
−52.695
1.00
185.01
C

ATOM
690
C
THR
A
1
4.970
52.120
−51.488
1.00
185.01
C

ATOM
691
O
THR
A
1
5.217
50.918
−51.336
1.00
185.01
O

ATOM
692
CB
THR
A
1
7.056
52.621
−52.911
1.00
185.01
C

ATOM
693
N
GLN
A
2
4.183
52.835
−50.641
1.00
180.26
N

ATOM
694
CA
GLN
A
2
3.446
52.356
−49.469
1.00
180.26
C

ATOM
695
C
GLN
A
2
1.944
52.225
−49.832
1.00
180.26
C

ATOM
696
O
GLN
A
2
1.080
52.324
−48.959
1.00
180.26
O

ATOM
697
CB
GLN
A
2
3.665
53.304
−48.267
1.00
180.26
C

ATOM
698
N
ASP
A
3
1.646
52.010
−51.131
1.00
174.34
N

ATOM
699
CA
ASP
A
3
0.285
51.826
−51.612
1.00
174.34
C

ATOM
700
C
ASP
A
3
0.061
50.363
−51.975
1.00
174.34
C

ATOM
701
O
ASP
A
3
1.013
49.581
−52.010
1.00
174.34
O

ATOM
702
CB
ASP
A
3
−0.025
52.747
−52.792
1.00
174.34
C

ATOM
703
N
CYS
A
4
−1.205
49.987
−52.205
1.00
170.58
N

ATOM
704
CA
CYS
A
4
−1.605
48.619
−52.524
1.00
170.58
C

ATOM
705
C
CYS
A
4
−2.976
48.619
−53.170
1.00
170.58
C

ATOM
706
O
CYS
A
4
−3.971
48.863
−52.498
1.00
170.58
O

ATOM
707
CB
CYS
A
4
−1.593
47.758
−51.261
1.00
170.58
C

ATOM
708
SG
CYS
A
4
−2.029
46.023
−51.540
1.00
170.58
S

ATOM
709
N
SER
A
5
−3.030
48.351
−54.465
1.00
169.59
N

ATOM
710
CA
SER
A
5
−4.276
48.306
−55.223
1.00
169.59
C

ATOM
711
C
SER
A
5
−4.051
47.507
−56.482
1.00
169.59
C

ATOM
712
O
SER
A
5
−2.911
47.415
−56.947
1.00
169.59
O

ATOM
713
CB
SER
A
5
−4.740
49.713
−55.580
1.00
169.59
C

ATOM
714
OG
SER
A
5
−3.864
50.295
−56.533
1.00
169.59
O

ATOM
715
N
PHE
A
6
−5.124
46.944
−57.050
1.00
166.76
N

ATOM
716
CA
PHE
A
6
−5.011
46.128
−58.253
1.00
166.76
C

ATOM
717
C
PHE
A
6
−5.940
46.647
−59.320
1.00
166.76
C

ATOM
718
O
PHE
A
6
−7.147
46.740
−59.092
1.00
166.76
O

ATOM
719
CB
PHE
A
6
−5.313
44.657
−57.931
1.00
166.76
C

ATOM
720
CG
PHE
A
6
−4.504
44.124
−56.772
1.00
166.76
C

ATOM
721
CD1
PHE
A
6
−3.361
43.363
−56.990
1.00
166.76
C

ATOM
722
CD2
PHE
A
6
−4.860
44.421
−55.459
1.00
166.76
C

ATOM
723
CE1
PHE
A
6
−2.594
42.897
−55.915
1.00
166.76
C

ATOM
724
CE2
PHE
A
6
−4.089
43.968
−54.388
1.00
166.76
C

ATOM
725
CZ
PHE
A
6
−2.964
43.204
−54.622
1.00
166.76
C

ATOM
726
N
GLN
A
7
−5.374
46.987
−60.497
1.00
163.88
N

ATOM
727
CA
GLN
A
7
−6.110
47.520
−61.655
1.00
163.88
C

ATOM
728
C
GLN
A
7
−6.991
46.436
−62.292
1.00
163.88
C

ATOM
729
O
GLN
A
7
−8.166
46.688
−62.565
1.00
163.88
O

ATOM
730
CB
GLN
A
7
−5.140
48.116
−62.699
1.00
163.88
C

ATOM
731
N
HIS
A
8
−6.422
45.235
−62.505
1.00
159.70
N

ATOM
732
CA
HIS
A
8
−7.101
44.085
−63.095
1.00
159.70
C

ATOM
733
C
HIS
A
8
−7.144
42.968
−62.064
1.00
159.70
C

ATOM
734
O
HIS
A
8
−6.228
42.877
−61.249
1.00
159.70
O

ATOM
735
CB
HIS
A
8
−6.393
43.655
−64.389
1.00
159.70
C

ATOM
736
CG
HIS
A
8
−6.235
44.774
−65.383
1.00
159.70
C

ATOM
737
CD2
HIS
A
8
−5.180
45.598
−65.604
1.00
159.70
C

ATOM
738
ND1
HIS
A
8
−7.268
45.136
−66.237
1.00
159.70
N

ATOM
739
CE1
HIS
A
8
−6.801
46.147
−66.957
1.00
159.70
C

ATOM
740
NE2
HIS
A
8
−5.547
46.453
−66.621
1.00
159.70
N

ATOM
741
N
SER
A
9
−8.228
42.155
−62.074
1.00
154.92
N

ATOM
742
CA
SER
A
9
−8.541
41.065
−61.129
1.00
154.92
C

ATOM
743
C
SER
A
9
−7.425
40.033
−60.991
1.00
154.92
C

ATOM
744
O
SER
A
9
−7.248
39.212
−61.895
1.00
154.92
O

ATOM
745
CB
SER
A
9
−9.830
40.353
−61.531
1.00
154.92
C

ATOM
746
N
PRO
A
10
−6.686
40.027
−59.855
1.00
150.62
N

ATOM
747
CA
PRO
A
10
−5.606
39.043
−59.689
1.00
150.62
C

ATOM
748
C
PRO
A
10
−6.133
37.708
−59.186
1.00
150.62
C

ATOM
749
O
PRO
A
10
−5.368
36.748
−59.045
1.00
150.62
O

ATOM
750
CB
PRO
A
10
−4.690
39.707
−58.667
1.00
150.62
C

ATOM
751
CG
PRO
A
10
−5.594
40.487
−57.822
1.00
150.62
C

ATOM
752
CD
PRO
A
10
−6.765
40.918
−58.683
1.00
150.62
C

ATOM
753
N
ILE
A
11
−7.441
37.660
−58.911
1.00
146.11
N

ATOM
754
CA
ILE
A
11
−8.132
36.464
−58.472
1.00
146.11
C

ATOM
755
C
ILE
A
11
−8.739
35.810
−59.711
1.00
146.11
C

ATOM
756
O
ILE
A
11
−9.515
36.446
−60.433
1.00
146.11
O

ATOM
757
CB
ILE
A
11
−9.172
36.820
−57.395
1.00
146.11
C

ATOM
758
CG1
ILE
A
11
−8.481
37.471
−56.186
1.00
146.11
C

ATOM
759
CG2
ILE
A
11
−9.972
35.580
−56.987
1.00
146.11
C

ATOM
760
CD1
ILE
A
11
−9.327
38.396
−55.412
1.00
146.11
C

ATOM
761
N
SER
A
12
−8.347
34.555
−59.977
1.00
142.13
N

ATOM
762
CA
SER
A
12
−8.834
33.808
−61.129
1.00
142.13
C

ATOM
763
C
SER
A
12
−10.236
33.272
−60.869
1.00
142.13
C

ATOM
764
O
SER
A
12
−10.688
33.277
−59.728
1.00
142.13
O

ATOM
765
CB
SER
A
12
−7.889
32.657
−61.448
1.00
142.13
C

ATOM
766
OG
SER
A
12
−8.167
31.538
−60.624
1.00
142.13
O

ATOM
767
N
SER
A
13
−10.921
32.805
−61.924
1.00
139.08
N

ATOM
768
CA
SER
A
13
−12.252
32.214
−61.818
1.00
139.08
C

ATOM
769
C
SER
A
13
−12.118
30.762
−61.319
1.00
139.08
C

ATOM
770
O
SER
A
13
−12.832
30.356
−60.402
1.00
139.08
O

ATOM
771
CB
SER
A
13
−12.990
32.270
−63.161
1.00
139.08
C

ATOM
772
OG
SER
A
13
−13.289
33.582
−63.611
1.00
139.08
O

ATOM
773
N
ASP
A
14
−11.156
30.009
−61.881
1.00
137.46
N

ATOM
774
CA
ASP
A
14
−10.945
28.594
−61.600
1.00
137.46
C

ATOM
775
C
ASP
A
14
−9.926
28.348
−60.490
1.00
137.46
C

ATOM
776
O
ASP
A
14
−9.169
27.378
−60.566
1.00
137.46
O

ATOM
777
CB
ASP
A
14
−10.510
27.875
−62.888
1.00
137.46
C

ATOM
778
N
PHE
A
15
−9.926
29.185
−59.443
1.00
135.86
N

ATOM
779
CA
PHE
A
15
−8.999
29.006
−58.323
1.00
135.86
C

ATOM
780
C
PHE
A
15
−9.483
27.901
−57.394
1.00
135.86
C

ATOM
781
O
PHE
A
15
−8.676
27.258
−56.722
1.00
135.86
O

ATOM
782
CB
PHE
A
15
−8.832
30.317
−57.542
1.00
135.86
C

ATOM
783
CG
PHE
A
15
−10.051
30.729
−56.756
1.00
135.86
C

ATOM
784
CD1
PHE
A
15
−10.346
30.136
−55.533
1.00
135.86
C

ATOM
785
CD2
PHE
A
15
−10.900
31.713
−57.230
1.00
135.86
C

ATOM
786
CE1
PHE
A
15
−11.479
30.508
−54.807
1.00
135.86
C

ATOM
787
CE2
PHE
A
15
−12.023
32.101
−56.495
1.00
135.86
C

ATOM
788
CZ
PHE
A
15
−12.301
31.498
−55.285
1.00
135.86
C

ATOM
789
N
ALA
A
16
−10.811
27.707
−57.337
1.00
133.89
N

ATOM
790
CA
ALA
A
16
−11.448
26.716
−56.479
1.00
133.89
C

ATOM
791
C
ALA
A
16
−11.138
25.319
−56.977
1.00
133.89
C

ATOM
792
O
ALA
A
16
−11.240
24.344
−56.235
1.00
133.89
O

ATOM
793
CB
ALA
A
16
−12.949
26.950
−56.459
1.00
133.89
C

ATOM
794
N
VAL
A
17
−10.740
25.231
−58.233
1.00
130.93
N

ATOM
795
CA
VAL
A
17
−10.433
23.967
−58.841
1.00
130.93
C

ATOM
796
C
VAL
A
17
−9.027
23.551
−58.457
1.00
130.93
C

ATOM
797
O
VAL
A
17
−8.817
22.388
−58.131
1.00
130.93
O

ATOM
798
CB
VAL
A
17
−10.676
24.000
−60.361
1.00
130.93
C

ATOM
799
CG1
VAL
A
17
−11.739
25.029
−60.746
1.00
130.93
C

ATOM
800
CG2
VAL
A
17
−9.407
24.164
−61.184
1.00
130.93
C

ATOM
801
N
LYS
A
18
−8.081
24.504
−58.437
1.00
132.98
N

ATOM
802
CA
LYS
A
18
−6.692
24.233
−58.081
1.00
132.98
C

ATOM
803
C
LYS
A
18
−6.570
23.852
−56.616
1.00
132.98
C

ATOM
804
O
LYS
A
18
−5.620
23.165
−56.245
1.00
132.98
O

ATOM
805
CB
LYS
A
18
−5.809
25.450
−58.382
1.00
132.98
C

ATOM
806
N
ILE
A
19
−7.535
24.274
−55.792
1.00
135.42
N

ATOM
807
CA
ILE
A
19
−7.520
23.990
−54.361
1.00
135.42
C

ATOM
808
C
ILE
A
19
−8.127
22.624
−54.103
1.00
135.42
C

ATOM
809
O
ILE
A
19
−7.710
21.933
−53.174
1.00
135.42
O

ATOM
810
CB
ILE
A
19
−8.224
25.098
−53.552
1.00
135.42
C

ATOM
811
CG1
ILE
A
19
−7.597
26.455
−53.861
1.00
135.42
C

ATOM
812
CG2
ILE
A
19
−8.150
24.809
−52.044
1.00
135.42
C

ATOM
813
CD1
ILE
A
19
−8.245
27.604
−53.171
1.00
135.42
C

ATOM
814
N
ARG
A
20
−9.111
22.235
−54.917
1.00
141.42
N

ATOM
815
CA
ARG
A
20
−9.749
20.934
−54.794
1.00
141.42
C

ATOM
816
C
ARG
A
20
−8.778
19.888
−55.295
1.00
141.42
C

ATOM
817
O
ARG
A
20
−8.769
18.768
−54.797
1.00
141.42
O

ATOM
818
CB
ARG
A
20
−11.068
20.896
−55.581
1.00
141.42
C

ATOM
819
N
GLU
A
21
−7.935
20.270
−56.260
1.00
145.72
N

ATOM
820
CA
GLU
A
21
−6.944
19.373
−56.826
1.00
145.72
C

ATOM
821
C
GLU
A
21
−5.814
19.152
−55.849
1.00
145.72
C

ATOM
822
O
GLU
A
21
−5.315
18.034
−55.759
1.00
145.72
O

ATOM
823
CB
GLU
A
21
−6.420
19.916
−58.148
1.00
145.72
C

ATOM
824
CG
GLU
A
21
−7.133
19.312
−59.346
1.00
145.72
C

ATOM
825
CD
GLU
A
21
−7.273
20.227
−60.555
1.00
145.72
C

ATOM
826
OE1
GLU
A
21
−6.230
20.699
−61.072
1.00
145.72
O

ATOM
827
OE2
GLU
A
21
−8.424
20.449
−61.007
1.00
145.72
O + 1

ATOM
828
N
LEU
A
22
−5.415
20.201
−55.110
1.00
147.14
N

ATOM
829
CA
LEU
A
22
−4.363
20.083
−54.107
1.00
147.14
C

ATOM
830
C
LEU
A
22
−4.871
19.221
−52.977
1.00
147.14
C

ATOM
831
O
LEU
A
22
−4.189
18.292
−52.553
1.00
147.14
O

ATOM
832
CB
LEU
A
22
−3.935
21.461
−53.585
1.00
147.14
C

ATOM
833
CG
LEU
A
22
−3.155
21.473
−52.275
1.00
147.14
C

ATOM
834
CD1
LEU
A
22
−1.800
20.894
−52.445
1.00
147.14
C

ATOM
835
CD2
LEU
A
22
−3.012
22.852
−51.747
1.00
147.14
C

ATOM
836
N
SER
A
23
−6.087
19.514
−52.517
1.00
151.36
N

ATOM
837
CA
SER
A
23
−6.723
18.790
−51.435
1.00
151.36
C

ATOM
838
C
SER
A
23
−6.881
17.299
−51.755
1.00
151.36
C

ATOM
839
O
SER
A
23
−6.873
16.483
−50.839
1.00
151.36
O

ATOM
840
CB
SER
A
23
−8.077
19.407
−51.131
1.00
151.36
C

ATOM
841
OG
SER
A
23
−8.804
18.608
−50.217
1.00
151.36
O

ATOM
842
N
ASP
A
24
−6.989
16.935
−53.038
1.00
156.26
N

ATOM
843
CA
ASP
A
24
−7.142
15.538
−53.448
1.00
156.26
C

ATOM
844
C
ASP
A
24
−5.890
14.695
−53.227
1.00
156.26
C

ATOM
845
O
ASP
A
24
−5.969
13.469
−53.278
1.00
156.26
O

ATOM
846
CB
ASP
A
24
−7.542
15.468
−54.915
1.00
156.26
C

ATOM
847
CG
ASP
A
24
−9.020
15.682
−55.147
1.00
156.26
C

ATOM
848
OD1
ASP
A
24
−9.760
15.861
−54.147
1.00
156.26
O

ATOM
849
OD2
ASP
A
24
−9.441
15.693
−56.328
1.00
156.26
O + 1

ATOM
850
N
TYR
A
25
−4.752
15.336
−52.966
1.00
157.45
N

ATOM
851
CA
TYR
A
25
−3.499
14.637
−52.735
1.00
157.45
C

ATOM
852
C
TYR
A
25
−2.916
14.997
−51.375
1.00
157.45
C

ATOM
853
O
TYR
A
25
−1.769
14.674
−51.072
1.00
157.45
O

ATOM
854
CB
TYR
A
25
−2.519
14.975
−53.848
1.00
157.45
C

ATOM
855
CG
TYR
A
25
−2.914
14.402
−55.190
1.00
157.45
C

ATOM
856
CD1
TYR
A
25
−3.432
15.216
−56.193
1.00
157.45
C

ATOM
857
CD2
TYR
A
25
−2.744
13.050
−55.472
1.00
157.45
C

ATOM
858
CE1
TYR
A
25
−3.726
14.711
−57.460
1.00
157.45
C

ATOM
859
CE2
TYR
A
25
−3.033
12.533
−56.736
1.00
157.45
C

ATOM
860
CZ
TYR
A
25
−3.530
13.367
−57.726
1.00
157.45
C

ATOM
861
OH
TYR
A
25
−3.826
12.873
−58.973
1.00
157.45
O

ATOM
862
N
LEU
A
26
−3.719
15.635
−50.539
1.00
161.33
N

ATOM
863
CA
LEU
A
26
−3.274
16.036
−49.222
1.00
161.33
C

ATOM
864
C
LEU
A
26
−3.778
15.147
−48.125
1.00
161.33
C

ATOM
865
O
LEU
A
26
−4.883
14.587
−48.193
1.00
161.33
O

ATOM
866
CB
LEU
A
26
−3.739
17.464
−48.912
1.00
161.33
C

ATOM
867
CG
LEU
A
26
−2.907
18.595
−49.467
1.00
161.33
C

ATOM
868
CD1
LEU
A
26
−3.439
19.914
−48.993
1.00
161.33
C

ATOM
869
CD2
LEU
A
26
−1.481
18.497
−49.018
1.00
161.33
C

ATOM
870
N
LEU
A
27
−2.946
15.057
−47.093
1.00
164.65
N

ATOM
871
CA
LEU
A
27
−3.244
14.462
−45.810
1.00
164.65
C

ATOM
872
C
LEU
A
27
−3.843
15.607
−45.017
1.00
164.65
C

ATOM
873
O
LEU
A
27
−3.119
16.489
−44.541
1.00
164.65
O

ATOM
874
CB
LEU
A
27
−1.971
13.896
−45.173
1.00
164.65
C

ATOM
875
CG
LEU
A
27
−1.357
12.683
−45.865
1.00
164.65
C

ATOM
876
CD1
LEU
A
27
−0.112
12.234
−45.177
1.00
164.65
C

ATOM
877
CD2
LEU
A
27
−2.318
11.530
−45.914
1.00
164.65
C

ATOM
878
N
GLN
A
28
−5.176
15.647
−44.972
1.00
166.78
N

ATOM
879
CA
GLN
A
28
−6.001
16.720
−44.421
1.00
166.78
C

ATOM
880
C
GLN
A
28
−5.640
17.254
−43.026
1.00
166.78
C

ATOM
881
O
GLN
A
28
−5.956
18.412
−42.751
1.00
166.78
O

ATOM
882
CB
GLN
A
28
−7.459
16.270
−44.378
1.00
166.78
C

ATOM
883
CG
GLN
A
28
−8.310
16.740
−45.541
1.00
166.78
C

ATOM
884
CD
GLN
A
28
−7.764
16.314
−46.871
1.00
166.78
C

ATOM
885
NE2
GLN
A
28
−7.570
17.290
−47.753
1.00
166.78
N

ATOM
886
OE1
GLN
A
28
−7.489
15.126
−47.107
1.00
166.78
O

ATOM
887
N
ASP
A
29
−5.034
16.453
−42.142
1.00
168.93
N

ATOM
888
CA
ASP
A
29
−4.747
16.968
−40.801
1.00
168.93
C

ATOM
889
C
ASP
A
29
−3.297
17.389
−40.629
1.00
168.93
C

ATOM
890
O
ASP
A
29
−2.814
17.482
−39.491
1.00
168.93
O

ATOM
891
CB
ASP
A
29
−5.156
15.964
−39.706
1.00
168.93
C

ATOM
892
CG
ASP
A
29
−4.336
14.679
−39.658
1.00
168.93
C

ATOM
893
OD1
ASP
A
29
−3.656
14.357
−40.669
1.00
168.93
O

ATOM
894
OD2
ASP
A
29
−4.395
13.977
−38.623
1.00
168.93
O + 1

ATOM
895
N
TYR
A
30
−2.605
17.654
−41.748
1.00
166.35
N

ATOM
896
CA
TYR
A
30
−1.235
18.132
−41.683
1.00
166.35
C

ATOM
897
C
TYR
A
30
−1.263
19.426
−40.884
1.00
166.35
C

ATOM
898
O
TYR
A
30
−2.086
20.292
−41.187
1.00
166.35
O

ATOM
899
CB
TYR
A
30
−0.652
18.339
−43.084
1.00
166.35
C

ATOM
900
CG
TYR
A
30
0.729
18.961
−43.084
1.00
166.35
C

ATOM
901
CD1
TYR
A
30
1.865
18.180
−43.254
1.00
166.35
C

ATOM
902
CD2
TYR
A
30
0.898
20.334
−42.927
1.00
166.35
C

ATOM
903
CE1
TYR
A
30
3.133
18.747
−43.289
1.00
166.35
C

ATOM
904
CE2
TYR
A
30
2.165
20.907
−42.913
1.00
166.35
C

ATOM
905
CZ
TYR
A
30
3.280
20.109
−43.111
1.00
166.35
C

ATOM
906
OH
TYR
A
30
4.538
20.657
−43.130
1.00
166.35
O

ATOM
907
N
PRO
A
31
−0.410
19.602
−39.863
1.00
162.90
N

ATOM
908
CA
PRO
A
31
−0.506
20.824
−39.065
1.00
162.90
C

ATOM
909
C
PRO
A
31
0.118
22.071
−39.698
1.00
162.90
C

ATOM
910
O
PRO
A
31
1.309
22.131
−40.006
1.00
162.90
O

ATOM
911
CB
PRO
A
31
0.174
20.453
−37.757
1.00
162.90
C

ATOM
912
CG
PRO
A
31
1.141
19.407
−38.127
1.00
162.90
C

ATOM
913
CD
PRO
A
31
0.618
18.689
−39.334
1.00
162.90
C

ATOM
914
N
VAL
A
32
−0.733
23.082
−39.866
1.00
161.19
N

ATOM
915
CA
VAL
A
32
−0.391
24.423
−40.331
1.00
161.19
C

ATOM
916
C
VAL
A
32
−0.826
25.356
−39.203
1.00
161.19
C

ATOM
917
O
VAL
A
32
−1.238
24.871
−38.147
1.00
161.19
O

ATOM
918
CB
VAL
A
32
−1.059
24.783
−41.685
1.00
161.19
C

ATOM
919
CG1
VAL
A
32
−0.493
23.959
−42.832
1.00
161.19
C

ATOM
920
CG2
VAL
A
32
−2.572
24.642
−41.605
1.00
161.19
C

ATOM
921
N
THR
A
33
−0.772
26.675
−39.418
1.00
159.72
N

ATOM
922
CA
THR
A
33
−1.244
27.643
−38.430
1.00
159.72
C

ATOM
923
C
THR
A
33
−2.101
28.693
−39.085
1.00
159.72
C

ATOM
924
O
THR
A
33
−2.040
28.901
−40.294
1.00
159.72
O

ATOM
925
CB
THR
A
33
−0.103
28.323
−37.655
1.00
159.72
C

ATOM
926
CG2
THR
A
33
0.831
27.336
−37.008
1.00
159.72
C

ATOM
927
OG1
THR
A
33
0.620
29.210
−38.511
1.00
159.72
O

ATOM
928
N
VAL
A
34
−2.884
29.367
−38.273
1.00
161.95
N

ATOM
929
CA
VAL
A
34
−3.754
30.459
−38.675
1.00
161.95
C

ATOM
930
C
VAL
A
34
−3.652
31.500
−37.599
1.00
161.95
C

ATOM
931
O
VAL
A
34
−3.471
31.125
−36.436
1.00
161.95
O

ATOM
932
CB
VAL
A
34
−5.215
29.999
−38.871
1.00
161.95
C

ATOM
933
CG1
VAL
A
34
−5.400
29.296
−40.201
1.00
161.95
C

ATOM
934
CG2
VAL
A
34
−5.683
29.124
−37.719
1.00
161.95
C

ATOM
935
N
ALA
A
35
−3.775
32.789
−37.939
1.00
164.70
N

ATOM
936
CA
ALA
A
35
−3.743
33.798
−36.888
1.00
164.70
C

ATOM
937
C
ALA
A
35
−4.999
33.645
−36.068
1.00
164.70
C

ATOM
938
O
ALA
A
35
−6.080
33.479
−36.636
1.00
164.70
O

ATOM
939
CB
ALA
A
35
−3.645
35.191
−37.476
1.00
164.70
C

ATOM
940
N
SER
A
36
−4.857
33.632
−34.746
1.00
168.33
N

ATOM
941
CA
SER
A
36
−5.993
33.466
−33.862
1.00
168.33
C

ATOM
942
C
SER
A
36
−6.612
34.817
−33.475
1.00
168.33
C

ATOM
943
O
SER
A
36
−7.810
34.885
−33.250
1.00
168.33
O

ATOM
944
CB
SER
A
36
−5.588
32.680
−32.618
1.00
168.33
C

ATOM
945
OG
SER
A
36
−4.652
33.383
−31.817
1.00
168.33
O

ATOM
946
N
ASN
A
37
−5.827
35.895
−33.428
1.00
171.90
N

ATOM
947
CA
ASN
A
37
−6.369
37.179
−32.986
1.00
171.90
C

ATOM
948
C
ASN
A
37
−6.231
38.305
−34.016
1.00
171.90
C

ATOM
949
O
ASN
A
37
−5.745
39.394
−33.684
1.00
171.90
O

ATOM
950
CB
ASN
A
37
−5.685
37.584
−31.681
1.00
171.90
C

ATOM
951
CG
ASN
A
37
−4.222
37.915
−31.850
1.00
171.90
C

ATOM
952
ND2
ASN
A
37
−3.735
38.845
−31.050
1.00
171.90
N

ATOM
953
OD1
ASN
A
37
−3.538
37.385
−32.731
1.00
171.90
O

ATOM
954
N
LEU
A
38
−6.685
38.066
−35.248
1.00
174.92
N

ATOM
955
CA
LEU
A
38
−6.642
39.116
−36.258
1.00
174.92
C

ATOM
956
C
LEU
A
38
−7.794
40.052
−36.024
1.00
174.92
C

ATOM
957
O
LEU
A
38
−8.932
39.599
−35.894
1.00
174.92
O

ATOM
958
CB
LEU
A
38
−6.690
38.565
−37.692
1.00
174.92
C

ATOM
959
CG
LEU
A
38
−5.363
38.247
−38.370
1.00
174.92
C

ATOM
960
CD1
LEU
A
38
−5.607
37.620
−39.691
1.00
174.92
C

ATOM
961
CD2
LEU
A
38
−4.504
39.487
−38.554
1.00
174.92
C

ATOM
962
N
GLN
A
39
−7.500
41.353
−35.936
1.00
178.71
N

ATOM
963
CA
GLN
A
39
−8.503
42.387
−35.740
1.00
178.71
C

ATOM
964
C
GLN
A
39
−9.610
42.223
−36.757
1.00
178.71
C

ATOM
965
O
GLN
A
39
−9.341
41.848
−37.898
1.00
178.71
O

ATOM
966
CB
GLN
A
39
−7.850
43.771
−35.854
1.00
178.71
C

ATOM
967
CG
GLN
A
39
−8.828
44.923
−36.003
1.00
178.71
C

ATOM
968
CD
GLN
A
39
−8.116
46.202
−36.325
1.00
178.71
C

ATOM
969
NE2
GLN
A
39
−8.197
46.655
−37.573
1.00
178.71
N

ATOM
970
OE1
GLN
A
39
−7.473
46.798
−35.456
1.00
178.71
O

ATOM
971
N
ASP
A
40
−10.856
42.459
−36.340
1.00
183.25
N

ATOM
972
CA
ASP
A
40
−11.986
42.376
−37.260
1.00
183.25
C

ATOM
973
C
ASP
A
40
−11.959
43.592
−38.160
1.00
183.25
C

ATOM
974
O
ASP
A
40
−12.146
44.718
−37.692
1.00
183.25
O

ATOM
975
CB
ASP
A
40
−13.320
42.258
−36.510
1.00
183.25
C

ATOM
976
CG
ASP
A
40
−13.605
40.867
−35.960
1.00
183.25
C

ATOM
977
OD1
ASP
A
40
−12.921
39.906
−36.379
1.00
183.25
O

ATOM
978
OD2
ASP
A
40
−14.516
40.738
−35.108
1.00
183.25
O + 1

ATOM
979
N
ASP
A
41
−11.638
43.369
−39.435
1.00
182.51
N

ATOM
980
CA
ASP
A
41
−11.520
44.438
−40.418
1.00
182.51
C

ATOM
981
C
ASP
A
41
−12.039
43.939
−41.751
1.00
182.51
C

ATOM
982
O
ASP
A
41
−11.279
43.401
−42.555
1.00
182.51
O

ATOM
983
CB
ASP
A
41
−10.052
44.911
−40.526
1.00
182.51
C

ATOM
984
CG
ASP
A
41
−9.871
46.277
−41.151
1.00
182.51
C

ATOM
985
OD1
ASP
A
41
−10.709
46.660
−42.006
1.00
182.51
O

ATOM
986
OD2
ASP
A
41
−8.887
46.966
−40.793
1.00
182.51
O + 1

ATOM
987
N
GLU
A
42
−13.322
44.165
−42.011
1.00
179.48
N

ATOM
988
CA
GLU
A
42
−13.994
43.701
−43.221
1.00
179.48
C

ATOM
989
C
GLU
A
42
−13.332
44.110
−44.564
1.00
179.48
C

ATOM
990
O
GLU
A
42
−13.758
43.622
−45.614
1.00
179.48
O

ATOM
991
CB
GLU
A
42
−15.433
44.185
−43.200
1.00
179.48
C

ATOM
992
N
LEU
A
43
−12.272
44.931
−44.533
1.00
175.07
N

ATOM
993
CA
LEU
A
43
−11.585
45.340
−45.748
1.00
175.07
C

ATOM
994
C
LEU
A
43
−10.243
44.614
−45.884
1.00
175.07
C

ATOM
995
O
LEU
A
43
−9.891
44.175
−46.982
1.00
175.07
O

ATOM
996
CB
LEU
A
43
−11.390
46.876
−45.759
1.00
175.07
C

ATOM
997
CG
LEU
A
43
−10.245
47.435
−46.638
1.00
175.07
C

ATOM
998
CD1
LEU
A
43
−10.660
47.584
−48.097
1.00
175.07
C

ATOM
999
CD2
LEU
A
43
−9.725
48.747
−46.110
1.00
175.07
C

ATOM
1000
N
CYS
A
44
−9.500
44.500
−44.775
1.00
169.01
N

ATOM
1001
CA
CYS
A
44
−8.171
43.907
−44.779
1.00
169.01
C

ATOM
1002
C
CYS
A
44
−8.166
42.453
−44.337
1.00
169.01
C

ATOM
1003
O
CYS
A
44
−7.234
41.730
−44.673
1.00
169.01
O

ATOM
1004
CB
CYS
A
44
−7.222
44.731
−43.918
1.00
169.01
C

ATOM
1005
SG
CYS
A
44
−7.014
46.447
−44.467
1.00
169.01
S

ATOM
1006
N
GLY
A
45
−9.176
42.050
−43.576
1.00
162.29
N

ATOM
1007
CA
GLY
A
45
−9.323
40.705
−43.018
1.00
162.29
C

ATOM
1008
C
GLY
A
45
−8.874
39.527
−43.855
1.00
162.29
C

ATOM
1009
O
GLY
A
45
−7.887
38.869
−43.521
1.00
162.29
O

ATOM
1010
N
GLY
A
46
−9.599
39.261
−44.929
1.00
154.64
N

ATOM
1011
CA
GLY
A
46
−9.285
38.162
−45.829
1.00
154.64
C

ATOM
1012
C
GLY
A
46
−7.910
38.298
−46.442
1.00
154.64
C

ATOM
1013
O
GLY
A
46
−7.202
37.305
−46.612
1.00
154.64
O

ATOM
1014
N
LEU
A
47
−7.522
39.544
−46.744
1.00
146.30
N

ATOM
1015
CA
LEU
A
47
−6.234
39.899
−47.324
1.00
146.30
C

ATOM
1016
C
LEU
A
47
−5.087
39.544
−46.370
1.00
146.30
C

ATOM
1017
O
LEU
A
47
−4.068
39.027
−46.810
1.00
146.30
O

ATOM
1018
CB
LEU
A
47
−6.234
41.397
−47.644
1.00
146.30
C

ATOM
1019
CG
LEU
A
47
−5.251
41.889
−48.673
1.00
146.30
C

ATOM
1020
CD1
LEU
A
47
−5.491
41.236
−49.997
1.00
146.30
C

ATOM
1021
CD2
LEU
A
47
−5.366
43.378
−48.831
1.00
146.30
C

ATOM
1022
N
TRP
A
48
−5.263
39.795
−45.075
1.00
141.37
N

ATOM
1023
CA
TRP
A
48
−4.254
39.481
−44.074
1.00
141.37
C

ATOM
1024
C
TRP
A
48
−4.027
37.985
−43.999
1.00
141.37
C

ATOM
1025
O
TRP
A
48
−2.891
37.526
−43.953
1.00
141.37
O

ATOM
1026
CB
TRP
A
48
−4.700
40.004
−42.702
1.00
141.37
C

ATOM
1027
CG
TRP
A
48
−4.556
41.486
−42.482
1.00
141.37
C

ATOM
1028
CD1
TRP
A
48
−3.685
42.330
−43.104
1.00
141.37
C

ATOM
1029
CD2
TRP
A
48
−5.274
42.283
−41.525
1.00
141.37
C

ATOM
1030
CE2
TRP
A
48
−4.788
43.602
−41.632
1.00
141.37
C

ATOM
1031
CE3
TRP
A
48
−6.282
42.007
−40.583
1.00
141.37
C

ATOM
1032
NE1
TRP
A
48
−3.823
43.603
−42.605
1.00
141.37
N

ATOM
1033
CZ2
TRP
A
48
−5.279
44.646
−40.840
1.00
141.37
C

ATOM
1034
CZ3
TRP
A
48
−6.770
43.041
−39.804
1.00
141.37
C

ATOM
1035
CH2
TRP
A
48
−6.277
44.343
−39.940
1.00
141.37
C

ATOM
1036
N
ARG
A
49
−5.118
37.228
−43.998
1.00
139.14
N

ATOM
1037
CA
ARG
A
49
−5.087
35.774
−43.911
1.00
139.14
C

ATOM
1038
C
ARG
A
49
−4.531
35.159
−45.190
1.00
139.14
C

ATOM
1039
O
ARG
A
49
−3.867
34.127
−45.123
1.00
139.14
O

ATOM
1040
CB
ARG
A
49
−6.487
35.219
−43.608
1.00
139.14
C

ATOM
1041
CG
ARG
A
49
−7.300
36.057
−42.612
1.00
139.14
C

ATOM
1042
CD
ARG
A
49
−8.808
35.810
−42.650
1.00
139.14
C

ATOM
1043
NE
ARG
A
49
−9.224
34.727
−41.747
1.00
139.14
N

ATOM
1044
CZ
ARG
A
49
−9.348
34.841
−40.421
1.00
139.14
C

ATOM
1045
NH1
ARG
A
49
−9.073
35.993
−39.816
1.00
139.14
N + 1

ATOM
1046
NH2
ARG
A
49
−9.729
33.797
−39.690
1.00
139.14
N

ATOM
1047
N
LEU
A
50
−4.802
35.784
−46.348
1.00
134.28
N

ATOM
1048
CA
LEU
A
50
−4.278
35.308
−47.622
1.00
134.28
C

ATOM
1049
C
LEU
A
50
−2.779
35.487
−47.647
1.00
134.28
C

ATOM
1050
O
LEU
A
50
−2.069
34.635
−48.158
1.00
134.28
O

ATOM
1051
CB
LEU
A
50
−4.914
36.043
−48.799
1.00
134.28
C

ATOM
1052
CG
LEU
A
50
−6.263
35.524
−49.279
1.00
134.28
C

ATOM
1053
CD1
LEU
A
50
−6.772
36.364
−50.412
1.00
134.28
C

ATOM
1054
CD2
LEU
A
50
−6.181
34.075
−49.716
1.00
134.28
C

ATOM
1055
N
VAL
A
51
−2.295
36.580
−47.067
1.00
130.97
N

ATOM
1056
CA
VAL
A
51
−0.864
36.853
−46.971
1.00
130.97
C

ATOM
1057
C
VAL
A
51
−0.209
35.763
−46.139
1.00
130.97
C

ATOM
1058
O
VAL
A
51
0.855
35.267
−46.491
1.00
130.97
O

ATOM
1059
CB
VAL
A
51
−0.634
38.245
−46.343
1.00
130.97
C

ATOM
1060
CG1
VAL
A
51
0.827
38.454
−45.967
1.00
130.97
C

ATOM
1061
CG2
VAL
A
51
−1.109
39.351
−47.268
1.00
130.97
C

ATOM
1062
N
LEU
A
52
−0.854
35.408
−45.032
1.00
129.47
N

ATOM
1063
CA
LEU
A
52
−0.382
34.386
−44.115
1.00
129.47
C

ATOM
1064
C
LEU
A
52
−0.461
33.016
−44.749
1.00
129.47
C

ATOM
1065
O
LEU
A
52
0.484
32.235
−44.642
1.00
129.47
O

ATOM
1066
CB
LEU
A
52
−1.204
34.416
−42.826
1.00
129.47
C

ATOM
1067
CG
LEU
A
52
−1.102
35.682
−41.979
1.00
129.47
C

ATOM
1068
CD1
LEU
A
52
−2.122
35.672
−40.865
1.00
129.47
C

ATOM
1069
CD2
LEU
A
52
0.289
35.858
−41.401
1.00
129.47
C

ATOM
1070
N
ALA
A
53
−1.591
32.720
−45.412
1.00
131.94
N

ATOM
1071
CA
ALA
A
53
−1.811
31.453
−46.120
1.00
131.94
C

ATOM
1072
C
ALA
A
53
−0.776
31.322
−47.202
1.00
131.94
C

ATOM
1073
O
ALA
A
53
−0.227
30.245
−47.412
1.00
131.94
O

ATOM
1074
CB
ALA
A
53
−3.210
31.415
−46.721
1.00
131.94
C

ATOM
1075
N
GLN
A
54
−0.481
32.449
−47.861
1.00
134.96
N

ATOM
1076
CA
GLN
A
54
0.523
32.530
−48.908
1.00
134.96
C

ATOM
1077
C
GLN
A
54
1.883
32.183
−48.350
1.00
134.96
C

ATOM
1078
O
GLN
A
54
2.618
31.428
−48.981
1.00
134.96
O

ATOM
1079
CB
GLN
A
54
0.546
33.923
−49.536
1.00
134.96
C

ATOM
1080
CG
GLN
A
54
1.219
33.972
−50.906
1.00
134.96
C

ATOM
1081
CD
GLN
A
54
2.727
33.973
−50.832
1.00
134.96
C

ATOM
1082
NE2
GLN
A
54
3.352
33.388
−51.829
1.00
134.96
N

ATOM
1083
OE1
GLN
A
54
3.342
34.471
−49.879
1.00
134.96
O

ATOM
1084
N
ARG
A
55
2.229
32.731
−47.176
1.00
139.94
N

ATOM
1085
CA
ARG
A
55
3.505
32.423
−46.551
1.00
139.94
C

ATOM
1086
C
ARG
A
55
3.595
30.934
−46.256
1.00
139.94
C

ATOM
1087
O
ARG
A
55
4.688
30.385
−46.295
1.00
139.94
O

ATOM
1088
CB
ARG
A
55
3.715
33.246
−45.284
1.00
139.94
C

ATOM
1089
CG
ARG
A
55
4.449
34.557
−45.586
1.00
139.94
C

ATOM
1090
CD
ARG
A
55
5.092
35.149
−44.341
1.00
139.94
C

ATOM
1091
NE
ARG
A
55
5.426
36.565
−44.501
1.00
139.94
N

ATOM
1092
N
TRP
A
56
2.449
30.273
−46.025
1.00
143.91
N

ATOM
1093
CA
TRP
A
56
2.419
28.848
−45.748
1.00
143.91
C

ATOM
1094
C
TRP
A
56
2.637
28.007
−46.991
1.00
143.91
C

ATOM
1095
O
TRP
A
56
3.250
26.950
−46.891
1.00
143.91
O

ATOM
1096
CB
TRP
A
56
1.111
28.450
−45.079
1.00
143.91
C

ATOM
1097
CG
TRP
A
56
1.248
28.192
−43.607
1.00
143.91
C

ATOM
1098
CD1
TRP
A
56
0.677
28.906
−42.590
1.00
143.91
C

ATOM
1099
CD2
TRP
A
56
1.993
27.130
−42.984
1.00
143.91
C

ATOM
1100
CE2
TRP
A
56
1.832
27.269
−41.584
1.00
143.91
C

ATOM
1101
CE3
TRP
A
56
2.787
26.077
−43.474
1.00
143.91
C

ATOM
1102
NE1
TRP
A
56
1.030
28.365
−41.371
1.00
143.91
N

ATOM
1103
CZ2
TRP
A
56
2.444
26.399
−40.668
1.00
143.91
C

ATOM
1104
CZ3
TRP
A
56
3.385
25.214
−42.567
1.00
143.91
C

ATOM
1105
CH2
TRP
A
56
3.204
25.372
−41.183
1.00
143.91
C

ATOM
1106
N
MET
A
57
2.156
28.463
−48.158
1.00
147.65
N

ATOM
1107
CA
MET
A
57
2.321
27.736
−49.426
1.00
147.65
C

ATOM
1108
C
MET
A
57
3.776
27.617
−49.802
1.00
147.65
C

ATOM
1109
O
MET
A
57
4.164
26.641
−50.434
1.00
147.65
O

ATOM
1110
CB
MET
A
57
1.562
28.408
−50.574
1.00
147.65
C

ATOM
1111
CG
MET
A
57
0.071
28.290
−50.458
1.00
147.65
C

ATOM
1112
SD
MET
A
57
−0.497
26.589
−50.265
1.00
147.65
S

ATOM
1113
CE
MET
A
57
−2.159
26.913
−49.638
1.00
147.65
C

ATOM
1114
N
GLU
A
58
4.579
28.603
−49.406
1.00
151.17
N

ATOM
1115
CA
GLU
A
58
6.011
28.638
−49.672
1.00
151.17
C

ATOM
1116
C
GLU
A
58
6.733
27.657
−48.776
1.00
151.17
C

ATOM
1117
O
GLU
A
58
7.556
26.886
−49.268
1.00
151.17
O

ATOM
1118
CB
GLU
A
58
6.554
30.049
−49.453
1.00
151.17
C

ATOM
1119
CG
GLU
A
58
5.965
31.072
−50.416
1.00
151.17
C

ATOM
1120
CD
GLU
A
58
6.548
31.121
−51.820
1.00
151.17
C

ATOM
1121
OE1
GLU
A
58
5.945
31.808
−52.678
1.00
151.17
O

ATOM
1122
OE2
GLU
A
58
7.594
30.479
−52.068
1.00
151.17
O + 1

ATOM
1123
N
ARG
A
59
6.425
27.682
−47.458
1.00
153.37
N

ATOM
1124
CA
ARG
A
59
7.018
26.780
−46.468
1.00
153.37
C

ATOM
1125
C
ARG
A
59
6.733
25.357
−46.851
1.00
153.37
C

ATOM
1126
O
ARG
A
59
7.619
24.518
−46.766
1.00
153.37
O

ATOM
1127
CB
ARG
A
59
6.470
27.053
−45.058
1.00
153.37
C

ATOM
1128
CG
ARG
A
59
7.163
28.187
−44.312
1.00
153.37
C

ATOM
1129
CD
ARG
A
59
6.652
28.358
−42.875
1.00
153.37
C

ATOM
1130
NE
ARG
A
59
5.251
28.817
−42.806
1.00
153.37
N

ATOM
1131
CZ
ARG
A
59
4.858
30.080
−42.613
1.00
153.37
C

ATOM
1132
NH1
ARG
A
59
5.753
31.050
−42.467
1.00
153.37
N + 1

ATOM
1133
NH2
ARG
A
59
3.565
30.379
−42.572
1.00
153.37
N

ATOM
1134
N
LEU
A
60
5.501
25.092
−47.307
1.00
152.61
N

ATOM
1135
CA
LEU
A
60
5.060
23.761
−47.700
1.00
152.61
C

ATOM
1136
C
LEU
A
60
5.768
23.234
−48.934
1.00
152.61
C

ATOM
1137
O
LEU
A
60
6.030
22.033
−49.004
1.00
152.61
O

ATOM
1138
CB
LEU
A
60
3.557
23.734
−47.943
1.00
152.61
C

ATOM
1139
CG
LEU
A
60
2.652
23.798
−46.728
1.00
152.61
C

ATOM
1140
CD1
LEU
A
60
1.233
23.606
−47.146
1.00
152.61
C

ATOM
1141
CD2
LEU
A
60
3.033
22.772
−45.688
1.00
152.61
C

ATOM
1142
N
LYS
A
61
6.064
24.107
−49.908
1.00
151.58
N

ATOM
1143
CA
LYS
A
61
6.736
23.710
−51.141
1.00
151.58
C

ATOM
1144
C
LYS
A
61
8.106
23.111
−50.863
1.00
151.58
C

ATOM
1145
O
LYS
A
61
8.499
22.183
−51.563
1.00
151.58
O

ATOM
1146
CB
LYS
A
61
6.873
24.899
−52.081
1.00
151.58
C

ATOM
1147
CG
LYS
A
61
5.619
25.174
−52.895
1.00
151.58
C

ATOM
1148
CD
LYS
A
61
5.623
26.572
−53.486
1.00
151.58
C

ATOM
1149
CE
LYS
A
61
6.668
26.739
−54.558
1.00
151.58
C

ATOM
1150
NZ
LYS
A
61
6.908
28.171
−54.874
1.00
151.58
N + 1

ATOM
1151
N
THR
A
62
8.811
23.605
−49.824
1.00
150.95
N

ATOM
1152
CA
THR
A
62
10.164
23.158
−49.457
1.00
150.95
C

ATOM
1153
C
THR
A
62
10.227
21.734
−48.896
1.00
150.95
C

ATOM
1154
O
THR
A
62
11.280
21.100
−48.961
1.00
150.95
O

ATOM
1155
CB
THR
A
62
10.788
24.126
−48.456
1.00
150.95
C

ATOM
1156
CG2
THR
A
62
10.873
25.547
−49.002
1.00
150.95
C

ATOM
1157
OG1
THR
A
62
10.038
24.115
−47.242
1.00
150.95
O

ATOM
1158
N
VAL
A
63
9.125
21.243
−48.337
1.00
150.93
N

ATOM
1159
CA
VAL
A
63
9.066
19.906
−47.744
1.00
150.93
C

ATOM
1160
C
VAL
A
63
8.102
19.013
−48.522
1.00
150.93
C

ATOM
1161
O
VAL
A
63
7.673
17.969
−48.024
1.00
150.93
O

ATOM
1162
CB
VAL
A
63
8.671
19.984
−46.253
1.00
150.93
C

ATOM
1163
CG1
VAL
A
63
9.609
20.905
−45.488
1.00
150.93
C

ATOM
1164
CG2
VAL
A
63
7.223
20.435
−46.090
1.00
150.93
C

ATOM
1165
N
ALA
A
64
7.748
19.437
−49.732
1.00
150.82
N

ATOM
1166
CA
ALA
A
64
6.825
18.708
−50.586
1.00
150.82
C

ATOM
1167
C
ALA
A
64
7.562
17.895
−51.616
1.00
150.82
C

ATOM
1168
O
ALA
A
64
8.649
18.281
−52.051
1.00
150.82
O

ATOM
1169
CB
ALA
A
64
5.892
19.678
−51.279
1.00
150.82
C

ATOM
1170
N
GLY
A
65
6.958
16.782
−52.012
1.00
152.23
N

ATOM
1171
CA
GLY
A
65
7.521
15.909
−53.031
1.00
152.23
C

ATOM
1172
C
GLY
A
65
7.427
16.552
−54.390
1.00
152.23
C

ATOM
1173
O
GLY
A
65
6.631
17.474
−54.581
1.00
152.23
O

ATOM
1174
N
SER
A
66
8.229
16.077
−55.341
1.00
154.59
N

ATOM
1175
CA
SER
A
66
8.268
16.613
−56.705
1.00
154.59
C

ATOM
1176
C
SER
A
66
6.882
16.951
−57.277
1.00
154.59
C

ATOM
1177
O
SER
A
66
6.698
18.042
−57.810
1.00
154.59
O

ATOM
1178
CB
SER
A
66
8.960
15.629
−57.636
1.00
154.59
C

ATOM
1179
OG
SER
A
66
8.174
14.458
−57.776
1.00
154.59
O

ATOM
1180
N
LYS
A
67
5.912
16.043
−57.141
1.00
157.07
N

ATOM
1181
CA
LYS
A
67
4.582
16.272
−57.699
1.00
157.07
C

ATOM
1182
C
LYS
A
67
3.669
17.100
−56.774
1.00
157.07
C

ATOM
1183
O
LYS
A
67
2.771
17.773
−57.271
1.00
157.07
O

ATOM
1184
CB
LYS
A
67
3.911
14.951
−58.069
1.00
157.07
C

ATOM
1185
CG
LYS
A
67
4.657
14.178
−59.149
1.00
157.07
C

ATOM
1186
CD
LYS
A
67
3.895
12.903
−59.526
1.00
157.07
C

ATOM
1187
CE
LYS
A
67
4.588
12.025
−60.550
1.00
157.07
C

ATOM
1188
NZ
LYS
A
67
3.905
10.696
−60.680
1.00
157.07
N + 1

ATOM
1189
N
MET
A
68
3.891
17.066
−55.451
1.00
156.90
N

ATOM
1190
CA
MET
A
68
3.101
17.862
−54.510
1.00
156.90
C

ATOM
1191
C
MET
A
68
3.512
19.305
−54.652
1.00
156.90
C

ATOM
1192
O
MET
A
68
2.708
20.202
−54.436
1.00
156.90
O

ATOM
1193
CB
MET
A
68
3.301
17.364
−53.068
1.00
156.90
C

ATOM
1194
CG
MET
A
68
2.567
18.168
−51.999
1.00
156.90
C

ATOM
1195
SD
MET
A
68
0.915
18.725
−52.446
1.00
156.90
S

ATOM
1196
CE
MET
A
68
−0.063
17.270
−52.105
1.00
156.90
C

ATOM
1197
N
GLN
A
69
4.769
19.525
−55.040
1.00
159.73
N

ATOM
1198
CA
GLN
A
69
5.320
20.854
−55.260
1.00
159.73
C

ATOM
1199
C
GLN
A
69
4.539
21.598
−56.337
1.00
159.73
C

ATOM
1200
O
GLN
A
69
4.331
22.805
−56.202
1.00
159.73
O

ATOM
1201
CB
GLN
A
69
6.785
20.763
−55.664
1.00
159.73
C

ATOM
1202
CG
GLN
A
69
7.747
20.754
−54.492
1.00
159.73
C

ATOM
1203
CD
GLN
A
69
9.160
21.056
−54.935
1.00
159.73
C

ATOM
1204
NE2
GLN
A
69
10.057
21.240
−53.974
1.00
159.73
N

ATOM
1205
OE1
GLN
A
69
9.472
21.115
−56.136
1.00
159.73
O

ATOM
1206
N
GLY
A
70
4.105
20.872
−57.375
1.00
158.09
N

ATOM
1207
CA
GLY
A
70
3.327
21.426
−58.480
1.00
158.09
C

ATOM
1208
C
GLY
A
70
1.935
21.847
−58.060
1.00
158.09
C

ATOM
1209
O
GLY
A
70
1.450
22.895
−58.486
1.00
158.09
O

ATOM
1210
N
LEU
A
71
1.297
21.044
−57.201
1.00
153.93
N

ATOM
1211
CA
LEU
A
71
−0.042
21.333
−56.706
1.00
153.93
C

ATOM
1212
C
LEU
A
71
−0.016
22.509
−55.760
1.00
153.93
C

ATOM
1213
O
LEU
A
71
−0.942
23.316
−55.781
1.00
153.93
O

ATOM
1214
CB
LEU
A
71
−0.636
20.113
−56.013
1.00
153.93
C

ATOM
1215
CG
LEU
A
71
−0.800
18.872
−56.872
1.00
153.93
C

ATOM
1216
CD1
LEU
A
71
−1.197
17.694
−56.021
1.00
153.93
C

ATOM
1217
CD2
LEU
A
71
−1.817
19.103
−57.984
1.00
153.93
C

ATOM
1218
N
LEU
A
72
1.049
22.613
−54.938
1.00
149.29
N

ATOM
1219
CA
LEU
A
72
1.231
23.713
−53.994
1.00
149.29
C

ATOM
1220
C
LEU
A
72
1.489
24.969
−54.754
1.00
149.29
C

ATOM
1221
O
LEU
A
72
0.788
25.957
−54.565
1.00
149.29
O

ATOM
1222
CB
LEU
A
72
2.391
23.446
−53.030
1.00
149.29
C

ATOM
1223
CG
LEU
A
72
2.190
22.369
−51.973
1.00
149.29
C

ATOM
1224
CD1
LEU
A
72
3.449
22.141
−51.217
1.00
149.29
C

ATOM
1225
CD2
LEU
A
72
1.088
22.744
−51.001
1.00
149.29
C

ATOM
1226
N
GLU
A
73
2.475
24.917
−55.648
1.00
144.34
N

ATOM
1227
CA
GLU
A
73
2.836
26.042
−56.485
1.00
144.34
C

ATOM
1228
C
GLU
A
73
1.634
26.561
−57.269
1.00
144.34
C

ATOM
1229
O
GLU
A
73
1.454
27.772
−57.365
1.00
144.34
O

ATOM
1230
CB
GLU
A
73
3.967
25.665
−57.442
1.00
144.34
C

ATOM
1231
CG
GLU
A
73
4.363
26.770
−58.413
1.00
144.34
C

ATOM
1232
CD
GLU
A
73
4.584
28.151
−57.809
1.00
144.34
C

ATOM
1233
OE1
GLU
A
73
4.193
29.150
−58.455
1.00
144.34
O

ATOM
1234
OE2
GLU
A
73
5.123
28.239
−56.683
1.00
144.34
O + 1

ATOM
1235
N
ARG
A
74
0.805
25.660
−57.802
1.00
137.64
N

ATOM
1236
CA
ARG
A
74
−0.373
26.048
−58.571
1.00
137.64
C

ATOM
1237
C
ARG
A
74
−1.432
26.737
−57.711
1.00
137.64
C

ATOM
1238
O
ARG
A
74
−2.233
27.506
−58.235
1.00
137.64
O

ATOM
1239
CB
ARG
A
74
−0.977
24.837
−59.270
1.00
137.64
C

ATOM
1240
CG
ARG
A
74
−0.178
24.413
−60.494
1.00
137.64
C

ATOM
1241
CD
ARG
A
74
−0.639
23.076
−61.076
1.00
137.64
C

ATOM
1242
NE
ARG
A
74
−1.875
23.169
−61.853
1.00
137.64
N

ATOM
1243
N
VAL
A
75
−1.455
26.459
−56.410
1.00
133.57
N

ATOM
1244
CA
VAL
A
75
−2.382
27.113
−55.481
1.00
133.57
C

ATOM
1245
C
VAL
A
75
−1.716
28.381
−55.031
1.00
133.57
C

ATOM
1246
O
VAL
A
75
−2.380
29.365
−54.731
1.00
133.57
O

ATOM
1247
CB
VAL
A
75
−2.751
26.207
−54.293
1.00
133.57
C

ATOM
1248
CG1
VAL
A
75
−3.424
26.994
−53.180
1.00
133.57
C

ATOM
1249
CG2
VAL
A
75
−3.645
25.068
−54.745
1.00
133.57
C

ATOM
1250
N
ASN
A
76
−0.387
28.347
−54.989
1.00
132.81
N

ATOM
1251
CA
ASN
A
76
0.428
29.480
−54.613
1.00
132.81
C

ATOM
1252
C
ASN
A
76
0.221
30.598
−55.612
1.00
132.81
C

ATOM
1253
O
ASN
A
76
0.076
31.740
−55.205
1.00
132.81
O

ATOM
1254
CB
ASN
A
76
1.899
29.089
−54.536
1.00
132.81
C

ATOM
1255
CG
ASN
A
76
2.783
30.166
−53.979
1.00
132.81
C

ATOM
1256
ND2
ASN
A
76
4.009
30.236
−54.460
1.00
132.81
N

ATOM
1257
OD1
ASN
A
76
2.389
30.940
−53.108
1.00
132.81
O

ATOM
1258
N
THR
A
77
0.152
30.265
−56.911
1.00
134.70
N

ATOM
1259
CA
THR
A
77
−0.052
31.237
−57.994
1.00
134.70
C

ATOM
1260
C
THR
A
77
−1.394
31.967
−57.875
1.00
134.70
C

ATOM
1261
O
THR
A
77
−1.492
33.144
−58.236
1.00
134.70
O

ATOM
1262
CB
THR
A
77
0.023
30.562
−59.364
1.00
134.70
C

ATOM
1263
CG2
THR
A
77
1.385
29.982
−59.657
1.00
134.70
C

ATOM
1264
OG1
THR
A
77
−0.983
29.552
−59.445
1.00
134.70
O

ATOM
1265
N
GLU
A
78
−2.421
31.260
−57.372
1.00
137.89
N

ATOM
1266
CA
GLU
A
78
−3.762
31.804
−57.205
1.00
137.89
C

ATOM
1267
C
GLU
A
78
−3.809
32.909
−56.166
1.00
137.89
C

ATOM
1268
O
GLU
A
78
−4.723
33.735
−56.215
1.00
137.89
O

ATOM
1269
CB
GLU
A
78
−4.750
30.694
−56.817
1.00
137.89
C

ATOM
1270
CG
GLU
A
78
−4.938
29.641
−57.887
1.00
137.89
C

ATOM
1271
CD
GLU
A
78
−5.387
30.202
−59.217
1.00
137.89
C

ATOM
1272
OE1
GLU
A
78
−6.094
31.236
−59.209
1.00
137.89
O

ATOM
1273
OE2
GLU
A
78
−5.031
29.613
−60.264
1.00
137.89
O + 1

ATOM
1274
N
ILE
A
79
−2.849
32.918
−55.217
1.00
139.92
N

ATOM
1275
CA
ILE
A
79
−2.817
33.885
−54.115
1.00
139.92
C

ATOM
1276
C
ILE
A
79
−1.478
34.611
−53.998
1.00
139.92
C

ATOM
1277
O
ILE
A
79
−1.269
35.315
−53.015
1.00
139.92
O

ATOM
1278
CB
ILE
A
79
−3.163
33.181
−52.779
1.00
139.92
C

ATOM
1279
CG1
ILE
A
79
−2.137
32.100
−52.437
1.00
139.92
C

ATOM
1280
CG2
ILE
A
79
−4.565
32.607
−52.816
1.00
139.92
C

ATOM
1281
CD1
ILE
A
79
−2.145
31.687
−51.010
1.00
139.92
C

ATOM
1282
N
HIS
A
80
−0.583
34.459
−54.981
1.00
146.78
N

ATOM
1283
CA
HIS
A
80
0.753
35.074
−54.952
1.00
146.78
C

ATOM
1284
C
HIS
A
80
0.704
36.572
−55.125
1.00
146.78
C

ATOM
1285
O
HIS
A
80
1.663
37.247
−54.756
1.00
146.78
O

ATOM
1286
CB
HIS
A
80
1.621
34.476
−56.076
1.00
146.78
C

ATOM
1287
CG
HIS
A
80
3.113
34.479
−55.871
1.00
146.78
C

ATOM
1288
CD2
HIS
A
80
4.001
33.464
−56.023
1.00
146.78
C

ATOM
1289
ND1
HIS
A
80
3.813
35.642
−55.594
1.00
146.78
N

ATOM
1290
CE1
HIS
A
80
5.086
35.283
−55.513
1.00
146.78
C

ATOM
1291
NE2
HIS
A
80
5.247
33.983
−55.768
1.00
146.78
N

ATOM
1292
N
PHE
A
81
−0.385
37.100
−55.696
1.00
150.80
N

ATOM
1293
CA
PHE
A
81
−0.520
38.534
−55.943
1.00
150.80
C

ATOM
1294
C
PHE
A
81
−0.376
39.385
−54.656
1.00
150.80
C

ATOM
1295
O
PHE
A
81
−0.010
40.553
−54.753
1.00
150.80
O

ATOM
1296
CB
PHE
A
81
−1.858
38.834
−56.628
1.00
150.80
C

ATOM
1297
CG
PHE
A
81
−3.081
38.501
−55.799
1.00
150.80
C

ATOM
1298
CD1
PHE
A
81
−3.520
39.361
−54.799
1.00
150.80
C

ATOM
1299
CD2
PHE
A
81
−3.806
37.341
−56.034
1.00
150.80
C

ATOM
1300
CE1
PHE
A
81
−4.640
39.051
−54.028
1.00
150.80
C

ATOM
1301
CE2
PHE
A
81
−4.942
37.042
−55.272
1.00
150.80
C

ATOM
1302
CZ
PHE
A
81
−5.345
37.893
−54.270
1.00
150.80
C

ATOM
1303
N
VAL
A
82
−0.650
38.804
−53.468
1.00
152.56
N

ATOM
1304
CA
VAL
A
82
−0.557
39.515
−52.188
1.00
152.56
C

ATOM
1305
C
VAL
A
82
0.855
40.035
−51.925
1.00
152.56
C

ATOM
1306
O
VAL
A
82
1.035
40.946
−51.121
1.00
152.56
O

ATOM
1307
CB
VAL
A
82
−1.015
38.648
−51.003
1.00
152.56
C

ATOM
1308
CG1
VAL
A
82
−2.481
38.265
−51.131
1.00
152.56
C

ATOM
1309
CG2
VAL
A
82
−0.129
37.420
−50.837
1.00
152.56
C

ATOM
1310
N
THR
A
83
1.852
39.450
−52.586
1.00
158.41
N

ATOM
1311
CA
THR
A
83
3.247
39.852
−52.424
1.00
158.41
C

ATOM
1312
C
THR
A
83
3.580
41.038
−53.325
1.00
158.41
C

ATOM
1313
O
THR
A
83
4.684
41.579
−53.251
1.00
158.41
O

ATOM
1314
CB
THR
A
83
4.177
38.674
−52.686
1.00
158.41
C

ATOM
1315
CG2
THR
A
83
3.971
37.548
−51.695
1.00
158.41
C

ATOM
1316
OG1
THR
A
83
3.976
38.192
−54.013
1.00
158.41
O

ATOM
1317
N
LYS
A
84
2.625
41.449
−54.170
1.00
164.53
N

ATOM
1318
CA
LYS
A
84
2.794
42.613
−55.021
1.00
164.53
C

ATOM
1319
C
LYS
A
84
2.790
43.845
−54.134
1.00
164.53
C

ATOM
1320
O
LYS
A
84
3.229
44.910
−54.559
1.00
164.53
O

ATOM
1321
CB
LYS
A
84
1.675
42.687
−56.063
1.00
164.53
C

ATOM
1322
N
CYS
A
85
2.307
43.684
−52.884
1.00
170.17
N

ATOM
1323
CA
CYS
A
85
2.205
44.745
−51.890
1.00
170.17
C

ATOM
1324
C
CYS
A
85
3.190
44.533
−50.758
1.00
170.17
C

ATOM
1325
O
CYS
A
85
3.366
43.409
−50.271
1.00
170.17
O

ATOM
1326
CB
CYS
A
85
0.780
44.850
−51.360
1.00
170.17
C

ATOM
1327
SG
CYS
A
85
−0.451
45.319
−52.605
1.00
170.17
S

ATOM
1328
N
ALA
A
86
3.784
45.645
−50.305
1.00
170.29
N

ATOM
1329
CA
ALA
A
86
4.766
45.689
−49.225
1.00
170.29
C

ATOM
1330
C
ALA
A
86
4.126
45.488
−47.836
1.00
170.29
C

ATOM
1331
O
ALA
A
86
4.149
46.392
−46.989
1.00
170.29
O

ATOM
1332
CB
ALA
A
86
5.524
47.006
−49.273
1.00
170.29
C

ATOM
1333
N
PHE
A
87
3.581
44.283
−47.602
1.00
170.15
N

ATOM
1334
CA
PHE
A
87
2.976
43.930
−46.321
1.00
170.15
C

ATOM
1335
C
PHE
A
87
4.050
43.874
−45.250
1.00
170.15
C

ATOM
1336
O
PHE
A
87
5.083
43.233
−45.454
1.00
170.15
O

ATOM
1337
CB
PHE
A
87
2.251
42.582
−46.420
1.00
170.15
C

ATOM
1338
CG
PHE
A
87
0.876
42.655
−47.032
1.00
170.15
C

ATOM
1339
CD1
PHE
A
87
0.685
42.401
−48.386
1.00
170.15
C

ATOM
1340
CD2
PHE
A
87
−0.232
42.953
−46.252
1.00
170.15
C

ATOM
1341
CE1
PHE
A
87
−0.588
42.460
−48.948
1.00
170.15
C

ATOM
1342
CE2
PHE
A
87
−1.502
43.008
−46.814
1.00
170.15
C

ATOM
1343
CZ
PHE
A
87
−1.672
42.763
−48.158
1.00
170.15
C

ATOM
1344
N
GLN
A
88
3.822
44.559
−44.124
1.00
172.23
N

ATOM
1345
CA
GLN
A
88
4.768
44.587
−43.014
1.00
172.23
C

ATOM
1346
C
GLN
A
88
4.770
43.263
−42.263
1.00
172.23
C

ATOM
1347
O
GLN
A
88
3.745
42.579
−42.239
1.00
172.23
O

ATOM
1348
CB
GLN
A
88
4.443
45.726
−42.048
1.00
172.23
C

ATOM
1349
N
PRO
A
89
5.908
42.884
−41.633
1.00
174.22
N

ATOM
1350
CA
PRO
A
89
5.948
41.613
−40.899
1.00
174.22
C

ATOM
1351
C
PRO
A
89
4.923
41.586
−39.774
1.00
174.22
C

ATOM
1352
O
PRO
A
89
4.578
42.649
−39.237
1.00
174.22
O

ATOM
1353
CB
PRO
A
89
7.386
41.552
−40.362
1.00
174.22
C

ATOM
1354
CG
PRO
A
89
7.853
42.954
−40.363
1.00
174.22
C

ATOM
1355
CD
PRO
A
89
7.215
43.567
−41.569
1.00
174.22
C

ATOM
1356
N
PRO
A
90
4.409
40.378
−39.433
1.00
174.34
N

ATOM
1357
CA
PRO
A
90
3.402
40.289
−38.371
1.00
174.34
C

ATOM
1358
C
PRO
A
90
3.832
40.981
−37.070
1.00
174.34
C

ATOM
1359
O
PRO
A
90
5.010
40.922
−36.696
1.00
174.34
O

ATOM
1360
CB
PRO
A
90
3.223
38.774
−38.170
1.00
174.34
C

ATOM
1361
CG
PRO
A
90
3.594
38.172
−39.472
1.00
174.34
C

ATOM
1362
CD
PRO
A
90
4.700
39.041
−40.005
1.00
174.34
C

ATOM
1363
N
PRO
A
91
2.873
41.669
−36.397
1.00
174.64
N

ATOM
1364
CA
PRO
A
91
3.181
42.334
−35.118
1.00
174.64
C

ATOM
1365
C
PRO
A
91
3.372
41.314
−33.991
1.00
174.64
C

ATOM
1366
O
PRO
A
91
2.948
40.158
−34.098
1.00
174.64
O

ATOM
1367
CB
PRO
A
91
1.959
43.223
−34.877
1.00
174.64
C

ATOM
1368
CG
PRO
A
91
0.856
42.537
−35.585
1.00
174.64
C

ATOM
1369
CD
PRO
A
91
1.448
41.823
−36.760
1.00
174.64
C

ATOM
1370
N
SER
A
92
4.024
41.746
−32.912
1.00
174.69
N

ATOM
1371
CA
SER
A
92
4.305
40.916
−31.740
1.00
174.69
C

ATOM
1372
C
SER
A
92
3.030
40.374
−31.104
1.00
174.69
C

ATOM
1373
O
SER
A
92
3.046
39.289
−30.518
1.00
174.69
O

ATOM
1374
CB
SER
A
92
5.075
41.731
−30.704
1.00
174.69
C

ATOM
1375
OG
SER
A
92
4.359
42.903
−30.338
1.00
174.69
O

ATOM
1376
N
CYS
A
93
1.935
41.146
−31.217
1.00
172.90
N

ATOM
1377
CA
CYS
A
93
0.635
40.834
−30.633
1.00
172.90
C

ATOM
1378
C
CYS
A
93
−0.060
39.672
−31.328
1.00
172.90
C

ATOM
1379
O
CYS
A
93
−0.990
39.104
−30.749
1.00
172.90
O

ATOM
1380
CB
CYS
A
93
−0.263
42.069
−30.630
1.00
172.90
C

ATOM
1381
SG
CYS
A
93
−0.591
42.777
−32.275
1.00
172.90
S

ATOM
1382
N
LEU
A
94
0.331
39.358
−32.577
1.00
173.60
N

ATOM
1383
CA
LEU
A
94
−0.321
38.318
−33.363
1.00
173.60
C

ATOM
1384
C
LEU
A
94
−0.002
36.916
−32.870
1.00
173.60
C

ATOM
1385
O
LEU
A
94
1.156
36.486
−32.897
1.00
173.60
O

ATOM
1386
CB
LEU
A
94
0.041
38.458
−34.840
1.00
173.60
C

ATOM
1387
CG
LEU
A
94
−0.938
37.872
−35.853
1.00
173.60
C

ATOM
1388
CD1
LEU
A
94
−2.379
38.218
−35.505
1.00
173.60
C

ATOM
1389
CD2
LEU
A
94
−0.611
38.373
−37.243
1.00
173.60
C

ATOM
1390
N
ARG
A
95
−1.053
36.215
−32.419
1.00
174.62
N

ATOM
1391
CA
ARG
A
95
−0.976
34.848
−31.918
1.00
174.62
C

ATOM
1392
C
ARG
A
95
−1.397
33.882
−32.995
1.00
174.62
C

ATOM
1393
O
ARG
A
95
−2.395
34.116
−33.675
1.00
174.62
O

ATOM
1394
CB
ARG
A
95
−1.868
34.671
−30.683
1.00
174.62
C

ATOM
1395
CG
ARG
A
95
−1.189
33.912
−29.552
1.00
174.62
C

ATOM
1396
CD
ARG
A
95
−1.214
34.665
−28.221
1.00
174.62
C

ATOM
1397
NE
ARG
A
95
−0.992
36.117
−28.342
1.00
174.62
N

ATOM
1398
CZ
ARG
A
95
0.201
36.711
−28.414
1.00
174.62
C

ATOM
1399
NH1
ARG
A
95
1.317
35.988
−28.401
1.00
174.62
N + 1

ATOM
1400
NH2
ARG
A
95
0.286
38.032
−28.512
1.00
174.62
N

ATOM
1401
N
PHE
A
96
−0.656
32.787
−33.144
1.00
172.75
N

ATOM
1402
CA
PHE
A
96
−0.978
31.775
−34.141
1.00
172.75
C

ATOM
1403
C
PHE
A
96
−1.472
30.518
−33.453
1.00
172.75
C

ATOM
1404
O
PHE
A
96
−1.036
30.216
−32.341
1.00
172.75
O

ATOM
1405
CB
PHE
A
96
0.234
31.482
−35.035
1.00
172.75
C

ATOM
1406
CG
PHE
A
96
0.683
32.679
−35.837
1.00
172.75
C

ATOM
1407
CD1
PHE
A
96
0.093
32.980
−37.056
1.00
172.75
C

ATOM
1408
CD2
PHE
A
96
1.688
33.516
−35.365
1.00
172.75
C

ATOM
1409
CE1
PHE
A
96
0.496
34.106
−37.785
1.00
172.75
C

ATOM
1410
CE2
PHE
A
96
2.095
34.638
−36.098
1.00
172.75
C

ATOM
1411
CZ
PHE
A
96
1.498
34.924
−37.303
1.00
172.75
C

ATOM
1412
N
VAL
A
97
−2.397
29.802
−34.100
1.00
171.81
N

ATOM
1413
CA
VAL
A
97
−2.965
28.578
−33.554
1.00
171.81
C

ATOM
1414
C
VAL
A
97
−2.858
27.457
−34.588
1.00
171.81
C

ATOM
1415
O
VAL
A
97
−3.121
27.682
−35.773
1.00
171.81
O

ATOM
1416
CB
VAL
A
97
−4.417
28.803
−33.060
1.00
171.81
C

ATOM
1417
CG1
VAL
A
97
−5.426
28.793
−34.204
1.00
171.81
C

ATOM
1418
CG2
VAL
A
97
−4.793
27.781
−32.002
1.00
171.81
C

ATOM
1419
N
GLN
A
98
−2.472
26.255
−34.135
1.00
170.10
N

ATOM
1420
CA
GLN
A
98
−2.321
25.089
−35.007
1.00
170.10
C

ATOM
1421
C
GLN
A
98
−3.656
24.580
−35.544
1.00
170.10
C

ATOM
1422
O
GLN
A
98
−4.581
24.317
−34.771
1.00
170.10
O

ATOM
1423
CB
GLN
A
98
−1.626
23.965
−34.263
1.00
170.10
C

ATOM
1424
CG
GLN
A
98
−1.301
22.781
−35.138
1.00
170.10
C

ATOM
1425
CD
GLN
A
98
−0.012
22.241
−34.632
1.00
170.10
C

ATOM
1426
NE2
GLN
A
98
1.088
22.872
−35.039
1.00
170.10
N

ATOM
1427
OE1
GLN
A
98
0.011
21.320
−33.821
1.00
170.10
O

ATOM
1428
N
THR
A
99
−3.742
24.405
−36.865
1.00
165.60
N

ATOM
1429
CA
THR
A
99
−4.961
23.931
−37.506
1.00
165.60
C

ATOM
1430
C
THR
A
99
−4.615
23.004
−38.656
1.00
165.60
C

ATOM
1431
O
THR
A
99
−3.489
23.029
−39.133
1.00
165.60
O

ATOM
1432
CB
THR
A
99
−5.803
25.132
−37.974
1.00
165.60
C

ATOM
1433
CG2
THR
A
99
−5.071
26.004
−38.977
1.00
165.60
C

ATOM
1434
OG1
THR
A
99
−7.003
24.643
−38.556
1.00
165.60
O

ATOM
1435
N
ASN
A
100
−5.587
22.205
−39.110
1.00
163.03
N

ATOM
1436
CA
ASN
A
100
−5.428
21.301
−40.243
1.00
163.03
C

ATOM
1437
C
ASN
A
100
−5.253
22.079
−41.525
1.00
163.03
C

ATOM
1438
O
ASN
A
100
−5.892
23.114
−41.718
1.00
163.03
O

ATOM
1439
CB
ASN
A
100
−6.633
20.383
−40.364
1.00
163.03
C

ATOM
1440
CG
ASN
A
100
−6.664
19.293
−39.327
1.00
163.03
C

ATOM
1441
ND2
ASN
A
100
−7.724
18.477
−39.364
1.00
163.03
N

ATOM
1442
OD1
ASN
A
100
−5.730
19.137
−38.516
1.00
163.03
O

ATOM
1443
N
ILE
A
101
−4.400
21.570
−42.416
1.00
160.68
N

ATOM
1444
CA
ILE
A
101
−4.154
22.195
−43.713
1.00
160.68
C

ATOM
1445
C
ILE
A
101
−5.452
22.249
−44.523
1.00
160.68
C

ATOM
1446
O
ILE
A
101
−5.597
23.101
−45.393
1.00
160.68
O

ATOM
1447
CB
ILE
A
101
−3.041
21.461
−44.485
1.00
160.68
C

ATOM
1448
CG1
ILE
A
101
−2.555
22.318
−45.661
1.00
160.68
C

ATOM
1449
CG2
ILE
A
101
−3.519
20.086
−44.957
1.00
160.68
C

ATOM
1450
CD1
ILE
A
101
−1.404
21.763
−46.393
1.00
160.68
C

ATOM
1451
N
SER
A
102
−6.383
21.335
−44.230
1.00
158.66
N

ATOM
1452
CA
SER
A
102
−7.678
21.300
−44.878
1.00
158.66
C

ATOM
1453
C
SER
A
102
−8.465
22.530
−44.468
1.00
158.66
C

ATOM
1454
O
SER
A
102
−9.235
23.061
−45.268
1.00
158.66
O

ATOM
1455
CB
SER
A
102
−8.430
20.039
−44.483
1.00
158.66
C

ATOM
1456
OG
SER
A
102
−8.880
20.109
−43.138
1.00
158.66
O

ATOM
1457
N
ARG
A
103
−8.276
22.973
−43.205
1.00
157.48
N

ATOM
1458
CA
ARG
A
103
−8.936
24.161
−42.676
1.00
157.48
C

ATOM
1459
C
ARG
A
103
−8.363
25.385
−43.361
1.00
157.48
C

ATOM
1460
O
ARG
A
103
−9.120
26.250
−43.806
1.00
157.48
O

ATOM
1461
CB
ARG
A
103
−8.783
24.264
−41.154
1.00
157.48
C

ATOM
1462
CG
ARG
A
103
−9.149
25.652
−40.622
1.00
157.48
C

ATOM
1463
CD
ARG
A
103
−10.421
25.702
−39.811
1.00
157.48
C

ATOM
1464
NE
ARG
A
103
−10.251
26.650
−38.710
1.00
157.48
N

ATOM
1465
CZ
ARG
A
103
−11.142
26.894
−37.752
1.00
157.48
C

ATOM
1466
NH1
ARG
A
103
−10.860
27.755
−36.783
1.00
157.48
N + 1

ATOM
1467
NH2
ARG
A
103
−12.316
26.270
−37.750
1.00
157.48
N

ATOM
1468
N
LEU
A
104
−7.030
25.442
−43.465
1.00
155.21
N

ATOM
1469
CA
LEU
A
104
−6.343
26.551
−44.101
1.00
155.21
C

ATOM
1470
C
LEU
A
104
−6.762
26.694
−45.556
1.00
155.21
C

ATOM
1471
O
LEU
A
104
−7.037
27.811
−46.008
1.00
155.21
O

ATOM
1472
CB
LEU
A
104
−4.827
26.368
−44.000
1.00
155.21
C

ATOM
1473
CG
LEU
A
104
−3.969
27.497
−44.582
1.00
155.21
C

ATOM
1474
CD1
LEU
A
104
−4.161
28.802
−43.810
1.00
155.21
C

ATOM
1475
CD2
LEU
A
104
−2.510
27.118
−44.606
1.00
155.21
C

ATOM
1476
N
LEU
A
105
−6.820
25.567
−46.282
1.00
156.56
N

ATOM
1477
CA
LEU
A
105
−7.196
25.555
−47.698
1.00
156.56
C

ATOM
1478
C
LEU
A
105
−8.632
26.004
−47.909
1.00
156.56
C

ATOM
1479
O
LEU
A
105
−8.922
26.717
−48.875
1.00
156.56
O

ATOM
1480
CB
LEU
A
105
−7.009
24.162
−48.307
1.00
156.56
C

ATOM
1481
CG
LEU
A
105
−5.597
23.683
−48.572
1.00
156.56
C

ATOM
1482
CD1
LEU
A
105
−5.618
22.647
−49.636
1.00
156.56
C

ATOM
1483
CD2
LEU
A
105
−4.701
24.811
−48.999
1.00
156.56
C

ATOM
1484
N
GLN
A
106
−9.526
25.574
−47.009
1.00
158.45
N

ATOM
1485
CA
GLN
A
106
−10.932
25.909
−47.078
1.00
158.45
C

ATOM
1486
C
GLN
A
106
−11.130
27.394
−46.871
1.00
158.45
C

ATOM
1487
O
GLN
A
106
−11.867
28.032
−47.625
1.00
158.45
O

ATOM
1488
CB
GLN
A
106
−11.730
25.097
−46.049
1.00
158.45
C

ATOM
1489
CG
GLN
A
106
−13.231
25.384
−46.045
1.00
158.45
C

ATOM
1490
CD
GLN
A
106
−13.967
25.121
−47.357
1.00
158.45
C

ATOM
1491
NE2
GLN
A
106
−13.249
24.881
−48.458
1.00
158.45
N

ATOM
1492
OE1
GLN
A
106
−15.204
25.153
−47.410
1.00
158.45
O

ATOM
1493
N
GLU
A
107
−10.457
27.941
−45.862
1.00
158.86
N

ATOM
1494
CA
GLU
A
107
−10.541
29.358
−45.546
1.00
158.86
C

ATOM
1495
C
GLU
A
107
−9.980
30.195
−46.686
1.00
158.86
C

ATOM
1496
O
GLU
A
107
−10.599
31.188
−47.072
1.00
158.86
O

ATOM
1497
CB
GLU
A
107
−9.808
29.662
−44.240
1.00
158.86
C

ATOM
1498
CG
GLU
A
107
−10.650
29.399
−42.999
1.00
158.86
C

ATOM
1499
CD
GLU
A
107
−9.917
29.556
−41.677
1.00
158.86
C

ATOM
1500
OE1
GLU
A
107
−8.832
30.185
−41.668
1.00
158.86
O

ATOM
1501
OE2
GLU
A
107
−10.432
29.059
−40.647
1.00
158.86
O + 1

ATOM
1502
N
THR
A
108
−8.834
29.769
−47.254
1.00
154.65
N

ATOM
1503
CA
THR
A
108
−8.209
30.464
−48.377
1.00
154.65
C

ATOM
1504
C
THR
A
108
−9.202
30.598
−49.523
1.00
154.65
C

ATOM
1505
O
THR
A
108
−9.304
31.671
−50.121
1.00
154.65
O

ATOM
1506
CB
THR
A
108
−6.956
29.734
−48.829
1.00
154.65
C

ATOM
1507
CG2
THR
A
108
−6.447
30.243
−50.161
1.00
154.65
C

ATOM
1508
OG1
THR
A
108
−5.941
29.903
−47.843
1.00
154.65
O

ATOM
1509
N
SER
A
109
−9.930
29.510
−49.828
1.00
153.07
N

ATOM
1510
CA
SER
A
109
−10.930
29.525
−50.889
1.00
153.07
C

ATOM
1511
C
SER
A
109
−11.982
30.566
−50.567
1.00
153.07
C

ATOM
1512
O
SER
A
109
−12.272
31.424
−51.402
1.00
153.07
O

ATOM
1513
CB
SER
A
109
−11.569
28.152
−51.053
1.00
153.07
C

ATOM
1514
OG
SER
A
109
−12.182
28.019
−52.325
1.00
153.07
O

ATOM
1515
N
GLU
A
110
−12.505
30.528
−49.332
1.00
153.58
N

ATOM
1516
CA
GLU
A
110
−13.522
31.462
−48.863
1.00
153.58
C

ATOM
1517
C
GLU
A
110
−13.068
32.910
−48.980
1.00
153.58
C

ATOM
1518
O
GLU
A
110
−13.833
33.756
−49.453
1.00
153.58
O

ATOM
1519
CB
GLU
A
110
−13.881
31.166
−47.417
1.00
153.58
C

ATOM
1520
CG
GLU
A
110
−14.620
29.858
−47.210
1.00
153.58
C

ATOM
1521
CD
GLU
A
110
−14.851
29.494
−45.752
1.00
153.58
C

ATOM
1522
OE1
GLU
A
110
−14.426
30.265
−44.859
1.00
153.58
O

ATOM
1523
OE2
GLU
A
110
−15.463
28.429
−45.503
1.00
153.58
O + 1

ATOM
1524
N
GLN
A
111
−11.823
33.194
−48.567
1.00
151.63
N

ATOM
1525
CA
GLN
A
111
−11.265
34.544
−48.619
1.00
151.63
C

ATOM
1526
C
GLN
A
111
−11.137
35.047
−50.041
1.00
151.63
C

ATOM
1527
O
GLN
A
111
−11.334
36.238
−50.282
1.00
151.63
O

ATOM
1528
CB
GLN
A
111
−9.904
34.593
−47.944
1.00
151.63
C

ATOM
1529
CG
GLN
A
111
−9.926
34.256
−46.454
1.00
151.63
C

ATOM
1530
CD
GLN
A
111
−8.597
33.690
−45.970
1.00
151.63
C

ATOM
1531
NE2
GLN
A
111
−7.524
33.862
−46.741
1.00
151.63
N

ATOM
1532
OE1
GLN
A
111
−8.506
33.081
−44.895
1.00
151.63
O

ATOM
1533
N
LEU
A
112
−10.818
34.149
−50.983
1.00
146.44
N

ATOM
1534
CA
LEU
A
112
−10.707
34.532
−52.380
1.00
146.44
C

ATOM
1535
C
LEU
A
112
−12.075
34.901
−52.927
1.00
146.44
C

ATOM
1536
O
LEU
A
112
−12.200
35.884
−53.652
1.00
146.44
O

ATOM
1537
CB
LEU
A
112
−10.078
33.415
−53.207
1.00
146.44
C

ATOM
1538
CG
LEU
A
112
−8.564
33.318
−53.140
1.00
146.44
C

ATOM
1539
CD1
LEU
A
112
−8.096
32.055
−53.771
1.00
146.44
C

ATOM
1540
CD2
LEU
A
112
−7.905
34.503
−53.831
1.00
146.44
C

ATOM
1541
N
VAL
A
113
−13.102
34.143
−52.542
1.00
143.55
N

ATOM
1542
CA
VAL
A
113
−14.473
34.379
−52.973
1.00
143.55
C

ATOM
1543
C
VAL
A
113
−14.960
35.734
−52.505
1.00
143.55
C

ATOM
1544
O
VAL
A
113
−15.475
36.498
−53.319
1.00
143.55
O

ATOM
1545
CB
VAL
A
113
−15.413
33.259
−52.486
1.00
143.55
C

ATOM
1546
CG1
VAL
A
113
−16.881
33.678
−52.572
1.00
143.55
C

ATOM
1547
CG2
VAL
A
113
−15.181
31.982
−53.282
1.00
143.55
C

ATOM
1548
N
ALA
A
114
−14.808
36.026
−51.203
1.00
140.81
N

ATOM
1549
CA
ALA
A
114
−15.258
37.284
−50.615
1.00
140.81
C

ATOM
1550
C
ALA
A
114
−14.487
38.483
−51.168
1.00
140.81
C

ATOM
1551
O
ALA
A
114
−15.045
39.572
−51.306
1.00
140.81
O

ATOM
1552
CB
ALA
A
114
−15.117
37.226
−49.108
1.00
140.81
C

ATOM
1553
N
LEU
A
115
−13.222
38.272
−51.504
1.00
139.88
N

ATOM
1554
CA
LEU
A
115
−12.341
39.313
−52.002
1.00
139.88
C

ATOM
1555
C
LEU
A
115
−12.509
39.591
−53.489
1.00
139.88
C

ATOM
1556
O
LEU
A
115
−12.216
40.698
−53.939
1.00
139.88
O

ATOM
1557
CB
LEU
A
115
−10.908
38.875
−51.720
1.00
139.88
C

ATOM
1558
CG
LEU
A
115
−9.811
39.911
−51.641
1.00
139.88
C

ATOM
1559
CD1
LEU
A
115
−10.218
41.104
−50.806
1.00
139.88
C

ATOM
1560
CD2
LEU
A
115
−8.595
39.298
−51.027
1.00
139.88
C

ATOM
1561
N
LYS
A
116
−12.971
38.593
−54.247
1.00
142.54
N

ATOM
1562
CA
LYS
A
116
−13.135
38.666
−55.700
1.00
142.54
C

ATOM
1563
C
LYS
A
116
−13.928
39.908
−56.180
1.00
142.54
C

ATOM
1564
O
LYS
A
116
−13.472
40.573
−57.122
1.00
142.54
O

ATOM
1565
CB
LYS
A
116
−13.778
37.380
−56.238
1.00
142.54
C

ATOM
1566
N
PRO
A
117
−15.078
40.268
−55.571
1.00
147.62
N

ATOM
1567
CA
PRO
A
117
−15.813
41.438
−56.084
1.00
147.62
C

ATOM
1568
C
PRO
A
117
−15.214
42.797
−55.694
1.00
147.62
C

ATOM
1569
O
PRO
A
117
−15.630
43.819
−56.248
1.00
147.62
O

ATOM
1570
CB
PRO
A
117
−17.208
41.272
−55.479
1.00
147.62
C

ATOM
1571
CG
PRO
A
117
−17.008
40.444
−54.261
1.00
147.62
C

ATOM
1572
CD
PRO
A
117
−15.825
39.584
−54.490
1.00
147.62
C

ATOM
1573
N
TRP
A
118
−14.239
42.821
−54.776
1.00
150.16
N

ATOM
1574
CA
TRP
A
118
−13.698
44.088
−54.289
1.00
150.16
C

ATOM
1575
C
TRP
A
118
−12.227
44.326
−54.560
1.00
150.16
C

ATOM
1576
O
TRP
A
118
−11.784
45.468
−54.451
1.00
150.16
O

ATOM
1577
CB
TRP
A
118
−13.929
44.179
−52.782
1.00
150.16
C

ATOM
1578
CG
TRP
A
118
−15.369
44.005
−52.424
1.00
150.16
C

ATOM
1579
CD1
TRP
A
118
−16.006
42.835
−52.135
1.00
150.16
C

ATOM
1580
CD2
TRP
A
118
−16.368
45.025
−52.404
1.00
150.16
C

ATOM
1581
CE2
TRP
A
118
−17.590
44.404
−52.068
1.00
150.16
C

ATOM
1582
CE3
TRP
A
118
−16.346
46.417
−52.610
1.00
150.16
C

ATOM
1583
NE1
TRP
A
118
−17.340
43.066
−51.912
1.00
150.16
N

ATOM
1584
CZ2
TRP
A
118
−18.781
45.123
−51.950
1.00
150.16
C

ATOM
1585
CZ3
TRP
A
118
−17.526
47.128
−52.505
1.00
150.16
C

ATOM
1586
CH2
TRP
A
118
−18.731
46.479
−52.209
1.00
150.16
C

ATOM
1587
N
ILE
A
119
−11.472
43.285
−54.903
1.00
157.85
N

ATOM
1588
CA
ILE
A
119
−10.031
43.388
−55.118
1.00
157.85
C

ATOM
1589
C
ILE
A
119
−9.574
44.574
−56.009
1.00
157.85
C

ATOM
1590
O
ILE
A
119
−8.466
45.092
−55.805
1.00
157.85
O

ATOM
1591
CB
ILE
A
119
−9.477
42.067
−55.676
1.00
157.85
C

ATOM
1592
CG1
ILE
A
119
−7.945
42.040
−55.583
1.00
157.85
C

ATOM
1593
CG2
ILE
A
119
−9.978
41.786
−57.110
1.00
157.85
C

ATOM
1594
CD1
ILE
A
119
−7.387
42.230
−54.214
1.00
157.85
C

ATOM
1595
N
THR
A
120
−10.409
44.992
−56.978
1.00
164.58
N

ATOM
1596
CA
THR
A
120
−10.030
46.057
−57.904
1.00
164.58
C

ATOM
1597
C
THR
A
120
−10.697
47.394
−57.618
1.00
164.58
C

ATOM
1598
O
THR
A
120
−10.336
48.386
−58.254
1.00
164.58
O

ATOM
1599
CB
THR
A
120
−10.334
45.641
−59.331
1.00
164.58
C

ATOM
1600
CG2
THR
A
120
−9.574
44.398
−59.733
1.00
164.58
C

ATOM
1601
OG1
THR
A
120
−11.742
45.439
−59.463
1.00
164.58
O

ATOM
1602
N
ARG
A
121
−11.649
47.433
−56.670
1.00
170.17
N

ATOM
1603
CA
ARG
A
121
−12.373
48.664
−56.330
1.00
170.17
C

ATOM
1604
C
ARG
A
121
−11.789
49.381
−55.105
1.00
170.17
C

ATOM
1605
O
ARG
A
121
−12.009
50.580
−54.920
1.00
170.17
O

ATOM
1606
CB
ARG
A
121
−13.854
48.354
−56.074
1.00
170.17
C

ATOM
1607
CG
ARG
A
121
−14.703
48.161
−57.320
1.00
170.17
C

ATOM
1608
CD
ARG
A
121
−14.844
46.697
−57.647
1.00
170.17
C

ATOM
1609
NE
ARG
A
121
−15.695
46.486
−58.814
1.00
170.17
N

ATOM
1610
CZ
ARG
A
121
−15.829
45.321
−59.442
1.00
170.17
C

ATOM
1611
NH1
ARG
A
121
−15.165
44.249
−59.020
1.00
170.17
N + 1

ATOM
1612
NH2
ARG
A
121
−16.623
45.220
−60.503
1.00
170.17
N

ATOM
1613
N
GLN
A
122
−11.056
48.648
−54.274
1.00
173.38
N

ATOM
1614
CA
GLN
A
122
−10.527
49.201
−53.045
1.00
173.38
C

ATOM
1615
C
GLN
A
122
−9.061
49.516
−53.105
1.00
173.38
C

ATOM
1616
O
GLN
A
122
−8.293
48.928
−53.871
1.00
173.38
O

ATOM
1617
CB
GLN
A
122
−10.768
48.236
−51.870
1.00
173.38
C

ATOM
1618
CG
GLN
A
122
−12.215
47.778
−51.692
1.00
173.38
C

ATOM
1619
CD
GLN
A
122
−13.138
48.858
−51.152
1.00
173.38
C

ATOM
1620
NE2
GLN
A
122
−12.575
49.958
−50.641
1.00
173.38
N

ATOM
1621
OE1
GLN
A
122
−14.377
48.722
−51.182
1.00
173.38
O

ATOM
1622
N
ASN
A
123
−8.698
50.472
−52.261
1.00
174.37
N

ATOM
1623
CA
ASN
A
123
−7.342
50.891
−51.966
1.00
174.37
C

ATOM
1624
C
ASN
A
123
−6.968
50.146
−50.680
1.00
174.37
C

ATOM
1625
O
ASN
A
123
−7.594
50.332
−49.631
1.00
174.37
O

ATOM
1626
CB
ASN
A
123
−7.247
52.417
−51.830
1.00
174.37
C

ATOM
1627
CG
ASN
A
123
−5.859
52.925
−51.513
1.00
174.37
C

ATOM
1628
ND2
ASN
A
123
−5.671
54.228
−51.555
1.00
174.37
N

ATOM
1629
OD1
ASN
A
123
−4.940
52.164
−51.235
1.00
174.37
O

ATOM
1630
N
PHE
A
124
−5.984
49.263
−50.775
1.00
172.43
N

ATOM
1631
CA
PHE
A
124
−5.616
48.409
−49.653
1.00
172.43
C

ATOM
1632
C
PHE
A
124
−4.347
48.853
−48.973
1.00
172.43
C

ATOM
1633
O
PHE
A
124
−3.703
48.060
−48.285
1.00
172.43
O

ATOM
1634
CB
PHE
A
124
−5.471
46.972
−50.150
1.00
172.43
C

ATOM
1635
CG
PHE
A
124
−6.758
46.416
−50.702
1.00
172.43
C

ATOM
1636
CD1
PHE
A
124
−7.025
46.458
−52.069
1.00
172.43
C

ATOM
1637
CD2
PHE
A
124
−7.714
45.870
−49.856
1.00
172.43
C

ATOM
1638
CE1
PHE
A
124
−8.219
45.942
−52.578
1.00
172.43
C

ATOM
1639
CE2
PHE
A
124
−8.905
45.355
−50.364
1.00
172.43
C

ATOM
1640
CZ
PHE
A
124
−9.149
45.389
−51.721
1.00
172.43
C

ATOM
1641
N
SER
A
125
−4.009
50.131
−49.126
1.00
170.68
N

ATOM
1642
CA
SER
A
125
−2.811
50.710
−48.542
1.00
170.68
C

ATOM
1643
C
SER
A
125
−2.758
50.545
−47.018
1.00
170.68
C

ATOM
1644
O
SER
A
125
−1.673
50.323
−46.491
1.00
170.68
O

ATOM
1645
CB
SER
A
125
−2.699
52.184
−48.911
1.00
170.68
C

ATOM
1646
OG
SER
A
125
−2.619
52.347
−50.318
1.00
170.68
O

ATOM
1647
N
ARG
A
126
−3.907
50.608
−46.321
1.00
168.27
N

ATOM
1648
CA
ARG
A
126
−3.945
50.488
−44.864
1.00
168.27
C

ATOM
1649
C
ARG
A
126
−3.897
49.032
−44.362
1.00
168.27
C

ATOM
1650
O
ARG
A
126
−3.842
48.799
−43.153
1.00
168.27
O

ATOM
1651
CB
ARG
A
126
−5.195
51.187
−44.327
1.00
168.27
C

ATOM
1652
N
CYS
A
127
−3.901
48.065
−45.277
1.00
168.87
N

ATOM
1653
CA
CYS
A
127
−3.865
46.641
−44.941
1.00
168.87
C

ATOM
1654
C
CYS
A
127
−2.447
46.164
−44.735
1.00
168.87
C

ATOM
1655
O
CYS
A
127
−2.234
45.062
−44.222
1.00
168.87
O

ATOM
1656
CB
CYS
A
127
−4.547
45.833
−46.036
1.00
168.87
C

ATOM
1657
SG
CYS
A
127
−6.252
46.321
−46.353
1.00
168.87
S

ATOM
1658
N
LEU
A
128
−1.474
46.978
−45.155
1.00
167.63
N

ATOM
1659
CA
LEU
A
128
−0.057
46.638
−45.105
1.00
167.63
C

ATOM
1660
C
LEU
A
128
0.444
46.384
−43.691
1.00
167.63
C

ATOM
1661
O
LEU
A
128
1.410
45.641
−43.515
1.00
167.63
O

ATOM
1662
CB
LEU
A
128
0.765
47.720
−45.801
1.00
167.63
C

ATOM
1663
CG
LEU
A
128
0.467
47.893
−47.303
1.00
167.63
C

ATOM
1664
CD1
LEU
A
128
1.602
48.589
−47.992
1.00
167.63
C

ATOM
1665
CD2
LEU
A
128
0.222
46.551
−47.993
1.00
167.63
C

ATOM
1666
N
GLU
A
129
−0.240
46.948
−42.689
1.00
165.82
N

ATOM
1667
CA
GLU
A
129
0.091
46.711
−41.294
1.00
165.82
C

ATOM
1668
C
GLU
A
129
−0.970
45.811
−40.711
1.00
165.82
C

ATOM
1669
O
GLU
A
129
−2.124
46.228
−40.590
1.00
165.82
O

ATOM
1670
CB
GLU
A
129
0.197
48.028
−40.519
1.00
165.82
C

ATOM
1671
N
LEU
A
130
−0.599
44.558
−40.405
1.00
164.53
N

ATOM
1672
CA
LEU
A
130
−1.519
43.585
−39.822
1.00
164.53
C

ATOM
1673
C
LEU
A
130
−1.851
44.018
−38.421
1.00
164.53
C

ATOM
1674
O
LEU
A
130
−0.956
44.435
−37.694
1.00
164.53
O

ATOM
1675
CB
LEU
A
130
−0.909
42.183
−39.828
1.00
164.53
C

ATOM
1676
CG
LEU
A
130
−1.122
41.349
−41.090
1.00
164.53
C

ATOM
1677
CD1
LEU
A
130
−0.316
41.888
−42.277
1.00
164.53
C

ATOM
1678
CD2
LEU
A
130
−0.760
39.901
−40.842
1.00
164.53
C

ATOM
1679
N
GLN
A
131
−3.125
43.965
−38.044
1.00
167.56
N

ATOM
1680
CA
GLN
A
131
−3.537
44.430
−36.723
1.00
167.56
C

ATOM
1681
C
GLN
A
131
−4.198
43.337
−35.884
1.00
167.56
C

ATOM
1682
O
GLN
A
131
−4.838
42.443
−36.426
1.00
167.56
O

ATOM
1683
CB
GLN
A
131
−4.482
45.626
−36.853
1.00
167.56
C

ATOM
1684
CG
GLN
A
131
−3.775
46.940
−37.188
1.00
167.56
C

ATOM
1685
CD
GLN
A
131
−4.663
47.913
−37.939
1.00
167.56
C

ATOM
1686
NE2
GLN
A
131
−4.749
49.141
−37.443
1.00
167.56
N

ATOM
1687
OE1
GLN
A
131
−5.254
47.593
−38.980
1.00
167.56
O

ATOM
1688
N
CYS
A
132
−4.041
43.420
−34.549
1.00
172.25
N

ATOM
1689
CA
CYS
A
132
−4.608
42.469
−33.571
1.00
172.25
C

ATOM
1690
C
CYS
A
132
−5.870
43.095
−32.905
1.00
172.25
C

ATOM
1691
O
CYS
A
132
−5.887
44.313
−32.721
1.00
172.25
O

ATOM
1692
CB
CYS
A
132
−3.564
42.049
−32.533
1.00
172.25
C

ATOM
1693
SG
CYS
A
132
−1.950
41.573
−33.218
1.00
172.25
S

ATOM
1694
N
GLN
A
133
−6.982
42.324
−32.687
1.00
169.42
N

ATOM
1695
CA
GLN
A
133
−8.271
42.876
−32.175
1.00
169.42
C

ATOM
1696
C
GLN
A
133
−8.228
43.303
−30.707
1.00
169.42
C

ATOM
1697
O
GLN
A
133
−9.029
44.145
−30.296
1.00
169.42
O

ATOM
1698
CB
GLN
A
133
−9.437
41.891
−32.372
1.00
169.42
C

END

CRYSTAL STRUCTURE OF FLT3 LIGAND-RECEPTOR COMPLEX

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Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

Claims

Priority Claims (1)

PCT Information