Identification of receptor and heparin binding sites in FGF4 by structure-based mutagenesis

Abstract

The present invention provides methods and compositions that may be used to modulate binding of a fibroblast growth factor (FGF) polypeptide to an FGF receptor (FGFR). In preferred embodiments, the methods and compositions of the invention modulate binding of a particular FGF polypeptide, the FGF4 polypeptide, to its receptor. The invention provides, in particular, variant FGF polypeptides that have at least one amino acid residue substitutions, insertion or deletion which alters the polypeptdies' binding affinity for an FGFR. The invention also provides models for the three-dimensional structure of a dimerized complex of FGF-FGFR-heparin. Using these models, key amino acid residues are identified and novel compounds (including novel variants of FGF and FGFR) can be identified which modulate FGF binding to its receptor. Such new compounds are therefore useful, e.g., as agonist and antagonist for FGF signaling and for bioactivities associated therewith.

Description

FIELD OF THE INVENTION

[0003] The present invention relates to a class of proteins known as fibroblast growth factor (FGF) proteins or FGF ligands. The invention also relates to receptors, known as fibroblast growth factor receptors (FGFRs), that recognize and specifically bind to FGF proteins. The invention further relates to a particular FGF polypeptide, referred to as FGF4. The methods and compositions of the invention relate to novel methods and compositions, including novel polypeptides and other compounds, that modulate FGF binding to its receptor and may be used, e.g., as agonists or antagonists to modulate FGF receptor activity and/or biological activities that are associated with FGF.

2. BACKGROUND OF THE INVENTION

[0004] The fibroblast growth factor (FGF) family of proteins comprises at least 22 polypeptides, referred to as FGF1-FGF22, with diverse biological activities. For reviews, see, McKeehan et al., Prog. Nucleic Acid Res. Mol. Biol. 1998, 59:135-176; Nishimura et al., Biochim Biophys. Acta. 2000, 1492:203-206; Yamashita et al., Biochem. Biophys. Res. Commun. 2000, 277:494-498. For example, FGF polypeptides modulate the proliferation and differentiation of a variety of cells of mesenchymal and neuro-ectodermal origin (Basilico & Moscatelli, Adv. Cancer Res. 1992, 59:115-165). FGF polypeptides also play critical roles during embryonic processes such as mesoderm induction, post-implantation blastocyst development, and limb and lung development (Goldfarb, Cytokine Growth Factor Rev. 1996, 7:311-325; Xu et al., Cell Tissue Res. 1999, 296:33-43). Increased FGF signaling leads to a variety of human skeletal disorders, including dwarfism and craniosynostosis syndromes (McIntosh et al., Cell Struct. Funct. 2000, 25:85-96; Naski & Omitz, Front. Biosci. 1998, 3:D781-D794; Wilkie, Hum. Mol. Genet. 1997, 6:1647-1656). In adult organisms FGFs are thought to be involved in physiological angiogenesis and wound healing as well as in pathological angiogenesis such as in tumor neovascularization and diabetic retinopathy (Basilico & Moscatelli, Adv. Cancer Res. 1992, 59:115-165).

[0005] The diverse effects of FGFs are mediated by at least four receptor tyrosine kinases, which are referred to collectively as the FGF receptor (FGFR) polypeptides and are known individually as FGFR1-FGFR4. The FGFR polypeptides comprise an extracellular domain, a single transmembrane helix and a cytoplasmic portion. The extracellular domain binds to the FGF polypeptide ligand, and may be subdivided into at least three distinct three immunoglobulin (Ig)-like domains, known as D1-D3, with each domain being connected by a “linker” polypeptide sequence. Ligand binding and specificity resides in the D2 and D3 domains and the short D2-D3 linker (Plotnikov et al., Cell 1999, 98:641-650; Plotnikov et al., Cell 2000, 101:413-424; Stauber et al., Proc. Natl. Acad. Sci. U.S.A. 2000, 97:49-54).

[0006] FGFR dimerization is prerequisite for FGF signaling and requires heparin or heparan sulfate proteoglycans (HSPG) (Ornitz, Bioessays 2000, 22:108-112; Schlessinger, Cell 2000, 103:211-225). The recent crystal structure of a ternary FGF2-FGFR1-heparin complex has provided a mechanistic view of the process by which heparin aids FGF polypeptides to induce FGFR dimerization (Schlessinger et al., Mol. Cell 2000, 6:743-750). According to this “two end” model, heparin interacts via its non-reducing end with the heparin binding sites of FGF and FGFR and promotes the formation of a ternary 1:1:1 FGF:FGFR:heparin complex. A second ternary 1:1:1 FGF:FGFR:heparin complex is then recruited to the first ternary complex via interactions of FGFR, FGF and heparin in one ternary complex with the FGFR in the adjoining ternary complex.

[0007] A fundamentally different model for FGFR dimerization has emerged from the recent crystal structure of a dimeric FGF1-FGFR2-heparin ternary complex (Pellegrini et al., Nature 2000, 407:1029-1034). In this structure, a single heparin molecule links two FGF ligands into a dimer that bridges between two receptor chains. The asymmetric heparin binding involves contacts with both FGF molecules but only one receptor chain. There is essentially no protein-protein interface between the two 1:1 FGF-FGFR complexes in the dimer.

[0008] With the exception of FGF1, which is the universal ligand for all FGFRs, most FGF polypeptides exhibit specific, albeit promiscuous, patterns of receptor binding affinity (Ornitz et al., J. Biol. Chem. 1996, 271:15292-15297). Comparison of the crystal structures of FGF1-FGFR1, FGF2-FGFR1 and FGF2-FGFR2 complexes defined a general binding interface for FGF-FGFR complexes involving contacts made by FGF to D2 and to the D2-D3 linker (Plotnikov et al., Cell 2000, 101:413-424). It has also been shown that specificity is achieved through interactions of the FGF N-terminal (i.e., the amino acid sequence immediately preceding the FGF polypeptide's β-trefoil core domain) and central regions with FGFR D3. These structures have also provided a molecular basis for how alternative splicing in FGFR modulates specificity. In both FGF2-FGFR1 and FGF2-FGFR2 structures, FGF2 makes specific contacts with the βC′-βE loop in D3, which is subject to alternative splicing. Consequently, FGF2 discriminates between the IIIc and IIIb variants of FGFRs. In contrast, FGF1 does not interact with the βC′-βE loop and therefore can bind all FGFRs irrespective of alternative splicing in D3 (Plotnikov et al., Cell 2000, 101:413-424).

[0009] FGF4 shares about 30% sequence identity with the prototypical members of the FGF family, FGF1 and FGF2 (Delli Bovi et al., Cell 1987, 50:729-737). FGF4, unlike FGF1 and FGF2, has a classical signal peptidp and thus is efficiently secreted from cells (Bellosta et al., J. Cell Biol. 1993, 121:705-713). Most receptor binding studies indicate that FGF4 binds and activates the IIIc splice forms of FGFR1-3 to comparable levels, but it shows little activity towards the IIIb splice forms of FGFR1-3 as well as towards FGFR4 (Ornitz et al., J. Biol. Chem. 1996, 271:15292-15297; Vainikka et al., EMBO J. 1993, 11:4273-4280). As for FGF1 and FGF2, heparin greatly augments the biological activity of FGF4 on cells lacking endogenous cell surface HSPG (Mansukhani et al., Proc. Natl. Acad. Sci. U.S.A. 1992, 89:3305-3309). However, employing selectively O-desulfated heparins, Guimond et al. (J. Biol. Chem. 1993, 268:23906-23914) have shown that both 2-O-and 6-O-desulfated heparin were able to support the mitogenic activity of FGF4, while neither of these heparins could support the biological activity of FGF1 and FGF2. It has therefore been suggested the sulfation motifs in heparin required for FGF4 activity may differ from those required for FGF1 and FGF2 actions (Guimond et al., supra; Ishihara, Glycobiology 1994, 4:817-824).

[0010] In summary, the exact interactions that stabilize complexes of the FGF4 polypeptide with its receptor and/or heparin remain poorly understood. Yet, given the range of biological disorders associated with FGF signaling, there is an urgent need to identify and characterize these interactions. There exist, moreover, a need to identify compounds that modulate binding of FGF4 to either an FGF receptor or heparin, including mutant or variant forms of the FGF4 polypeptide that have altered binding affinities, as well as other compounds that may be agonists or antagonists of FGF4 binding and/or activity.

3. SUMMARY OF THE INVENTION

[0011] The present invention provides mutant FGF4 polypeptides, wherein at least one amino acid residue in the primary binding site, the secondary binding site or the heparin binding site is different from the wild-type FGF4 molecule.

[0012] In preferred embodiments, the invention provides the following mutant FGF4 polypeptides, each containing the substitution at the indicated residue with alanine: tyrosine at amino acid residue 87; phenylalanine at amino acid residue 129; phenylalanine at amino acid residue 151; glutamic acid at amino acid residue 159; phenylalanine at amino acid residue 166; leucine at amino acid residue 203; arginine at amino acid residue 205; asparagine at amino acid residue 89; lysine at amino acid residue 198; asparagine at amino acid residue 89; lysine at amino acid residue 183; lysine at amino acid residue 188; lysine at amino acid residue 183; arginine at amino acid residue 103; lysine at amino acid residue 144; and arginine at amino acid residue 103.

[0013] In another preferred embodiment, the invention provides a mutant FGF4 polypeptide containing two alanine substitutions at lysine residue 144 and arginine residue 103.

[0014] The invention further provides a crystal of FGF4, the crystal belonging to the orthorhombic space group P212121 and having unit cell dimensions a=40.37, b=53.3 and c=56.23.

[0015] In a preferred embodiment, the crystal comprises an FGF4 polypeptide having the amino acid sequence depicted in FIG. 2 (top line).

[0016] The present invention also provides a method for producing mutant FGF4 polypeptides, and methods of testing mutant FGF4 polypeptides for increased or decreased binding and/or activity.

[0017] Crystalline forms of FGF4 are also provided by the present invention.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 is a ribbon diagram illustrating the tertiary structure for an FGF4 polypeptide whose amino acid sequence is set forth in FIG. 2, below. The secondary structure assignments were assigned using the program PRODCHECK (Laskowski et al., J. Appl. Cryst. 1993, 26:283-291), and the figure created using the programs Molscript (Kraulis, J. Appl. Crystallogr. 1991, 24:946-950) and Raster3D (Merritt & Bacon, Methods Enzymol. 1997, 277:505-524). β-strands are labeled according to conventional strand nomenclature for FGF1 and FGF2 (see, Faham, Curr. Opin. Struct. Biol. 1998, 8:578-586). NT and CT denote the amino- and carbyoxy-termini, respectfully.

[0019] FIG. 2A-F provides a structure based sequence alignment of the FGF4 polypeptide amino acid sequence (top line) whose structure is illustrated in FIG. 1A, and related human FGF polypeptides FGF6, FGF1 and FGF2. The alignment was generated using the program CLUSTALW (Stauber et al., Proc. Natl. Acad. Sci. U.S.A. 2000, 97:49-54). The location and length of the β-strands and α-helices of the proteins' structures are indicated on top of the alignment, and the box indicates boundaries of the β-trefoil core region or domain. Periods in the aligned sequences indicated amino acid residues that are identical to the amino acid residue of FGF4 at that position of the alignment, whereas dashes represent gaps introduced to optimize the alignment. Residues with the β-trefoil core region are labeled to indicate the region of FGFR1 with which they interact: residues that interact with D2 are shaded (2B), residues that interact with the FGFR1 linker region (2F), residues that interact with the D3 domain of FGFR1 (2A), and FGFR residues that interact with the βC′-βE loop in the FGFR1 D3 domain are also shaded (2E). Also, amino acid residues that constitute the conventional “low” and “high” affinity heparin binding sites (2C) and FGF4 amino acid residues that localize to the periphery of the “high” affinity heparin binding site (and may, therefore interact with heparin-2D) are also indicated. A star indicates those amino acid residues that are tested in the Examples, infra, by site-directed mutagenesis.

[0020] FIG. 3 provides a representative model (generated using the Molscript and Raster3D programs) for the three-dimensional structure of an FGF4-FGFR1 complex generated by superimposition of Cα traces within the β-trefoil of a structure for unbound FGF4 onto corresponding Cα traces of FGF2 in a structure for an FGF2-FGFR1 complex (see the Examples, infra). FGF4, D2 of FGFR1, D3 of FGFR1, and the D2-D3 are labeled. NT and CT denote the amino- and carboxy-termini, respectfully.

[0021] FIG. 4 shows a plot comparing the binding affinities of various FGF4 mutants twoards FGFP2 Data are expressed as percent inhibition of wild-type FGF4 binding to FGFR2 by the indicated amount of unlabeled mutant FGF4.

[0022] FIGS. 5A-B show plots illustrating the differential effect of heparin on stimulation of DNA synthesis in NIH 3T3 cells by different mutant FGF4 polypeptides that are described in the Examples, infra. FIG. 5A plots data from a representative experiments using the E159A and L203A mutants. FIG. 5B plots data from representative experiments using the K183A/K188A and the N89A/K198A double mutants.

[0023] FIG. 6 provides a representative model (generated using the Molscript and Raster3D programs) for a dimeric complex of FGF4-FGFR1-heparin. The model was created by superimposition of the Cα traces for two FGF4 structures onto Cα traces of the two FGF2 molecules in a ternary structure for FGF2-FGFR1-heparin (see, the Examples, infra). NT and CT denote the amino- and carboxy-termini, respectively, of the FGFR1 polypeptide. The FGF4, FGFR1, D2 and D3 domains are labeled. Heparin oligosaccharides are rendered in ball-and-stick.

[0024] FIG. 7A-B shows the primary binding site, i.e., the surface through which FGF4 binds to FGFR in the context of the FGF4-FGFR-heparin dimer. The shaded region in the three-dimensional model (7A), and in the amino acid sequence (7B) depicts the primary binding sites. The primary binding sites in the amino acid sequences are also shown for FGF6 and FGF1 in 7B.

[0025] FIG. 8A-B shows the secondary binding site, i.e., the surface which binds to a second FGFR molecule in the context of the FGF4-FGFR-heparin dimer. The secondary binding site is depicted as shaded in both the three-dimensional model (8A), and the amino acid sequence (8B). The secondary binding sites in the amino acid sequences are also shown for FGF6 and FGF1 in 8B.

[0026] FIG. 9A-B shows the heparin binding site, i.e., the surface which binds to heparin in the context of the FGF4-FGFR-heparin dimer. The primary binding site is depicted as shaded in both the three-dimensional model (9A), and the amino acid sequence (9B). The heparin binding sites in the amino acid sequences are also shown for FGF6 and FGF1 in 9B.

5. DETAILED DESCRIPTION OF THE INVENTION

[0027] The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them.

5.1 General Definitions

[0028] As used herein, the term “isolated” means that the referenced material is removed from the environment in which it is normally found. Thus, an isolated biological material can be free of cellular components, i.e., components of the cells in which the material is found or produced. In the case of nucleic acid molecules, an isolated nucleic acid includes a PCR product, an isolated mRNA, a cDNA, or a restriction fragment. In another embodiment, an isolated nucleic acid is preferably excised from the chromosome in which it may be found, and more preferably is no longer joined to non-regulatory, non-coding regions, or to other genes, located upstream or downstream of the gene contained by the isolated nucleic acid molecule when found in the chromosome. In yet another embodiment, the isolated nucleic acid lacks one or more introns. Isolated nucleic acid molecules include sequences inserted into plasmids, cosmids, artificial chromosomes, and the like. Thus, in a specific embodiment, a recombinant nucleic acid is an isolated nucleic acid. An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein. An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism. An isolated material may be, but need not be, purified.

[0029] The term “purified” as used herein refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, i.e., contaminants, including native materials from which the material is obtained. For example, a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell; a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell. As used herein, the term “substantially free” is used operationally, in the context of analytical testing of the material. Preferably, purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.

[0030] Methods for purification are well-known in the art. For example, nucleic acids can be purified by precipitation, chromatography (including preparative solid phase chromatography, oligonucleotide hybridization, and triple helix chromatography), ultracentrifrigation, and other means. Polypeptides and proteins can be purified by various methods including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, precipitation and salting-out chromatography, extraction, and countercurrent distribution. For some purposes, it is preferable to produce the polypeptide in a recombinant system in which the protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence, or a sequence that specifically binds to an antibody, such as FLAG and GST. The polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix. Alternatively, antibodies produced against the protein or against peptides derived therefrom can be used as purification reagents. Cells can be purified by various techniques, including centrifugation, matrix separation (e.g., nylon wool separation), panning and other immunoselection techniques, depletion (e.g., complement depletion of contaminating cells), and cell sorting (e.g., fluorescence activated cell sorting [FACS]). Other purification methods are possible. A purified material may contain less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the cellular components with which it was originally associated. The “substantially pure” indicates the highest degree of purity which can be achieved using conventional purification techniques known in the art.

[0031] A “sample” as used herein refers to a biological material which can be tested, e.g., for the presence of an FGF polypeptide or FGF nucleic acid or, alternatively, for the presence of an FGFR polypeptide or nucleic acid (e.g., to identify cells that specifically express either FGF or FGFR). Such samples can be obtained from any source, including tissue, blood and blood cells, and cell culture.

[0032] Non-human animals include, without limitation, laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, etc.; domestic animals such as dogs and cats; and farm animals such as sheep, goats, pigs, horses, and cows.

[0033] In preferred embodiments, the terms “about” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.

[0034] The term “molecule” means any distinct or distinguishable structural unit of matter comprising one or more atoms, and includes, for example, polypeptides and polynucleotides.

[0035] The term “therapeutically effective dose” refers to that amount of a compound or compositions that is sufficient to result in a desired activity.

[0036] The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar adverse reaction (for example, gastric upset, dizziness and the like) when administered to an individual. Preferably, and particularly where a vaccine is used in humans, the term “pharmaceutically acceptable” may mean approved by a regulatory agency (for example, the U.S. Food and Drug Administration) or listed in a generally recognized pharmacopeia for use in animals (for example, the U.S. Pharmacopeia).

[0037] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Sterile water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Exemplary suitable pharmaceutical carriers are described in “Reminington's Pharmaceutical Sciences” by E. W. Martin.

5.2 Molecular Biology Definitions

[0038] In accordance with the present invention, there may be employed conventional molecular biology, microbiology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook, Fitsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (referred to herein as “Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins, eds. 1984); Animal Cell Culture (R. I. Freshney, ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. E. Perbal, A Practical Guide to Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0039] The term “polymer” means any substance or compound that is composed of two or more building blocks (‘mers’) that are repetitively linked together. For example, a “dimer” is a compound in which two building blocks have been joined togther; a “trimer” is a compound in which three building blocks have been joined together; etc.

[0040] The term “polynucleotide” or “nucleic acid molecule” as used herein refers to a polymeric molecule having a backbone that supports bases capable of hydrogen bonding to typical polynucleotides, wherein the polymer backbone presents the bases in a manner to permit such hydrogen bonding in a specific fashion between the polymeric molecule and a typical polynucleotide (e.g., single-stranded DNA). Such bases are typically inosine, adenosine, guanosine, cytosine, uracil and thymidine. Polymeric molecules include “double stranded” and “single stranded” DNA and RNA, as well as backbone modifications thereof (for example, methylphosphonate linkages).

[0041] Thus, a “polynucleotide” or “nucleic acid” sequence is a series of nucleotide bases (also called “nucleotides”), generally in DNA and RNA, and means any chain of two or more nucleotides. A nucleotide sequence frequently carries genetic information, including the information used by cellular machinery to make proteins and enzymes. The terms include genomic DNA, cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and antisense polynucleotides. This includes single- and double-stranded molecules; i.e., DNA-DNA, DNA-RNA, and RNA-RNA hybrids as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases, for example, thio-uracil, thio-guanine and fluoro-uracil.

[0042] The polynucleotides herein may be flanked by natural regulatory sequences, or may be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5′- and 3′-non-coding regions and the like. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Polynucleotides may contain one or more additional covalently linked moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.) and alkylators to name a few. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidite linkage. Furthermore, the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin and the like. Other non-limiting examples of modification which may be made are provided, below, in the description of the present invention.

[0043] A “polypeptide” is a chain of chemical building blocks called amino acids that are linked together by chemical bonds called “peptide bonds”. The term “protein” refers to polypeptides that contain the amino acid residues encoded by a gene or by a nucleic acid molecule (e.g., an mRNA or a cDNA) transcribed from that gene either directly or indirectly. Optionally, a protein may lack certain amino acid residues that are encoded by a gene or by an mRNA. For example, a gene or mRNA molecule may encode a sequence of amino acid residues on the N-terminus of a protein (i.e., a signal sequence) that is cleaved from, and therefore may not be part of, the final protein. A protein or polypeptide, including an enzyme, may be a “native” or “wild-type”, meaning that it occurs in nature; or it may be a “mutant”, “variant” or “modified”, meaning that it has been made, altered, derived, or is in some way different or changed from a native protein or from another mutant. See Appendix A for the three letter and one letter abbreviations for 20 amino acids.

[0044] A “ligand” is, broadly speaking, any molecule that binds to another molecule. In preferred embodiments, the ligand is either a soluble molecule or the smaller of the two molecules or both. The other molecule is referred to as a “receptor”. In preferred embodiments, both a ligand and its receptor are molecules (preferably proteins or polypeptides) produced by cells. Preferably, a ligand is a soluble molecule and the receptor is an integral membrane protein (i.e., a protein expressed on the surface of a cell). In a particularly preferred embodiment of the invention the ligand is a fibroblast growth factor (FGF) and the receptor is a fibroblast growth factor receptor (FGFR).

[0045] The binding of a ligand to its receptor is frequently a step of signal transduction with a cell. For example, in preferred embodiments where a ligand is an FGF polypeptide and a receptor is an FGFR polypeptide, the binding of FGF to the FGFR polypeptide may lead to activation of a tyrosine kinase activity within the FGFR polypeptide. Activation of the tyrosine kinase activity may, in turn, initiate other activities associated with FGF signaling, including but not limited to mitogenesis and angiogensis. Other exemplary ligand-receptor interactions include, but are not limited to, binding of a hormone to a hormone receptor (for example, the binding of estrogen to the estrogen receptor) and the binding of a neurotransmitter to a receptor on the surface of a neuron.

[0046] “Amplification” of a polynucleotide, as used herein, denotes the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixture of DNA sequences. For a description of PCR see Saiki et al., Science 1988, 239:487.

[0047] “Chemical sequencing” of DNA denotes methods such as that of Maxam and Gilbert (Maxam-Gilbert sequencing; see Maxam & Gilbert, Proc. Natl. Acad. Sci. U.S.A. 1977, 74:560), in which DNA is cleaved using individual base-specific reactions.

[0048] “Enzymatic sequencing” of DNA denotes methods such as that of Sanger (Sanger et al., Proc. Natl. Acad. Sci. U.S.A. 1977, 74:5463) and variations thereof well known in the art, in a single-stranded DNA is copied and randomly terminated using DNA polymerase.

[0049] A “gene” is a sequence of nucleotides which code for a functional “gene product”. Generally, a gene product is a functional protein. However, a gene product can also be another type of molecule in a cell, such as an RNA (e.g., a tRNA or a rRNA). For the purposes of the present invention, a gene product also refers to an mRNA sequence which may be found in a cell. For example, measuring gene expression levels according to the invention may correspond to measuring mRNA levels. A gene may also comprise regulatory (i.e., non-coding) sequences as well as coding sequences. Exemplary regulatory sequences include promoter sequences, which determine, for example, the conditions under which the gene is expressed. The transcribed region of the gene may also include untranslated regions including introns, a 5′-untranslated region (5′-UTR) and a 3′-untranslated region (3′-UTR).

[0050] A “coding sequence” or a sequence “encoding” an expression product, such as an RNA, polypeptide, protein or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein or enzyme; i.e., the nucleotide sequence “encodes” that RNA or it encodes the amino acid sequence for that polypeptide, protein or enzyme.

[0051] A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently found, for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

[0052] A coding sequence is “under the control of” or is “operatively associated with” transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into RNA, which is then trans-RNA spliced (if it contains introns) and, if the sequence encodes a protein, is translated into that protein.

[0053] The term “express” and “expression” means allowing or causing the information in a gene or DNA sequence to become manifest, for example producing RNA (such as rRNA or mRNA) or a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed by a cell to form an “expression product” such as an RNA (e.g., a mRNA or a rRNA) or a protein. The expression product itself, e.g., the resulting RNA or protein, may also be said to be “expressed” by the cell.

[0054] The term “transfection” means the introduction of a foreign nucleic acid into a eukaryotic cell. The term “transformation” means the introduction of a “foreign” (i.e., extrinsic or extracellular) gene, DNA or RNA sequence into a prokaryotic host cell so that the host cell will express the introduced gene or sequence to produce a desired substance, in this invention typically an RNA coded by the introduced gene or sequence, but also a protein or an enzyme coded by the introduced gene or sequence. The introduced gene or sequence may also be called a “cloned” or “foreign” gene or sequence, may include regulatory or control sequences (e.g., start, stop, promoter, signal, secretion or other sequences used by a cell's genetic machinery). The gene or sequence may include nonfunctional sequences or sequences with no known function. A host cell that receives and expresses introduced DNA or RNA has been “transformed” and is a “transformant” or a “clone”. The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell or cells of a different genus or species.

[0055] The terms “vector”, “cloning vector” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence. Vectors may include plasmids, phages, viruses, etc. and are discussed in greater detail below.

[0056] A “cassette” refers to a DNA coding sequence or segment of DNA that codes for an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame. Generally, foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA. A segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a “DNA construct.” A common type of vector is a “plasmid”, which generally is a self-contained molecule of double-stranded DNA, usually of bacterial origin, that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. The term “host cell” means any cell of any organism that is selected, modified, transformed, grown or used or manipulated in any way for the production of a substance by the cell. For example, a host cell may be one that is manipulated to express a particular gene, a DNA or RNA sequence, a protein or an enzyme. Host cells can further be used for screening or other assays that are described infra. Host cells may be cultured in vitro or one or more cells in a non-human animal (e.g., a transgenic animal or a transiently transfected animal).

[0057] The term “expression system” means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells such as Sf9, Hi5 or S2 cells and Baculovirus vectors, Drosophila cells (Schneider cells) and expression systems, and mammalian host cells and vectors.

[0058] The term “heterologous” refers to a combination of elements not naturally occurring. For example, heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. Preferably, the heterologous DNA includes a gene foreign to the cell. A heterologous expression regulatory element is a such an element operatively associated with a different gene than the one it is operatively associated with in nature. In the context of the present invention, a gene encoding a protein of interest, e.g., an FGF4 gene, is heterologous to the vector DNA in which it is inserted for cloning or expression, and it is heterologous to a host cell containing such a vector, in which it is expressed, e.g., a CHO cell. with a different gene that the one it is operatively associated with in nature.

[0059] The terms “mutant” and “mutation” mean any detectable change in genetic material, e.g., DNA, or any process, mechanism or result of such a change. This includes gene mutations, in which the structure (e.g., DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (e.g., RNA, protein or enzyme) expressed by a modified gene or DNA sequence. The term “variant” may also be used to indicate a modified or altered gene, DNA sequence, RNA, enzyme, cell, etc.; i.e., any kind of mutant. For example, the present invention relates to altered or “chimeric” RNA molecules that comprise an rRNA sequence that is altered by inserting a heterologous RNA sequence that is not naturally part of that sequence or is not naturally located at the position of that rRNA sequence. Such chimeric RNA sequences, as well as DNA and genes that encode them, are also referred to herein as “mutant” sequences.

[0060] “Sequence-conservative variants” of a polynucleotide sequence are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position.

[0061] “Function-conservative variants” of a polypeptide or polynucleotide are those in which a given amino acid residue in the polypeptide, or the amino acid residue encoded by a codon of the polynucleotide, has been changed or altered without altering the overall conformation and function of the polypeptide. For example, function-conservative variants may include, but are not limited to, replacement of an amino acid with one having similar properties (for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic and the like). Amino acid residues with similar properties are well known in the art. For example, the amino acid residues arginine, histidine and lysine are hydrophilic, basic amino acid residues and may therefore be interchangeable. Similar, the amino acid residue isoleucine, which is a hydrophobic amino acid residue, may be replaced with leucine, methionine or valine. Such changes are expected to have little or no effect on the apparent molecular weight or isoelectric point of the polypeptide. Amino acid residues other than those indicated as conserved may also differ in a protein or enzyme so that the percent protein or amino acid sequence similarity (e.g., percent identity or homology) between any two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme such as the Cluster Method, wherein similarity is based on the MEGALIGN algorithm. “Function-conservative variants” of a given polypeptide also include polypeptides that have at least 60% amino acid sequence identity to the given polypeptide as determined, e.g., by the BLAST or FASTA algorithms. Preferably, function-conservative variants of a given polypeptide have at least 75%, more preferably at least 85% and still more preferably at least 90% amino acid sequence identity to the given polypeptide and, preferably, also have the same or substantially similar properties (e.g., of molecular weight and/or isoelectric point) or functions (e.g., biological functions or activities) as the native or parent polypeptide to which it is compared.

[0062] Thus, for example, in particular embodiments wherein the polypeptides are FGFR polypeptides, function-conservative variants may not only have between at least 75% and at least 90% amino acid sequence identity to a given FGFR, but preferably also have similar properties, such as conserved domains (e.g., as in a D1, D2 or D3 domain, described supra) and/or similar biological function or activities, such as a tyrosine kinase activity and/or the ability to stimulate activities associated with FGF signaling (e.g., mitogenesis or angiogenesis).

[0063] Similarly, in embodiments wherein a polypeptide is an FGF ligand, function-conservative variants may not only have between at least 75% and at least 90% amino acid sequence identity to a given FGF, but preferably also have similar properties. For example, a function-conservative variant of an FGF ligand preferably binds to the same FGF receptor as the FGF ligand. (preferably, but not necessarily with the same or a similar affinity; e.g., preferably with at least 50% of the binding affinity, more preferably with at least 70% of the binding affinity, and still more preferably with at least 80% or at least 90% of the binding affinity). Preferably, by binding to the FGFR polypeptide, a function-conservative variant will also stimulate a same biological function or activity that is associated with binding of the FGF ligand to the receptor, including any of the functions or activities described, supra, for an FGF receptor.

[0064] The term “homologous”, in all its grammatical forms and spelling variations, refers to the relationship between two proteins that possess a “common evolutionary origin”, including proteins from superfamilies (e.g., the immunoglobulin superfamily) in the same species of organism, as well as homologous proteins from different species of organism (for example, myosin light chain polypeptide, etc.; see, Reeck et al., Cell 1987, 50:667). Homologous proteins of the invention therefore include various FGF proteins and polypeptides derived from the same species of organism (i.e., the FGF family of polypeptides, including FGF1-FGF22), and also FGF proteins and polypeptides derived from different species of organisms. Similarly, homologous proteins of the invention also include various FGFR proteins and polypeptides derived from the same species (i.e., the FGFR family, including FGFR1-4) or from different species of organisms.

[0065] Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions. For instance, referring agin to particular embodiments where homologous polypeptides are FGF and/or FGFR polypeptides, homologous polypeptides in either the same or in closely related species of organisms (for example, between mammals such as mice and humans) typically share greater than 50% sequence identity, more preferably share at least about 60 to 65% sequence identity, and still more preferably share at least about 75% to 80% sequence identity. Homologous polypeptides between closely related species of organisms may also be cross reactive in both species of organisms. For example, an FGF from one species of organism may bind to and/or activate an FGF receptor polypeptide from a different species of organism and, moreover, an FGF receptor from a first species of organism may stimulate a activity associated with FGF signalling (e.g., mitogenesis or angiogenesis) in a cell from a different species of organism (for example, when the heterologous FGFR polypeptide is recombinantly expressed in that cell).

[0066] The term “sequence similarity”, in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin (see, Reeck et al., Cell 1987, 50:667). However, in common usage and in the instant application, the term “homologous”, particularly when modified with an adverb such as “highly”, may refer to sequence similarity and may or may not relate to a common evolutionary origin.

[0067] In specific embodiments, two nucleic acid sequences are “substantially homologous” or “substantially similar” when at least about 80%, and more preferably at least about 90% or at least about 95% of the nucleotides match over a defined length of the nucleic acid sequences, as determined by a sequence comparison algorithm known such as BLAST, FASTA, DNA Strider, CLUSTAL, etc. An example of such a sequence is an allelic or species variant of the specific genes of the present invention. Sequences that are substantially homologous may also be identified by hybridization, e.g., in a Southern hybridization experiment under, e.g., stringent conditions as defined for that particular system.

[0068] Similarly, in particular embodiments of the invention, two amino acid sequences are “substantially homologous” or “substantially similar” when greater than 80% of the amino acid residues are identical, or when greater than about 90% of the amino acid residues are similar (i.e., are functionally identical). Preferably the similar or homologous polypeptide sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison Wis.) pileup program, or using any of the programs and algorithms described above (e.g., BLAST, FASTA, CLUSTAL, etc.).

[0069] As used herein, the term “oligonucleotide” refers to a nucleic acid, generally of at least 10, preferably at least 15, and more preferably at least 20 nucleotides, preferably no more than 100 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest. Oligonucleotides can be labeled, e.g., with 32P-nucleotides or nucleotides to which a label, such as biotin or a fluorescent dye (for example, Cy3 or Cy5) has been covalently conjugated. In one embodiment, a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. In another embodiment, oligonucleotides (one or both of which may be labeled) can be used as PCR primers; e.g. for cloning full length or a fragment of either an FGF or an FGFR nucleic acid, or to detect the presence of nucleic acids encoding either an FGF or an FGFR polypeptide. In a further embodiment, an oligonucleotide of the invention can form a triple helix with a an FGF or an FGFR DNA or RNA molecule. Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.

5.3 X-ray Crystallography Definitions

[0070] The present invention also uses techniques of conventional X-ray crystallography. These techniques are well known and are within the routine skill of the art. Such techniques are described more fully in the literature. See, for example, Cantor & Schimmel, Biophysical Chemistry 1980 (Vols. I-III) W. H. Freeman and Company (particularly Chapters 1-13 in Vol. I, and Chapter 13 in Vol. II).

[0071] The term “crystal” refers, generally, to any ordered (or at least partially ordered) three-dimensional array of molecules. Preferably, the ordering of molecules within a crystal is at least sufficient to produce a sharp X-ray diffraction pattern so that the molecules' three-dimensional structure may be determined.

[0072] The molecules in a crystal may be of any type, and it will be understood that a crystal may contain molecules of only one type or may comprise a plurality of different types of molecules. In preferred embodiments, crystals of the present invention comprise at least one biomolecule, such as a protein, or a fragment thereof. Crystals of the invention may even comprise a complex or assembly of two or more proteins or other biomolecules. For example, a crystal may comprise two different proteins, such as a receptor and a ligand, or a crystal may comprise two more molecules of the same protein bound together, e.g., to form a dimer or other multimer complex. Typically, crystals that contain biological molecules such as proteins will contain other molecules as well, such molecules of solvent (e.g., water molecules) and/or salt. Other molecules such as drugs, drug candidates or compounds that bind to the protein may also be present in a crystal.

[0073] It will be understood by a skilled artisan that crystals of the invention comprises a “unit cell”, or basic parallelepiped shaped block defined by vectors denoted a, b and c. The entire volume of a crystal may be constructed by the regular assembly of such blocks or “lattices”. A crystal is also defined by the overall symmetry of elements (i.e., molecules) within the cell, which is referred to as the “space group.” Thus, a crystal's space group is defined by symmetry relations within the molecules making up the unit cell. The “asymmetric unit” is the smallest possible unit from which the crystal structure may be generated by making use of the symmetric relations defining the space group.

[0074] The term “structure coordinates” or “structure” refers to mathematical coordinates that define the position of atoms in a molecule or in an assembly of molecules in three-dimensional space (for example, within the asymmetric unit of a crystal). Structure coordinates may be computed or otherwise determined using any information related to the three dimensional arrangement of atoms in a molecule. However, in preferred embodiments of the invention a structure is derived from equations that are related to patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (which, in such embodiments, may also be referred to as “scattering centers”) in a crystal. Typically, such diffraction data is used to calculate an “electron density” map of the crystal's asymmetric unit, and these maps are used, in turn, to establish positions of the individual atoms.

[0075] “Heavy atom derivatization” refers to a method of producing chemically modified forms of a crystal (typically a crystal of a protein or other biopolymer), in which the crystal may be soaked in a solution containing heavy metal atom salts or organometallic compounds that can diffuse through the crystal and bind to the surface of the protein or biopolymer. The location(s) of one or more heavy meatl atoms in the crystal may then be determined by X-ray diffraction analysis of the soaked crystal, and this information may be used to facilitate construction of the three-dimension structure of the protein or other molecules contained in the crystal.

[0076] “Molecular replacement” refers to a method wherein a preliminary structure coordinates are generated for molecules in a crystal whose structure coordinates are not known. Generally, molecular replacement involves orienting and/or positioning another, preferably similar molecule (such as a homologous protein) whose structure coordinates are known. Phases for an X-ray diffraction pattern may then be determined for the preliminary structure, and these phases can then be combined with actual X-ray diffraction intensities that are observed for the crystal whose structure coordinates are not known, to determine its structure.

5.4 Detailed Description of Invention

[0077] The present invention relates to a particular FGF ligand, known in the art as FGF4 and described in the Examples, infra. Such FGF polypeptides generally comprise the amino acid sequence set forth in FIG. 2, or a similar (i.e., homologous or highly-homologous) amino acid sequence. In other preferred embodiments, the methods and compositions of the invention relate to certain variants of the FGF4 polypeptide, hereinafter alternatively referred to as FGF4 mutants or mutant polypeptides. Preferred variants of the FGF4 polypeptide are ones having altered binding affinities either for heparin, for an FGF receptor (e.g., FGFR1) or for both FGFR and heparin. For instance, the Examples, infra, describe several specific variants of FGF4 having one or more amino acid residue substitutions, insertions or deletions that alter the polypeptides' binding affinities for heparin and/or for an FGFR (in particular, for FGFR1). These variant FGF4 polypeptides are therefore considered part of the present invention.

[0078] The Examples also provide a description of model three-dimensional structures that describe the binding of FGF4 to an FGF receptor and to heparin. Using these models, a skilled artisan may readily identify amino acid residues of either FGF or FGFR that may be modified to produce molecules with greater or lower binding affinities that wild-type FGF4 and FGFR. These molecules are useful agonist and antagonist for FGF ligand-receptor binding and/or for bioactivities associated therewith. Accordingly, the compositions and methods of the invention include screening methods that identify such molecules, preferably using the models of the present invention and/or by modifying key amino acid residues identified in the Examples, below.

[0079] The present invention identifies three regions or domains, hereinafter referred to as (1) the primary binding site, which is the surface through which one FGF4 molecule binds to one FGFR molecule and forms a complex, (2) the secondary binding site, which is the surface which binds to a second FGFR molecule in the context of an FGF4: FGFR-heparin dimer and (3) the heparin binding site, which binds heparin. The primary binding site for the FGF4 polypeptide involves amino acid residue numbers (from FIG. 2) 80-82, 84, 87, 92, 95, 108, 117, 119-124, 126, 129, 136, 151, 159, 161, 165-167 and 203-205. The heparin binding site involves amino acid residue numbers 89, 90, 182-184, 186, 189, 192 and 197-199. The secondary binding site involves amino acid residue numbers 162, 163, 195, 196 and 201. These regions are shown in FIG. 6. Mutation in at least one of the amino acids in these regions may lead to the production of either antagonists or agonists. Such polypeptides can be tested for their ability to stimulate DNA synthesis, in the presence or in the absence of heparin, and/or for their ability to bind to receptors on CHO cells, as described below. Non-limiting examples of mutant FGF4 polypeptides produced pursuant to the present invention are shown in Table 2.

[0080] The identification of the three major domains or binding sites of FGF4 that are important for receptor binding and receptor activation could lead to the rational design of mutated FGF4 forms with altered biological activity or function. Such mutant forms of FGF4 could possess 1) higher receptor binding affinity and therefore higher biological potency, 2) the ability to bind to the receptor without inducing receptor dimerization, therefore acting as FGF receptor antagonists, or 3) the ability to interact with different forms of heparin or heparin sulfate proteoglycans, therefore possibly modifying its spectrum of action in a biological context.

[0081] This could be accomplished by mutating selected amino acids in the primary binding site, and the secondary binding site, respectively to amino acids which would make stronger bonds with the corresponding receptor region and thus create new FGF4s with higher biological activity. An example of such mutations culd be changing F129 to Y or I, R205 to M or L in the primary binding site domain A, or H201 to L in the secondary binding site. On the other hand, by mutating amino acids in the secondary binding site (the dimerization domain) to Alanine or other amino acids it should be possible to create mutants FGF4 poly-peptides which bind the receptor as a monomer, but are incapable of promoting receptor dimerization, and thus behave as FGF4 receptor antagonists. Furthermore, mutations of the amino acids making up the heparin binding domain would result in lower or higher heparin binding activity. Mutations in this domain could be combined with mutations in the primary binding site to produce a more active FGF4, or they could be combined with mutations in the secondary binding site to produce a better antagonist, since it is known that heparin (that binds to both receptor and FGF ligand) contributes to the formation of stable, active FGF4/FGFR dimers.

[0082] Alternatively, one could search databases of chemical or peptide libraries well known to those of ordinary skill in the art, to identify small molecules (peptides) which could bind to the primary or secondary binding sites of FGF4, and thus would inhibit binding of the FGF4 ligand to its receptors.

[0083] A further application of the findings presented herein is the screening of the above mentioned databases for molecules which mimic the structure of the primary or secondary binding sites of FGF4. Such molecules would be expected to bind to FGF4 receptors, but would be unable to activate signaling through the receptor. They would however compete with the binding of FGF4 to its receptor, or with its ability to induce receptor dimerization, and thus would act as FGFR antagonists.

[0084] FGF4 mutant polypeptides for use in the present invention can be produced as described below, e.g., by site-directed mutagenesis of the nucleic acid encoding FGF4. subdloned in an expression vector and purified as described below. Antagonists can be used to treat patients suffering from FGF4-mediated illnesses such as bladder cancer. Agonists can be employed when using FGF4 for its angiogenic or wound healing properties. The nucleic acid encoding FGF4 can be obtained as described in U.S. Pat. Nos. 5,750,659 and 5,459,250. Either full length FGF4 or truncated FGF4 may be used.

6. EXAMPLES

[0085] The present invention is also described by means of particular examples. However, the use of such examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, many modifications and variations of the invention will be apparent to those skilled in the art upon reading this specification and can be made without departing from its spirit and scope. The invention is therefore to be limited only by the terms of the appended claims along with the fill scope of equivalents to which the claims are entitled.

[0086] To explore the structural determinants of FGF4 involved in receptor and heparin binding, the crystal structure of FGF4 was determined, as described here, at 1.8 Å resolution. As expected, FGF4 adopts a β-trefoil fold similar to other FGFs. Superimposition of FGF4 structure onto FGF2 bound to FGFR1 and heparin allows the identification of receptor and heparin binding sites in FGF4. Mutation of several key FGF4 residues, observed to interact with FGFR1 in the model described here, produces variant FGF4 ligands that have reduced receptor binding affinities and extremely low mitogenic potential. Significantly, the observed interactions between FGF4 and FGFR1 D3 provide a potential basis for preferential affinity of FGF4 towards IIIc splice variants of FGFR1-3. Moreover, the presented modeling studies along with mutational data suggest a two step model for FGF-FGFR binding that involves initial formation of a crucial FGF-D2 interface stabilized by heparin binding followed by secondary FGF-D3 interactions.

6.1 Materials and Methods

[0087] Protein expression and purification of FGF4. DNA fragments generated by polymerase chain reaction (PCR) of human FGF4 cDNA (encoding residues Gly79 to Leu206) were subcloned into the pET-15b bacterial expression vector using NcoI and XhoI cloning sites. The resulting construct (FGF4-pET15b) was transformed into the BL21 (DE3) bacteria and FGF4 expression was induced with 1 mM Isopropyl-1-thio-β-D-galactopyranoside for 5 hours. The bacteria were then centrifuged and subsequently lysed in a 25 mM Na/K phosphate buffer (pH 7.5) containing 300 mM NaCl using a French Cell Press. The N-terminal truncated FGF4 (Gly79-Leu206) was found primarily in the insoluble fraction and was extracted in 25 mM Na/K phosphate buffer (pH 7.5) containing 1 M NaCl at 4° C. overnight. Following centrifugation, soluble FGF4 was diluted 5 times with 25 mM Na/K phosphate buffer (pH 7.5) and loaded onto a Source S column (Pharmacia). Bound FGF4 was eluted by a linear gradient of NaCl to 1 M in a 25 mM Na/K phosphate buffer (pH 7.5). Matrix-assisted laser desorption ionization mass spectrometry of the purified FGF4 gave a molecular mass of 14,244 daltons (Da) (calculated mass 14,409 Da). The mass difference was due to the cleavage of the initiation methionine and a point mutation (Ser182Gly) resulting from PCR. This mutation had no effect on FGF4 biological activity.

[0088] Crystallization and data collection. Crystals of FGF4 were grown by vapor diffusion at 20° C. using the hanging drop method. Briefly, 2 μl of protein solution (2 mg/ml in 25 mM HEPES-NaOH buffer (pH 7.5) containing 150 mM NaCl) were mixed with an equal volume of the crystallization buffer (30% Polyethylene glycol 8000, 0.2 M ammonium sulfate). FGF4 crystals thus obtained belong to the orthorhombic space group P212121 with unit cell dimensions a=40.37 Å, b=53.3 Å, and c=56.23 Å. There is one molecule of FGF4 in the asymmetric unit with a solvent content of approximately 43%. Diffraction data were collected from a flash-frozen (in a dry nitrogen stream using mother liquor containing 10% glycerol as cryo-protectant) crystal on an R-Axis IV image plate detector at Beamline X4-A at the National Synchrotron Light Source, Brookhaven National Laboratory. Data were processed using DENZO and SCALEPACK (see, Otwinowski & Minor, Methods Enzymol. 1997, 276:307-326).

[0089] Structure determination and refinement. A molecular replacement solution was found for one copy of FGF4 in the asymmetric unit using the program AmoRe (Navaza, Acta Crystallogr. Sect. A 1994, 50:157-163) and the structure of FGF2 (Protein Data Bank entry 2FGF; see also, Zhang et al., Proc. Natl. Acad. Sci. U.S.A. 1991, 88:3446-3450) as the search model. Simulated annealing and positional/B-factor refinement were performed using CNS (Bruenger et al, Acta Crystallogr. Sect. D 1998, 54:905-921). Bulk solvent and anisotropic B-factor corrections were applied. Model building into 2F0-Fc and Fo-Fc electron density maps was performed with the program O (Jones et al., Acta Crystallogr. Sect. A 1991, 47:110-119). The atomic model contains residues 79 to 206 of FGF4, 3 sulfate ions, and 96 water molecules. The average B-factor is 10.5 Å2 for the FGF4 molecule, 40.5 Å2 for the sulfate ions, and 17.5 Å2 for the water molecules.

[0090] Production of mutant FGF4 proteins. Alanine substitutions were introduced into the N-terminal truncated FGF4 (Gly79-Leu206) by PCR site-directed mutagenesis (Quik Change™, Stratagene) with the FGF4-pET15b expression plasmid (see above for description) as the template and the following mutant oligos as primers:

Y87A:    5′-AAGCGGCTGCGGCGGCTCGCATGCAACGTGGGCATCGGC-3′
F129A:   5′-GCGTGGTGAGCATCGCCGGCGTGGCCAGCCGG-3′
F151A    5′-CTATGGCTCGCCCTTCGCGACCGATGAGTGCACGTTC-3′
E159A:   5′-GATGAGTGCACGTTCAAGGCCATTCTCCTTCCCAAC-3′
Y166A:   5′-CTCCTTCCCAACAACGCGAACGCGTACGAGTCC-3′
L203:    5′-CCATGAAGGTCACCCACTTCGCCCCTAGGCTGTGACCC-3′
R205A:   5′-CCCACTTCCTCCCCGCGCTGTGACCTTCCAGAGG-3′
N89A:    5′-CGGCGGCTCTACTGCGCCGTGGGCATCGGCTTC-3′
K198A:   5′-GTGTCGCCCACCATGGCGGTCACCCACTTCCTC-3′
K183A/K1 5′-GCCCTGAGCGCGAATGGGAAGACCGCGAAGGGGAAC-3′
88A:
R103A:   5′-GCGCTCCCCGACGGCGCCATCGGCGGCGCGCAC-3′
K144A:   5′-ATGAGCAGCAAGGGCGCGCTCTATGGCTCGCCC-3′
N89A/K19 Alanine substitution were introduced into the N-
8A/K1893 terminally trunacted FGF4 N89A/K198A mutant as a
A/K188A: template by PCR site-directed mutagenesis with
         sense and antisense oligonucleotides containing
         K183A/K188A mutations:
         5′-GCCCTGAGCGCGAATGGGAAGACCGCGAAGGGGAAC-3′
E117A    5′-CGCGACAGCCTGCTGGCGCTCTCGCCCGTGGAG-3′
L162A    5′-TTCAAGGAGATTCTCGCTCCCAACAACTACAA-3′
H201A    5′-ACCATGAAGGTCACCGCCTTCCTCCCAGGCT-3′
P163A    5′-GGAGATTCTCCTTGCCAACAACTACAACGCC-3′
P195A    5′-GGGAACCGAGTGTCGGCCACCATGAAGGTCACC-3′
T196A    5′-GGGAACCGAGTGTCGCCGCCATGAAGGTCACCCACTTCC-3′

[0091] The presence of the mutations was confirmed by sequencing. Mutant FGF4-pET15b plasmids were transformed into E. coli strain BL21 (DE3). Expression of the FGF4 proteins was induced as described above. Following centrifugation, cells expressing wild type and various mutant FGF4 proteins were suspended in a 50 mM HEPES-NaOH buffer (pH 7.4) containing 1 M NaCl and protease inhibitors (100 μg/ml PMSF, and 2 μg/ml Aprotinin) and disrupted by sonication. Lysates were left at 4° C. overnight in order to salt-extract the FGF4 proteins from particulate fractions. Following centrifugation supernatants containing soluble FGF4 proteins were diluted 4 times with 50 mM HEPES-NaOH buffer (pH 7.4), and loaded onto heparin-Sepharose columns. After washing the columns with 50 mM HEPES-NaOH (pH 7.4) buffer containing 250 mM NaCl, the FGF4 proteins were eluted by a 50 mM HEPES buffer (pH 7.4) containing 1.5 M NaCl. Fractions were analyzed by SDS-PAGE (15%) and the purity of the FGF4 proteins was accessed by staining with Coomassie Blue R-250.

[0092] DNA synthesis assay. NIH3T3 cells were seeded at a density of 3×104 cells/well in 24-wells plates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum. The following day the medium was replaced with DMEM containing only 0.5% calf serum and the cells were allowed to reach quiescence for 48 hours. Thereafter, serial dilutions of wild-type full length FGF4 (Ala31-Leu206), N-terminal truncated wild type or mutant FGF4 (Gly79-Leu206) were added for 18 hours. Cells were then labeled with 1 μCi of 3H-thymidine for 6 hours, washed with Tris-HCl buffered saline (pH 7.5), and lysed with 0.5 M NaOH. The lysates were then neutralized with 0.5 HCl and the radioactivity incorporated into the acid-precipitable material was measured using a β-counter (LKB, Pharmacia). Each assay was performed in triplicate.

[0093] Receptor bintdinig assay. N-terminal truncated FGF4 (Gly79-Leu206) was radio-iodinated by the chloramine T method using a previously described protocol (Bellosta et al., J. Cell Biol. 1993, 121:705-713). The labeled FGF4 were separated from free iodine over a Sephadex G-25 column, which was previously equiliberated in phosphate buffered saline (PBS) containing 1% bovine serum albumin. CHO cells, over-expressing FGFR2 (Mansukhani et al., Proc. Natl. Acad. Sci. U.S.A. 1992, 89:3305-3309), were seeded at 1×106 cells/well in 6 well plates in DMEM containing 10% fetal calf serum. The following day the medium was removed and the cells were allowed to bind labeled FGF4 (specific activity 2.5×104 cpm/ng) in DMEM containing 25 mM HEPES-NaOH (pH 7.4), 15% gelatin, 10 μg/ml heparin, and increasing concentrations of the wild type or mutant FGF4 proteins for 2 hours at 4° C. Cells were then washed several times with ice-cold Tris-HCl buffered saline (pH 7.5) and the receptor-bound radio-labeled FGF4 was released using a 50 mM Sodium Acetate buffer (pH 4.0) containing 2 M NaCl. Radioactivity was measured using a γ-counter (LKB-Pharmacia). Binding assays were done in duplicate.

6.2 Results and Discussion

[0094] Structure determination. The mature, secreted form of human FGF4 spans amino acids Ala31 to Leu206 (Bellosta et al., J. Cell Biol. 1993, 121:705-713). Based on the crystal structure of FGF2 (Eriksson et al., Proc. Natl. Acad. Sci. U.S.A. 1991, 88:3441-3445; Zhang et al., Proc. Natl. Acad. Sci. U.S.A. 1991, 88:3446-3450; Zhu et al., Science 1991, 251:90-93), the β-trefoil core of FGF4 is expected to start at Leu83 (Pro29 in FGF2). Recent crystal structures of three different FGF-FGFR complexes have revealed that the residues immediately preceeding the β-trefoil core in FGF1 and FGF2 are involved in FGFR binding (Plotnikov et al., Cell 1999, 98:641-650; Plotnikov et al., Cell 2000, 101:413-424; Springer et al., J. Biol. Chem. 1994, 269:26879-26884). These residues correspond to amino acids Gly79 to Arg82 of FGF4 (FIG. 2). Thus, to maximize the likelihood of obtaining diffracting crystals without jeopardizing the biological activity of FGF4, an N-terminal truncated FGF4 polypeptide containing residues Gly79 to Leu206 (Gly79-Leu206) was crystallized. Truncated FGF4 was expressed in E. coli and purified to homogeneity (see, Subsection 6.1, supra). The mitogenic activity of the truncated FGF4 on NIH3T3 cells was only slightly lower than that of the mature FGF4, indicating that the truncated FGF4 contains the majority of receptor binding sites. Crystallization trials with FGF4 produced orthorhombic crystals with one molecule per asymmetric unit. The crystal structure of FGF4 was solved by molecular replacement as described, supra, and refined to 1.8 Å resolution with an R-value of 19.4% (free R-value of 20.7%). The atomic model for FGF4 consists of one FGF4 molecule (residues 79 to 206), 3 sulfate ions and 96 water molecules. The coordinates for the FGF4 crystal structure are given in the accompanying Appendix B, infra, in PDB file format. Data collection and refinement statistics are given below in Table 1.

TABLE 1
Summary of crystallographic analysis
I. Data C llection Statistics:
Resolution	Reflections	Completeness	Rsyma	Signal
(Å)	(total/unique)	(%)	(%)	(<51 /σ|>)
25.0-1.8	40562/11590	99.6 (995)b	5.1 (10.9)b	16.5
II. Refinement Statistics:c
+TC,19/32 Root-mean-square Deviations
Resolution		Rcryst/Rfreed	Bonds	Angles	B-factorse
(Å)	Reflections	(%)	(Å)	(°)	(Å2)
25.0-1.8	11488	19.4/20.7	0.005	1.31	1.00
1${}^a{R}_{\mathrm{sym}}=100\times \sum _{\mathrm{hkl}}\sum _i\&LeftBracketingBar;l_i\left(\mathrm{hkl}\right)-⟨l\left(\mathrm{hkl}\right)⟩\&RightBracketingBar;/\sum _{\mathrm{hkl}}\sum _i{l}_i\left(\mathrm{hkl}\right).$
bValue in parentheses is for the highest resolution shell: 1.86-1.8 Å.
cAtomic model: 994 protein atoms, 3 SO42−ions, and 42 water molecules.
2${}^d{R}_{\mathrm{cryst}/\mathrm{free}}=100\times \sum _{\mathrm{hkl}}\&LeftBracketingBar;\&LeftBracketingBar;F_o\left(\mathrm{hkl}\right)\&RightBracketingBar;-\&LeftBracketingBar;F_c\left(\mathrm{hkl}\right)\&RightBracketingBar;\&RightBracketingBar;/\sum _{\mathrm{hkl}}\&LeftBracketingBar;F_o\left(\mathrm{hkl}\right)\&RightBracketingBar;,$
where Fo (> 0 σ) and Fc are the observed and calculated structure
factors. 5% of the reflections were used for calculations of Rfree.
cFor bonded protein atoms.

[0095] Description of the structure. As expected by sequence similarity (Vainikka et al., EMBO J. 1993, 11:4273-4280), FGF4 adopts a β-trefoil fold conformation (FIG. 1). Superimposition of the Cα traces located within the β-trefoil core of FGF4 with those of FGF1 (Zhu et al., Science 1991, 251:90-93) and FGF2 (Zhang et al., Proc. Natl. Acad. Sci. U.S.A. 1991, 88:3446-3450) gives a root-mean-square (rms) deviation of only 0.86 Å (122 common Cα atoms) and 0.76 Å (123 common Cα atoms), respectively. Within the β-trefoil core, the major differences between the FGF4 structure and the structures of FGF1 and FGF2 are the conformations of the β1-β2, β3-β4 and β9-β10 loops. These loops vary in both length and sequence among the various members of the FGF family (FIG. 2). In FGF4, the β1-β2 loop is one residue longer than the corresponding loops of FGF1 and FGF2 (FIG. 2). In contrast, the β3-β4 loop in FGF4 is shorter by one residue than the corresponding loops in FGF1 and FGF2. Like FGF2, the β9-β10 loop in FGF4 is shorter by two residues than in FGF1 (FIG. 2).

[0096] As with the structures of free FGF1 and FGF2 (Zhu et al., Science 1991, 251:90-93), a sulfate ion is bound to the predicted high affinity heparin-binding site of FGF4. In addition, two other sulfate ions are coordinated by FGF4 residues, whose corresponding residues in FGF1 and FGF2 have not been observed to bind sulfate ions (see, FIG. 6).

[0097] Receptor binding sites and specificity. To identify potential receptor binding sites in FGF4, an FGF4-FGFR1 model was constructed by superimposing the FGF4 structure onto the FGF2 structure bound to the ligand binding portion of FGFR1 consisting of Ig-like domains 2 (D2) and 3 (D3) (FIG. 3). Careful inspection of the FGF4-FGFR1 interface showed that the majority of the interactions between FGF4 and FGFR1 in the FGF4-FGFR1 model could be accommodated with minor adjustments of FGF4 side chain rotamers. Three loop regions, the β1-β2 and β9-β10 loops (within the β-trefoil core) and the N-terminus (outside the β-trefoil core), sterically clash with the receptor. In the present crystal structure these loop regions are involved in crystal lattice contacts, implying that the present conformations of these loops are dictated by the lattice contacts. However, upon FGFR binding these regions are expected to undergo changes in backbone conformation to allow an engagement with FGFR1 to occur.

[0098] At the FGF4-D2 interface three highly conserved solvent-exposed FGF4 residues, Tyr87, Tyr166 and Leu203, are predicted to pack against a highly conserved hydrophobic surface consisting of Ala167, Pro169, and Val248 at the bottom of D2 in FGFR1 (data not shown). Significant differences between FGF4 and FGF2 at the FGF-D2 interface are the substitutions of Phe40 and Met151 in FGF2 with His95 and Arg205 in FGF4 (FIG. 2). These substitutions may indicate a weaker hydrophobic FGF4-D2 interface compared to the FGF2-D2 interface. At the FGF4-linker interface, Asn167, also highly conserved among FGFs (FIG. 2), is expected to make hydrogen bonds with the FGFR-invariant arginine (Arg250 in FGFR1) in the D2-D3 linker region.

[0099] To provide experimental support for the described interactions between FGF4 and FGFR1 at the FGF4-D2 interface, Tyr87, Tyr166, Leu203 and Arg205 were individually mutated to alanine in the N-terminal truncated FGF4 construct (Gly79-Leu206). Mutant FGF4 proteins were expressed in E. coli, purified to near homogeneity as described, supra, and were assayed for the ability to induce DNA synthesis in NIH 3T3 fibroblasts. As shown in FIG. 4 and in Table 2, below, the Y87A, Y166A and L203A mutant FGF4 proteins were severely compromised in their ability to induce DNA synthesis, while the R205A mutant showed a more modest decrease in the induction of DNA synthesis. Thus, these data confirm the observed interactions between FGF4 and D2 in the FGF4-FGFR1 model, and indeed the corresponding 4 residues in FGF2 had been previously shown to be also important for biological activity (Springer et al., J. Biol. Chem. 1994, 269:26879-26884).

TABLE 2
Summary of DNA Synthesis Activity and
Receptor Binding Affinity of Mutant FGF4 Polypeptides
	DNA Synthesis
	NIH 3T3 cells	DNA synthesis assay	Receptor Binding
	(ED50 mutant/	32D-FGFR2 cells	CHO-FGFR2 cells
Protein	ED50 wild-type)a	ED50mut/ED50 wt	(IC50 mutant/IC50 wild-type)b
(Gly79-Leu206)	−heparin	+heparin	−heparin	+heparin	+heparin
wild-type	1	1	0	1	1
Y87A	>500	500			>100
F129A	>500	>500			>100
F151A	1000	4	0	200	2.5
E159A	>1000	80	0	>1000	5.0
Y166A	500	>500			>100
L203A	>500	>500			>100
R205A	25	2.5			2.5
N89A/K198A	10	10	0	65	ND
K183A/K188A	70	13	0	125	ND
R103A/K144A	1	1			ND
N89A/K198A/K	>1333	275	0	>1000	>1000
183A/K188A
E117A	≈1	≈1	0	7	7
L162A	600	158	0	>1000	>1000
H201A	>666	125			>1000
P163A	10	4.6
P195A	8	4.6
T196A	5.5	4
aED50 (Effective Dose) is the dose of FGF4 necessary to reach 50% of maximum DNA synthesis obtained with the wild type FGF4.
bIC50 (Inhibitory Concentration) is the concentration of FGF4 required to compete 50% of binding of labeled wild type FGF4 to FGFR2.
cND (Not Determined).
d32D is a murine hematopoietic cell line, which does not express FGF receptors or heparin sulfate proteoglycans. As a consequence, even when transfected to express FGF receptors (as in the example, 32D-FGFR2), they require exogenous heparin to be stimulated to proliferate by FGF (Mansukhani et al, PNAS 89: 3305, 1992). In the absence of heparin, FGFs are not mitogenic in these cells (expressed as 0 activity).

[0100] Interactions between FGF4 and D3 occur at the upper part of D3 and involve mainly the βB′-βC, βC′-βE and βF-βG loops in D3. At the interface between FGF4 and the βB′-βC loop of D3, Glu159 of FGF4 (an FGF-invariant residue) (FIG. 2) is expected to make a hydrogen bond with Gln284 (an FGFR-invariant residue). This prediction is supported by a 1,000-fold reduction in the ability of the E159A mutant FGF4 to induce DNA synthesis in living cells (FIG. 5A and Table 2, above).

[0101] In contrast to the interface between FGF4 and the βB′-βC loop, interactions between FGF4 and the βC′-βE and βF-βG loops are variable. Significantly, FGF4, like FGF1, has a serine (Ser119) at the position homologous to Gln65 of FGF2 (FIG. 2). It has previously been shown that Gln65 of FGF2 makes two hydrogen bonds with Asp320/Asp321 in the βC′-βE loop of FGFR1/FGFR2, and as a result, the βC′-βE loop is ordered in both FGF2-FGFR1 and FGF2-FGFR2 structures (Plotnikov et al., Cell 1999, 98:641-650; Plotnikov et al., Cell 2000, 101;413-424). In contrast, in the FGF1-FGFR1 structure the βC′-βE loop is disordered because Ser62 of FGF1 cannot interact with Asp320 of FGFR1 in the βC′-βE loop (Plotnikov et al., Cell 2000, 101:413-424). Thus, by analogy a skilled artisan would expect that Ser119 of FGF4 will also not make hydrogen bonds with Asp320 located in the βC′-βE loop. However, based on the present model, the side chain of Glu117 in FGF4 could make hydrogen bonds with Lys321 located in the βC′-βE loop of FGFR1. This interaction may potentially compensate for the inability of FGF4 to engage Asp320 and lead to an ordered βC′-βE loop. Moreover, in the FGF4-FGFR1 model, several solvent-exposed hydrophobic residues in FGF4 (Val121, Phe129, Phe136, Phe151) are in the vicinity of the βC′-βE loop of FGFR1 and could engage in hydrophobic contacts with Val-316 in the βC′-βE loop of FGFR1. These hydrophobic interactions would further contribute to the ordering of the βC′-βE loop in an FGF4-FGFR1 structure. DNA synthesis assays performed using mutant FGF4 molecules support the aforementioned hypothesis. Substitutions of Phe129 and Phe151 with alanine in FGF4 reduced the ability of FGF4 to induce thymidine-incorporation in NIH3T3 cells about a thousand-fold (Table 2, above). Mutation of the residue corresponding to Phe151 in FGF2 was also shown to be important for FGFR binding (Zhu et al., Protein Eng. 1998, 11:937-940).

[0102] Thus, the FGF4-FGFR1 model presented shows that FGF4, like FGF2, may engage the βC′-βE loop of FGFR1. Consequently, sequence variations in the βC′-βE loop resulting from alternative splicing should affect FGF4-FGFR binding affinity. A sequence comparison of FGFRs at the βC′-βE loop region demonstrates that Lys321 is conserved only in the IIIc isoforms of FGFR1-FGFR3, providing a potential explanation for reduced affinity of FGF4 towards the IIIb splice variants of FGFR1-FRFR3 and FGFR4.

[0103] Binding of the FGF4 mutants to FGFR2. Mutant FGF4 proteins were tested in a receptor binding assay to confirm that the diminished capacity of the mutant FGF4 proteins to induce DNA synthesis is a result of the reduced ability of the mutant FGF4 to interact with FGFR. CHO cells over-expressing FGFR2 (Mansukhani et al., Proc. Natl. Acad. Sci. U.S.A. 1992, 89:3305-3309) were allowed to bind radio-labeled N-terminal truncated FGF4 (Gly79-Leu206) in the presence of increasing concentrations of unlabeled full length (Ala31-Leu206), N-terminal truncated wild-type or various N-terminal truncated mutant FGF4 proteins (FIG. 4). The N-terminal truncated wild-type FGF4 bound FGFR2 with only a slightly lower affinity than full length FGF4 (Ala31-Leu206), indicating that the majority of receptor binding sites is contained within the Gly79-Leu206 construct (FIG. 4) Substitutions of Tyr87, Tyr166 and Leu203 with alanine severely reduced the affinity of FGF4 towards FGFR2 (FIG. 4 and Table 2, supra), emphasizing the importance of the hydrophobic FGF4-D2 interface in providing FGF4-FGFR affinity. In contrast, the R205A mutant FGF4 showed only a slight reduction in FGFR2 binding affinity. The relative decrease in binding affinity of these mutants towards FGFR2 is consistent with the results of the DNA synthesis assay, thus implying that the impaired ability of these mutants to induce DNA synthesis is a consequence of loss of binding affinity to FGFR2.

[0104] Alanine substitutions of FGF4 residues predicted to interact with D3 also reduced the binding affinity of FGF4 for FGFR2. The F129A mutant showed a large decrease (more than 100-fold) in receptor binding affinity, which paralleled the severe impairment of this mutant in induction of DNA synthesis (FIG. 4 and Table 2, supra). In contrast, the F151A and E159A mutants were only slightly affected (2.5- and 5-fold, respectively) in FGFR2 binding (FIG. 4 and Table 2), yet these mutants were severely compromised in induction of DNA synthesis (see, Table 2, above). This was particularly unexpected for the E159A mutant as Glu159 is highly conserved among FGFs (FIG. 2) and the corresponding glutamic acid in FGF2 (Glu96) was shown to be critical for binding of FGF2 to FGFR1 (Zhu et al., J. Biol. Chem. 1995, 270:21869-21874).

[0105] This discrepancy between receptor binding and DNA synthesis data for the F151A and E159A mutants may be due to a difference between the experimental conditions used for the receptor binding and for DNA synthesis assays. Since NIH 3T3 cells naturally express cell surface HSPG in abundance, they do not require exogenous heparin to respond fully to FGF4. Therefore, exogenous heparin was not included in the DNA synthesis assays. In contrast, receptor binding assays were also performed in the presence of exogenous heparin to exclude binding of FGF to the abundantly expressed cell surface HSPG. In the absence of heparin, binding to these low affinity but very abundant receptors can not easily be distinguished from binding to FGFR.

[0106] Since heparin stabilizes FGF-FGFR interactions, it was possible that the presence of exogenous heparin in the receptor binding assay could have partially reversed the reduced ability of the F151A and E159A mutants to interact with FGFR. To test this possibility, the DNA synthesis assays were repeated in the presence of exogenous soluble heparin. While, as expected, heparin had no effect on the mitogenic ability of wild type FGF4, heparin dramatically enhanced the capacity of the F151A and E159A mutants to induce DNA synthesis (FIG. 5A and Table 2, above). In contrast, addition of heparin had no effect on the Y87A, F129A, Y166A and L203A mutants and enhanced the ability of R205A to induce DNA synthesis only by about ten-fold (shown in FIG. 5A, and in Table 2).

[0107] Analysis of the location of the various FGF4 mutations in the ternary FGF4-FGFR1-heparin model provides a potential explanation for the differential ability of heparin to rescue only some of the mutants. Both the F115A and E159A mutations, which display the greatest potentiation upon addition of heparin, affect FGF4 interaction with FGFR D3 (FIG. 2). In contrast, with the exception of the F129A mutant, all the mutations that are not rescued by heparin affect FGF4 interaction with FGFR D2. Upon binding of FGF to FGFR, a continuous negatively charged surface is formed by the heparin binding sites of FGF and FGFR D2, to which heparin binds (Plotnikov et al., Cell 1999, 98:641-650). Simultaneous binding of the same heparin polymer to both FGF and FGFR will clearly increase apparent FGF-FGFR affinity (Schlessinger et al., Mol. Cell 2000, 6:743-750). Mutations affecting the FGF-D2 interface will hamper a productive juxtaposition of the heparin binding sites of FGF and FGFR to form a continuous heparin binding surface, and thus heparin will not reverse the deleterious effects of these mutations. In contrast, mutations affecting the FGF-D3 interface will not interfere with the formation of a productive heparin binding surface by FGF and FGFR D2, and heparin can enhance FGF-FGFR affinity by interacting with both FGF and FGFR D2.

[0108] The mutagenesis data suggest that interactions of FGF with FGFR D2 provide the primary FGF-FGFR binding affinity. Indeed, Wang et al. (Biochemistry 1999, 38:160-171) have shown that several FGFs can bind to the isolated D2 domain of FGFR1 in vitro in the presence of heparin. Such teaching, coupled with what is taught, supra, in these examples, indicates that FGFR binds FGF first via D2. Heparin then may stabilize the FGF-D2 interaction and facilitates formation of a FGF-D3 interface.

[0109] Heparin binding sites. Recent biochemical and structural data demonstrate that FGF in the absence of heparin can form an initial low affinity complex with FGFR (Pantoliano et al., Biochemistry 1994, 33:10229-10248). In the presence of heparin, the low affinity complexes become stabilized which then in turn lead to stable 2:2 FGF:FGFR signaling complexes. The recent crystal structure of a dimeric 2:2:2 FGF2:FGFR1:heparin complex provides a molecular basis for how heparin enhances FGF-FGFR affinity and promotes dimerization (Schlessinger et al., Mol. Cell 2000, 6:743-750). Within each ternary 1:1:1 FGF:FGFR:heparin complex, heparin makes numerous contacts with the heparin binding residues of FGF and FGFR, thereby increasing the affinity of FGF towards FGFRs. In addition, heparin also interacts with the heparin binding residues in D2 of the adjoining FGFR, thereby augmenting the weak interactions of FGF and FGFR in one ternary complex with the FGFR in the adjoining ternary complex. Since FGFs differ in the primary sequences of heparin binding sites, each FGF may require different heparin motifs (e.g., different sulfation patterns and/or lengths) to exert its optimal biological activities (Faham et al., Curr. Opin. Struct. Biol. 1998, 8:578-586; Schlessinger et al., Mol. Cell 2000, 6:743-750).

[0110] To evaluate the potential heparin binding sites of FGF4, a dimeric FGF4-FGFR-heparin model was generated by superimposing two copies of the FGF4 structure onto the two copies of FGF2 in the dimeric FGF2-FGFR1-heparin ternary complex (FIG. 6). FGF4 residues corresponding to the heparin binding residues of FGF2 along with other FGF4 surface residues that could bind heparin were mapped onto the ribbon diagram of FGF4 (data not shown). With the exception of Lys188 (Lys134 in FGF2) and Lys198 (Lys144 in FGF2) the remainder of the heparin binding residues differs between FGF4 and FGF2 (FIG. 2). These differences are likely to determine the optimal sulfation motifs in heparin that are required to support FGF4 or FGF2 biological activities. Interestingly, Asn36 and Gln143, two critical heparin binding residues in FGF2, are substituted by hydrophobic residues Val90 and Met197 in FGF4 (FIG. 2). Therefore, these residues are unable to make hydrogen bonds with the hydroxyl group and N-sulfate group of ring D of heparin. Moreover, in the model the side chain of Val199 in FGF4 (Ala145 in FGF2) clashes with the N-sulfate of ring D (data not shown). These observations indicate that FGF4 may not require N-sulfate on ring D for heparin binding. Two other significant differences between FGF2 and FGF4 are the substitutions of Lys35 and Lys128 of FGF2 with Asn89 and Ser182, respectively, in FGF4 (FIG. 2). Based on the present model, Asn89 and Ser182 would engage better the 6-O-sulfate of ring B and the 2-O-sulfate group of ring E (data not shown).

[0111] A sulfate ion (provided by the crystallization buffer) is coordinated at the predicted high affinity heparin-binding site of FGF4 by Lys183 and Lys188 (data not shown). These two lysines are expected to bind the 2-O-sulfate group of ring E of heparin. In fact the sulfate ion in the FGF4 structure nearly colocalizes with the 2-O-sulfate group of ring E in the FGF4-heparin model (data not shown). To provide experimental support for the modeled FGF4-heparin interactions, mutant FGF4 proteins were generated in which FGF4 residues predicted to coordinate the 2-O-sulfate of ring E (K183 and K188) or the 6-O-sulfate of ring B (N89 and K198) are substituted with alanines. Both the doubly mutated K183A/K188A and N89A/K198A FGF4 proteins showed diminished ability to induce DNA synthesis in NIH3T3 cells (see, FIG. 5B and Table 2). Mutations in all four (4) amino acid residues abolishes all FGF4 activity (Table 2). Thus, as for FGF2, both the 6-O-sulfate of ring B and the 2-O-sulfate group of ring E of heparin may play important roles in promoting heparin-dependent FGF-FGFR interaction and dimerization. These data are consistent with findings that a high content in 6-O-sulfate groups in heparin is required for specific interaction with FGF4 (Ishihara, Blycolbiology 1994, 4:817-824).

[0112] Another sulfate ion is coordinated by the side chains of Arg103, Lys144 in the crystal structure of FGF4 (data not shown). Since bound sulfate ions in the crystal structures of free FGFs often indicate potential heparin binding sites in FGFs a doubly mutated R103/K144 FGF4 protein was also generated. This mutant FGF4 protein induced DNA synthesis in NIH3T3 cells to a level comparable to wild-type FGF4 (see, Table 2), suggesting that Arg103 and Lys144 most likely do not participate in heparin binding.

[0113] Experiments were also performed to determined whether exogenous heparin can compensate for the reduced ability of the K183A/K188A and N89A/K198A mutant FGF4 proteins in the DNA synthesis assay. As shown in FIG. 5B, exogenously added heparin significantly enhanced the ability of the K183A/K188A mutant FGF4 to induce DNA synthesis, but had no effect on the N89A/K198A mutant. A possible explanation for the differential effect of heparin on the activity of these two mutants lies in the heterogenous nature of commercial heparin. It is known that heparin is a mixture of oligosaccharides of different length and sulfate contents, generated by the polymerization of repeating disaccharide units consisting of D-glucoseamine (GlcN) and L-iduronic acid (IdoA). During biosynthesis, heparin is sulfated by the sequential actions of three different sulfotransferases, an N-sulfotransferase, a 2-O-sulfotransferase and a 6-O-sulfotransferase (Lindahl et al., J. Biol. Chem. 1998, 273:24979-24982). In general, these reactions proceed in the order indicated, but often fail to go to completion, resulting in tremendous chemical heterogeneity in sulfation pattern in heparin. The observation that addition of exogenous heparin partially rescues only the K183A/K188A mutant (predicted to coordinate 6-O-sulfate) and not the N89A/K198A mutant (predicted to coordinate 2-O-sulfate), is probably due to the fact that the heparin sub-fraction containing 2-O-sulfate is more abundant than the sub-fraction containing 6-O-sulfate.

[0114] The heterogeneity in sulfation pattern is even more profound in heparan sulfate moieties of cell surface HSPG, which are thought to cooperate with FGFs to induce FGFR dimerization and activation. The requirement for a specific sulfation motif in heparan sulfate for optimal FGF4 action may be a mechanism to fine tune FGF4-FGFR interactions and to restrict FGF4 signaling to a specific set of cells in a specific tissue during various stages of embryonic development, where spatial and temporal regulation by FGF is critically required.

[0115] Implications for tile general mode of FGF-FGFR binding. Those skilled in the art will note that some of the data presented here is not consistent with the model of FGF1-FGFR2 binding described in the recently published crystal structure of a ternary FGF1-FGFR2-heparin complex (Pellegrini et al., Nature 2000, 407:1029-1034). In the novel structure presented in Pellegrini, the FGFR-invariant Pro-253 located in the D2-D3 linker, is found in a cis configuration, while in all previously reported binary FGF-FGFR structures Pro253 is found only in a trans configuration (Plotnikov et al., Cell 1999, 98:641-650; Plotnikov et al., Cell 2000, 101:413-424; Stauber et al., Proc. Natl. Acad. Sci. U.S.A. 2000, 97:49-54). Consequently, relative to its position in all binary FGF-FGFR structures, the receptor D3 in the ternary FGF1-FGFR2-heparin structure is swiveled around the linker region by more than 160°. This creates a completely different set of interactions at the FGF-D3 interface. Pellegrini et al. (Nature 2000, 407:1029-1034) propose that this D3 rotation is caused by a heparin-mediated trans to cis isomerization of Pro253 in the D2-D3 linker region. However, the mutagenesis data presented here do not support this hypothesis. Based on this ternary FGF1-FGFR2-heparin structure (Pellegrini et al., supra), neither F129 nor F151 in FGF4 are predicted to make any contacts with D3 (FIG. 6). Thus, the drastically reduced mitogenic capacity of the F129A and-F151A mutants is in disagreement with the mode of FGF-FGFR binding described by those authors (i.e., by Pellegrini et al., supra) Surprisingly, the data presented here suggested that the cis isomerization of Pro253 observed in the FGF1-FGFR2-heparin structure (Pellegrini et al., supra) is probably the result of partial refolding of FGFR2.

[0116] The present invention is therefore based on a different model of FGF-FGFR binding, presented supra, in which interaction of FGF with the FGFR D2 domain provides the primary FGF-FGFR binding surface (FIG. 7A-B), and heparin facilitates the formation of an FGF-D3 interface by stabilizing the FGF-D2 interaction (FIG. 9A-B). This model may explain the exclusively heparin-dependent binding of FGF1 to an in vitro refolded FGFR2 described by Pellegrini et al. (supra). As discussed above, it is likely that the FGFR2 used by these authors was not properly refolded and consequently D3 is in a different position than the one observed in the previously reported FGF-FGFR crystal structures. Despite the lack of sufficient contact between FGF1 and FGFR2 D3, the FGF1-FGFR2 complex could still be captured in the presence of heparin as evident from the crystal structure (Pellegrini et al., supra).

[0117] The data presented here show that FGF4 adopts a typical β-trefoil fold similar to that adopted by other FGFs (see, e.g., Osslund et al., Protein Sci. 1998, 7:1681-1690; Plotnikov et al., J. Biol. Chem. 2001, 276:4322-4329; Zhu et al., J. Biol. Chem. 1995, 270:21869-21874). The ternary FGF4-FGFR1-heparin model constructed, above, by superimposing FGF4 onto FGF2 in the FGF2-FGFR1-heparin structure assists identification of several key residues in FGF4 involved in receptor and heparin binding. Substitution of several of these residues with alanine produces FGF4 molecules with reduced receptor binding and mitogenic potential, which may, at least in certain cases, be partially reversed by excess soluble heparin. Significantly, the modeling and mutagenesis data presented here show that FGF4 interacts with the βC′-βE loop in FGFR D3 and provide a molecular basis for why FGF4, like FGF2 but unlike FGF1, can discriminate between the IIIc and IIIb splice variants of FGFRs for binding. Based on these findings, a skilled artisan may readily identify specific FGF-FGFR interactions and, from these, design novel, variant FGF molecules with increased or altered binding specificity.

7. References Cited

[0118] Numerous references, including patents, patent applications and various publications, are cited and discussed in the description of this invention. The citation and/or discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any such reference is “prior art” to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety and to the same extent as if each reference was individually incorporated by reference.

8. Appendix A: One and Three Letter Designations for Amino Acids

[0119]

	Alanine	Ala	A	Isoleucine	Ile	I
	Arginine	Arg	R	Leucine	Leu	L
	Asparagine	Asn	N	Lysine	Lys	K
	Aspartic acid	Asp	D	Methionine	Met	M
	Cysteine	Cys	C	Phenylalanine	Phe	F
	Glutamine	Gln	Q	Proline	Pro	P
	Glutamic acid	Glu	E	Serine	Ser	S
	Glycine	Gly	G	Threonine	Thr	T
	Histidine	His	H	Tryptophan	Trp	W
	Tyrosine	Tyr	Y	Valine	Val	V

9. Appendix B: Crystal Structure Coordinates for FGF4

[0120]

REMARK coordinates from restrained individual B-factor refinement
REMARK refinement resolution: 30-1.8 A
REMARK starting r = 0.1944 free_r = 0.2070
REMARK final r = 0.1943 free_r = 0.2067
REMARK B rmsd for bonded mainchain atoms = 0.695 target = 1.5
REMARK B rmsd for bonded sidechain atoms = 1.228 target = 2.0
REMARK B rmsd for angle mainchain atoms = 1.094 target = 2.0
REMARK B rmsd for angle sidechain atoms = 1.939 target = 2.5
REMARK wa = 0.911288
REMARK rweight = 0.235168
REMARK target = mlf steps = 30
REMARK sg = P2(1)2(1)2 (1) a = 40.367 b = 53.295 c = 56.231 alpha = 90
beta = 90 gamma = 90
REMARK parameter file 1: CNS_TOPPAR: protein_rep.param
REMARK parameter file 2: CNS_TOPPAR: dna-rna.param
REMARK parameter file 3: CNS_TOPPAR: water_rep.param
REMARK parameter file 4: CNS_TOPPAR: ion.param
REMARK molecular structure file: fgf4_9.mtf
REMARK input coordinates: fgf4_9X.pdb
REMARK reflection file = fgf4.hklt
REMARK ncs = none
REMARK B-correction resolution: 6.0-1.8
REMARK initial B-factor correction applied to fobs:
REMARK B11 = −3.650 B22 = 0.683 B33 = 2.967
REMARK B12 = 0.000 B13 = 0.000 B23 = 0.000
REMARK B-factor correction applied to coordinate array B: 0.028
REMARK bulk solvent: density level = 0.570684 e/A{circumflex over ( )}3, B-factor = 74.852 A{circumflex over ( )}2
REMARK reflections with |Fobs|/sigma_F > 0.0 rejected
REMARK reflections with |Fobs| > 10000 * rms(Fobs) rejected
REMARK theoretical total number of refl. in resol. range:	11739 (100.0%)
REMARK number of unobserved reflections (no entry or |F| = 0):	251 (2.1%)
REMARK number of reflections rejected:	0 (0.0%)
REMARK total number of reflections used:	11488 (97.9%)
REMARK number of reflections in working set:	10894 (92.8%)
REMARK number of reflections in test set:	594 (5.1%)
CRYST1 40.367 53.295 56.231 90.00 90.00 90.00 P 21 21 21
REMARK FILENAME = “fgf4_9XB.pdb”
REMARK DATE: 25-Jan-00 22:33:40 created by user: mohammad
REMARK VERSION: 0.5
ATOM	1	C	GLY	79	15.981	20.788	35.818	1.00	14.10
ATOM	2	O	GLY	79	15.510	20.619	34.693	1.00	15.42
ATOM	3	N	GLY	79	13.808	21.802	36.441	1.00	15.71
ATOM	4	CA	GLY	79	15.211	21.566	36.865	1.00	14.54
ATOM	5	N	ILE	80	17.165	20.308	36.178	1.00	12.57
ATOM	6	CA	ILE	80	17.971	19.552	35.234	1.00	11.35
ATOM	7	CB	ILE	80	19.443	19.416	35.728	1.00	11.22
ATOM	8	CG2	ILE	80	20.089	20.790	35.818	1.00	10.86
ATOM	9	CG1	ILE	80	19.497	18.729	37.096	1.00	11.55
ATOM	10	CD1	ILE	80	19.441	17.210	37.040	1.00	12.63
ATOM	11	C	ILE	80	17.393	18.163	35.008	1.00	10.56
ATOM	12	O	ILE	80	16.667	17.637	35.852	1.00	9.79
ATOM	13	N	LYS	81	17.700	17.589	33.849	1.00	9.65
ATOM	14	CA	LYS	81	17.268	16.239	33.506	1.00	9.81
ATOM	15	CB	LYS	81	16.448	16.227	32.216	1.00	10.72
ATOM	16	CG	LYS	81	14.969	16.523	32.406	1.00	12.85
ATOM	17	CD	LYS	81	14.737	17.940	32.867	1.00	14.94
ATOM	18	CE	LYS	81	13.254	18.227	33.041	1.00	15.71
ATOM	19	NZ	LYS	81	13.025	19.662	33.354	1.00	17.00
ATOM	20	C	LYS	81	18.548	15.446	33.304	1.00	9.37
ATOM	21	O	LYS	81	19.457	15.896	32.613	1.00	9.66
ATOM	22	N	ARG	82	18.631	14.271	33.913	1.00	9.22
ATOM	23	CA	ARG	82	19.829	13.462	33.785	1.00	9.59
ATOM	24	CB	ARG	82	19.895	12.453	34.932	1.00	11.59
ATOM	25	CG	ARG	82	19.818	13.111	36.302	1.00	14.57
ATOM	26	CD	ARG	82	20.077	12.130	37.423	1.00	17.30
ATOM	27	NE	ARG	82	21.465	11.682	37.440	1.00	18.57
ATOM	28	CZ	ARG	82	21.982	10.905	38.384	1.00	20.23
ATOM	29	NH1	ARG	82	21.222	10.491	39.392	1.00	21.72
ATOM	30	NH2	ARG	82	23.258	10.545	38.325	1.00	20.18
ATOM	31	C	ARG	82	19.904	12.741	32.447	1.00	9.37
ATOM	32	O	ARG	82	18.891	12.279	31.920	1.00	8.91
ATOM	33	N	LEU	83	21.112	12.674	31.895	1.00	8.33
ATOM	34	CA	LEU	83	21.367	11.987	30.634	1.00	9.07
ATOM	35	CB	LEU	83	22.237	12.843	29.708	1.00	9.05
ATOM	36	CG	LEU	83	21.697	14.220	29.331	1.00	9.81
ATOM	37	CD1	LEU	83	22.633	14.866	28.318	1.00	9.99
ATOM	38	CD2	LEU	83	20.301	14.088	28.750	1.00	9.81
ATOM	39	C	LEU	83	22.133	10.747	31.055	1.00	10.07
ATOM	40	O	LEU	83	23.229	10.847	31.607	1.00	9.78
ATOM	41	N	ARG	84	21.561	9.576	30.812	1.00	9.78
ATOM	42	CA	ARG	84	22.220	8.354	31.234	1.00	11.27
ATOM	43	CB	ARG	84	21.635	7.907	32.573	1.00	13.30
ATOM	44	CG	ARG	84	21.624	9.002	33.617	1.00	16.93
ATOM	45	CD	ARG	84	22.305	8.543	34.881	1.00	18.68
ATOM	46	NE	ARG	84	21.347	8.127	35.893	1.00	21.27
ATOM	47	CZ	ARG	84	21.689	7.665	37.088	1.00	21.80
ATOM	48	NH1	ARG	84	22.971	7.558	37.415	1.00	23.39
ATOM	49	NH2	ARG	84	20.752	7.322	37.959	1.00	23.50
ATOM	50	C	ARG	84	22.098	7.212	30.253	1.00	9.55
ATOM	51	O	ARG	84	21.488	7.335	29.199	1.00	8.79
ATOM	52	N	ARG	85	22.712	6.095	30.617	1.00	9.72
ATOM	53	CA	ARG	85	22.632	4.886	29.823	1.00	9.53
ATOM	54	CB	ARG	85	24.026	4.410	29.393	1.00	11.00
ATOM	55	CG	ARG	85	24.690	5.336	28.390	1.00	13.65
ATOM	56	CD	ARG	85	25.824	4.654	27.640	1.00	15.37
ATOM	57	NE	ARG	85	26.269	5.472	26.517	1.00	18.74
ATOM	58	CZ	ARG	85	26.907	6.630	26.638	1.00	19.28
ATOM	59	NH1	ARG	85	27.193	7.113	27.837	1.00	20.48
ATOM	60	NH2	ARG	85	27.236	7.313	25.552	1.00	21.17
ATOM	61	C	ARG	85	21.981	3.873	30.751	1.00	9.35
ATOM	62	O	ARG	85	22.216	3.891	31.962	1.00	9.90
ATOM	63	N	LEU	86	21.128	3.024	30.194	1.00	8.17
ATOM	64	CA	LEU	86	20.452	2.000	30.978	1.00	7.93
ATOM	65	CB	LEU	86	18.945	2.045	30.711	1.00	7.25
ATOM	66	CG	LEU	86	18.309	3.365	31.168	1.00	7.91
ATOM	67	CD1	LEU	86	16.839	3.414	30.769	1.00	7.67
ATOM	68	CD2	LEU	86	18.448	3.496	32.681	1.00	7.54
ATOM	69	C	LEU	86	21.061	0.673	30.552	1.00	8.05
ATOM	70	O	LEU	86	20.845	0.199	29.437	1.00	7.14
ATOM	71	N	TYR	87	21.839	0.093	31.461	1.00	8.27
ATOM	72	CA	TYR	87	22.559	−1.150	31.218	1.00	9.30
ATOM	73	CB	TYR	87	23.936	−1.046	31.877	1.00	10.81
ATOM	74	CG	TYR	87	24.805	−2.278	31.762	1.00	13.81
ATOM	75	CD1	TYR	87	24.886	−3.200	32.808	1.00	14.83
ATOM	76	CE1	TYR	87	25.721	−4.317	32.720	1.00	16.91
ATOM	77	CD2	TYR	87	25.576	−2.503	30.621	1.00	15.00
ATOM	78	CE2	TYR	87	26.412	−3.615	30.522	1.00	16.14
ATOM	79	CZ	TYR	87	26.481	−4.514	31.575	1.00	16.64
ATOM	80	OH	TYR	87	27.319	−5.601	31.483	1.00	19.14
ATOM	81	C	TYR	87	21.850	−2.412	31.689	1.00	8.55
ATOM	82	O	TYR	87	21.529	−2.552	32.865	1.00	7.71
ATOM	83	N	CYS	88	21.613	−3.327	30.754	1.00	8.66
ATOM	84	CA	CYS	88	20.969	−4.598	31.058	1.00	8.99
ATOM	85	CB	CYS	88	19.961	−4.956	29.964	1.00	9.37
ATOM	86	SG	CYS	88	18.868	−6.332	30.382	1.00	10.25
ATOM	87	C	CYS	88	22.094	−5.630	31.102	1.00	9.81
ATOM	88	O	CYS	88	22.782	−5.850	30.105	1.00	9.57
ATOM	89	N	ASN	89	22.278	−6.262	32.256	1.00	10.42
ATOM	90	CA	ASN	89	23.353	−7.228	32.416	1.00	11.44
ATOM	91	CB	ASN	89	23.800	−7.259	33.882	1.00	12.56
ATOM	92	CG	ASN	89	25.097	−8.023	34.078	1.00	14.22
ATOM	93	OD1	ASN	89	25.975	−7.997	33.217	1.00	14.10
ATOM	94	ND2	ASN	89	25.229	−8.693	35.219	1.00	14.82
ATOM	95	C	ASN	89	23.021	−8.639	31.935	1.00	11.60
ATOM	96	O	ASN	89	23.029	−9.589	32.717	1.00	12.87
ATOM	97	N	VAL	90	22.724	−8.762	30.644	1.00	11.13
ATOM	98	CA	VAL	90	22.414	−10.052	30.030	1.00	11.34
ATOM	99	CB	VAL	90	20.936	−10.130	29.553	1.00	11.83
ATOM	100	CG1	VAL	90	19.998	−10.058	30.752	1.00	12.67
ATOM	101	CG2	VAL	90	20.636	−9.008	28.566	1.00	11.70
ATOM	102	C	VAL	90	23.344	−10.214	28.830	1.00	10.91
ATOM	103	O	VAL	90	23.743	−9.227	28.214	1.00	10.94
ATOM	104	N	GLY	91	23.689	−11.451	28.490	1.00	11.30
ATOM	105	CA	GLY	91	24.596	−11.655	27.377	1.00	11.22
ATOM	106	C	GLY	91	25.905	−10.960	27.710	1.00	11.54
ATOM	107	O	GLY	91	26.358	−11.021	28.851	1.00	11.96
ATOM	108	N	ILE	92	26.515	−10.295	26.735	1.00	11.95
ATOM	109	CA	ILE	92	27.770	−9.590	26.983	1.00	12.07
ATOM	110	CB	ILE	92	28.553	−9.333	25.673	1.00	12.90
ATOM	111	CG2	ILE	92	29.022	−10.653	25.075	1.00	13.76
ATOM	112	CG1	ILE	92	27.679	−8.557	24.684	1.00	12.69
ATOM	113	CD1	ILE	92	28.396	−8.175	23.403	1.00	13.90
ATOM	114	C	ILE	92	27.526	−8.241	27.661	1.00	11.76
ATOM	115	O	ILE	92	28.470	−7.516	27.974	1.00	12.10
ATOM	116	N	GLY	93	26.258	−7.912	27.888	1.00	11.33
ATOM	117	CA	GLY	93	25.921	−6.643	28.513	1.00	10.40
ATOM	118	C	GLY	93	25.518	−5.634	27.454	1.00	10.14
ATOM	119	O	GLY	93	26.279	−5.386	26.514	1.00	9.39
ATOM	120	N	PHE	94	24.330	−5.048	27.587	1.00	9.60
ATOM	121	CA	PHE	94	23.868	−4.081	26.591	1.00	9.48
ATOM	122	CB	PHE	94	22.881	−4.738	25.620	1.00	10.07
ATOM	123	CG	PHE	94	23.416	−5.959	24.932	1.00	11.72
ATOM	124	CD1	PHE	94	23.281	−7.215	25.512	1.00	11.80
ATOM	125	CD2	PHE	94	24.052	−5.851	23.699	1.00	12.37
ATOM	126	CE1	PHE	94	23.768	−8.350	24.872	1.00	12.80
ATOM	127	CE2	PHE	94	24.544	−6.977	23.052	1.00	13.09
ATOM	128	CZ	PHE	94	24.402	−8.229	23.637	1.00	13.50
ATOM	129	C	PHE	94	23.196	−2.837	27.162	1.00	9.07
ATOM	130	O	PHE	94	22.530	−2.898	28.194	1.00	9.31
ATOM	131	N	HIS	95	23.372	−1.712	26.473	1.00	9.61
ATOM	132	CA	HIS	95	22.739	−0.454	26.868	1.00	8.83
ATOM	133	CB	HIS	95	23.667	0.755	26.670	1.00	9.80
ATOM	134	CG	HIS	95	24.991	0.636	27.356	1.00	11.64
ATOM	135	CD2	HIS	95	25.374	0.950	28.614	1.00	11.84
ATOM	136	ND1	HIS	95	26.112	0.151	26.722	1.00	12.48
ATOM	137	CE1	HIS	95	27.133	0.170	27.559	1.00	12.86
ATOM	138	NE2	HIS	95	26.712	0.650	28.717	1.00	13.05
ATOM	139	C	HIS	95	21.544	−0.270	25.940	1.00	8.68
ATOM	140	O	HIS	95	21.663	−0.449	24.728	1.00	8.37
ATOM	141	N	LEU	96	20.397	0.086	26.505	1.00	7.77
ATOM	142	CA	LEU	96	19.198	0.312	25.711	1.00	7.43
ATOM	143	CB	LEU	96	18.028	0.661	26.634	1.00	7.74
ATOM	144	CG	LEU	96	16.658	0.911	26.000	1.00	7.64
ATOM	145	CD1	LEU	96	16.145	−0.363	25.333	1.00	8.64
ATOM	146	CD2	LEU	96	15.689	1.369	27.083	1.00	7.70
ATOM	147	C	LEU	96	19.485	1.479	24.763	1.00	7.66
ATOM	148	O	LEU	96	20.047	2.492	25.177	1.00	6.82
ATOM	149	N	GLN	97	19.120	1.341	23.493	1.00	7.35
ATOM	150	CA	GLN	97	19.369	2.430	22.556	1.00	8.13
ATOM	151	CB	GLN	97	20.675	2.190	21.787	1.00	9.41
ATOM	152	CG	GLN	97	20.610	1.130	20.698	1.00	11.56
ATOM	153	CD	GLN	97	21.901	1.049	19.892	1.00	13.36
ATOM	154	OE1	GLN	97	22.723	1.967	19.919	1.00	14.09
ATOM	155	NE2	GLN	97	22.076	−0.045	19.159	1.00	13.35
ATOM	156	C	GLN	97	18.231	2.650	21.571	1.00	7.87
ATOM	157	O	GLN	97	17.467	1.735	21.265	1.00	6.47
ATOM	158	N	ALA	98	18.119	3.884	21.091	1.00	7.19
ATOM	159	CA	ALA	98	17.094	4.242	20.122	1.00	7.41
ATOM	160	CB	ALA	98	16.259	5.399	20.650	1.00	6.51
ATOM	161	C	ALA	98	17.817	4.642	18.839	1.00	7.88
ATOM	162	O	ALA	98	18.651	5.549	18.846	1.00	7.94
ATOM	163	N	LEU	99	17.504	3.960	17.743	1.00	8.72
ATOM	164	CA	LEU	99	18.149	4.237	16.465	1.00	9.77
ATOM	165	CB	LEU	99	18.245	2.946	15.651	1.00	10.44
ATOM	166	CG	LEU	99	19.040	1.816	16.318	1.00	11.58
ATOM	167	CD1	LEU	99	19.132	0.639	15.367	1.00	13.27
ATOM	168	CD2	LEU	99	20.433	2.299	16.678	1.00	11.97
ATOM	169	C	LEU	99	17.441	5.321	15.657	1.00	9.49
ATOM	170	O	LEU	99	16.262	5.599	15.871	1.00	10.13
ATOM	171	N	PRO	100	18.159	5.943	14.709	1.00	9.88
ATOM	172	CD	PRO	100	19.583	5.707	14.403	1.00	10.62
ATOM	173	CA	PRO	100	17.615	7.006	13.858	1.00	10.16
ATOM	174	CB	PRO	100	18.847	7.512	13.105	1.00	10.38
ATOM	175	CG	PRO	100	19.707	6.290	13.012	1.00	11.95
ATOM	176	C	PRO	100	16.482	6.603	12.921	1.00	9.96
ATOM	177	O	PRO	100	15.811	7.471	12.358	1.00	9.98
ATOM	178	N	ASP	101	16.261	5.301	12.743	1.00	9.35
ATOM	179	CA	ASP	101	15.175	4.862	11.874	1.00	8.93
ATOM	180	CB	ASP	101	15.613	3.683	10.988	1.00	8.94
ATOM	181	CG	ASP	101	16.058	2.464	11.779	1.00	9.32
ATOM	182	OD1	ASP	101	16.005	2.478	13.028	1.00	7.56
ATOM	183	OD2	ASP	101	16.467	1.474	11.132	1.00	9.79
ATOM	184	C	ASP	101	13.923	4.499	12.668	1.00	8.49
ATOM	185	O	ASP	101	12.914	4.074	12.098	1.00	7.97
ATOM	186	N	GLY	102	13.992	4.681	13.985	1.00	7.41
ATOM	187	CA	GLY	102	12.851	4.386	14.835	1.00	6.68
ATOM	188	C	GLY	102	12.906	3.057	15.562	1.00	6.54
ATOM	189	O	GLY	102	12.011	2.740	16.344	1.00	6.36
ATOM	190	N	ARG	103	13.946	2.268	15.316	1.00	5.93
ATOM	191	CA	ARG	103	14.068	0.982	15.987	1.00	6.49
ATOM	192	CB	ARG	103	14.961	0.034	15.174	1.00	7.14
ATOM	193	CG	ARG	103	14.338	−0.417	13.854	1.00	8.91
ATOM	194	CD	ARG	103	15.206	−1.444	13.124	1.00	11.34
ATOM	195	NE	ARG	103	15.547	−2.579	13.979	1.00	13.84
ATOM	196	CZ	ARG	103	16.740	−2.764	14.540	1.00	15.57
ATOM	197	NH1	ARG	103	16.956	−3.823	15.309	1.00	16.83
ATOM	198	NH2	ARG	103	17.725	−1.901	14.317	1.00	15.44
ATOM	199	C	ARG	103	14.632	1.154	17.394	1.00	6.57
ATOM	200	O	ARG	103	15.241	2.175	17.713	1.00	7.34
ATOM	201	N	ILE	104	14.404	0.149	18.230	1.00	5.76
ATOM	202	CA	ILE	104	14.883	0.138	19.606	1.00	6.17
ATOM	203	CB	ILE	104	13.705	0.097	20.621	1.00	5.44
ATOM	204	CG2	ILE	104	14.243	0.072	22.047	1.00	5.86
ATOM	205	CG1	ILE	104	12.780	1.306	20.427	1.00	5.60
ATOM	206	CD1	ILE	104	13.424	2.652	20.740	1.00	6.40
ATOM	207	C	ILE	104	15.704	−1.146	19.752	1.00	6.60
ATOM	208	O	ILE	104	15.334	−2.190	19.208	1.00	6.24
ATOM	209	N	GLY	105	16.820	−1.071	20.468	1.00	6.98
ATOM	210	CA	GLY	105	17.641	−2.255	20.645	1.00	8.04
ATOM	211	C	GLY	105	18.680	−2.100	21.737	1.00	8.60
ATOM	212	O	GLY	105	18.540	−1.255	22.620	1.00	8.84
ATOM	213	N	GLY	106	19.723	−2.923	21.674	1.00	8.72
ATOM	214	CA	GLY	106	20.779	−2.861	22.667	1.00	9.01
ATOM	215	C	GLY	106	22.146	−2.743	22.021	1.00	8.89
ATOM	216	O	GLY	106	22.356	−3.219	20.905	1.00	8.84
ATOM	217	N	ALA	107	23.072	−2.092	22.715	1.00	8.61
ATOM	218	CA	ALA	107	24.432	−1.920	22.213	1.00	9.67
ATOM	219	CB	ALA	107	24.604	−0.528	21.620	1.00	10.06
ATOM	220	C	ALA	107	25.409	−2.124	23.364	1.00	10.28
ATOM	221	O	ALA	107	25.273	−1.508	24.418	1.00	9.28
ATOM	222	N	HIS	108	26.391	−2.995	23.161	1.00	11.70
ATOM	223	CA	HIS	108	27.374	−3.270	24.198	1.00	12.04
ATOM	224	CB	HIS	108	28.253	−4.455	23.786	1.00	13.11
ATOM	225	CG	HIS	108	29.307	−4.801	24.792	1.00	14.15
ATOM	226	CD2	HIS	108	30.657	−4.798	24.702	1.00	14.78
ATOM	227	ND1	HIS	108	29.008	−5.191	26.079	1.00	14.21
ATOM	228	CE1	HIS	108	30.129	−5.412	26.741	1.00	15.02
ATOM	229	NE2	HIS	108	31.146	−5.181	25.927	1.00	16.19
ATOM	230	C	HIS	108	28.244	−2.048	24.480	1.00	12.52
ATOM	231	O	HIS	108	28.554	−1.752	25.633	1.00	12.77
ATOM	232	N	ALA	109	28.631	−1.334	23.429	1.00	13.09
ATOM	233	CA	ALA	109	29.470	−0.150	23.587	1.00	13.18
ATOM	234	CB	ALA	109	30.199	0.147	22.280	1.00	14.30
ATOM	235	C	ALA	109	28.654	1.067	24.016	1.00	13.45
ATOM	236	O	ALA	109	27.423	1.040	23.985	1.00	12.92
ATOM	237	N	ASP	110	29.344	2.127	24.427	1.00	13.54
ATOM	238	CA	ASP	110	28.674	3.355	24.841	1.00	14.78
ATOM	239	CB	ASP	110	29.548	4.164	25.807	1.00	16.93
ATOM	240	CG	ASP	110	29.747	3.470	27.143	1.00	19.56
ATOM	241	OD1	ASP	110	28.758	2.968	27.715	1.00	20.96
ATOM	242	OD2	ASP	110	30.897	3.442	27.631	1.00	21.98
ATOM	243	C	ASP	110	28.375	4.190	23.599	1.00	13.85
ATOM	244	O	ASP	110	28.956	5.257	23.399	1.00	13.85
ATOM	245	N	THR	111	27.465	3.687	22.773	1.00	12.59
ATOM	246	CA	THR	111	27.066	4.351	21.532	1.00	11.75
ATOM	247	CB	THR	111	26.120	3.456	20.709	1.00	11.78
ATOM	248	OG1	THR	111	24.926	3.205	21.464	1.00	11.42
ATOM	249	CG2	THR	111	26.783	2.133	20.378	1.00	11.72
ATOM	250	C	THR	111	26.340	5.666	21.779	1.00	11.02
ATOM	251	O	THR	111	25.736	5.864	22.831	1.00	10.14
ATOM	252	N	ARG	112	26.397	6.565	20.799	1.00	10.53
ATOM	253	CA	ARG	112	25.715	7.845	20.918	1.00	10.82
ATOM	254	CB	ARG	112	25.941	8.701	19.669	1.00	12.11
ATOM	255	CG	ARG	112	27.380	9.110	19.432	1.00	14.73
ATOM	256	CD	ARG	112	27.470	9.990	18.198	1.00	17.34
ATOM	257	NE	ARG	112	28.842	10.372	17.886	1.00	19.56
ATOM	258	CZ	ARG	112	29.169	11.260	16.955	1.00	20.19
ATOM	259	NH1	ARG	112	28.218	11.857	16.248	1.00	21.18
ATOM	260	NH2	ARG	112	30.443	11.552	16.729	1.00	21.74
ATOM	261	C	ARG	112	24.220	7.604	21.083	1.00	10.26
ATOM	262	O	ARG	112	23.562	8.275	21.874	1.00	10.16
ATOM	263	N	ASP	113	23.694	6.635	20.335	1.00	9.55
ATOM	264	CA	ASP	113	22.270	6.319	20.385	1.00	9.39
ATOM	265	CB	ASP	113	21.875	5.410	19.214	1.00	9.84
ATOM	266	CG	ASP	113	21.993	6.103	17.867	1.00	11.80
ATOM	267	OD1	ASP	113	22.074	7.351	17.838	1.00	12.79
ATOM	268	OD2	ASP	113	21.991	5.402	16.833	1.00	11.25
ATOM	269	C	ASP	113	21.802	5.686	21.690	1.00	8.59
ATOM	270	O	ASP	113	20.604	5.473	21.876	1.00	7.24
ATOM	271	N	SER	114	22.730	5.386	22.595	1.00	8.92
ATOM	272	CA	SER	114	22.343	4.794	23.871	1.00	8.63
ATOM	273	CB	SER	114	23.294	3.650	24.256	1.00	9.68
ATOM	274	OG	SER	114	24.625	4.100	24.427	1.00	10.49
ATOM	275	C	SER	114	22.286	5.836	24.989	1.00	8.56
ATOM	276	O	SER	114	22.042	5.495	26.145	1.00	8.23
ATOM	277	N	LEU	115	22.520	7.103	24.651	1.00	7.61
ATOM	278	CA	LEU	115	22.445	8.164	25.652	1.00	7.24
ATOM	279	CB	LEU	115	23.260	9.386	25.230	1.00	8.19
ATOM	280	CG	LEU	115	23.344	10.488	26.292	1.00	8.61
ATOM	281	CD1	LEU	115	24.133	9.981	27.504	1.00	9.28
ATOM	282	CD2	LEU	115	24.015	11.717	25.703	1.00	9.34
ATOM	283	C	LEU	115	20.972	8.534	25.745	1.00	6.88
ATOM	284	O	LEU	115	20.374	8.976	24.762	1.00	7.36
ATOM	285	N	LEU	116	20.389	8.353	26.923	1.00	6.00
ATOM	286	CA	LEU	116	18.968	8.621	27.108	1.00	6.40
ATOM	287	CB	LEU	116	18.266	7.325	27.528	1.00	6.05
ATOM	288	CG	LEU	116	18.557	6.068	26.702	1.00	5.86
ATOM	289	CD1	LEU	116	17.940	4.851	27.392	1.00	6.33
ATOM	290	CD2	LEU	116	17.991	6.231	25.304	1.00	6.19
ATOM	291	C	LEU	116	18.663	9.703	28.137	1.00	7.23
ATOM	292	O	LEU	116	19.225	9.710	29.230	1.00	8.16
ATOM	293	N	GLU	117	17.771	10.621	27.785	1.00	7.97
ATOM	294	CA	GLU	117	17.384	11.669	28.723	1.00	8.82
ATOM	295	CB	GLU	117	17.043	12.971	27.990	1.00	10.69
ATOM	296	CG	GLU	117	16.520	14.065	28.914	1.00	12.86
ATOM	297	CD	GLU	117	16.478	15.433	28.258	1.00	16.05
ATOM	298	OE1	GLU	117	15.991	16.386	28.903	1.00	17.79
ATOM	299	OE2	GLU	117	16.937	15.562	27.105	1.00	17.80
ATOM	300	C	GLU	117	16.164	11.174	29.490	1.00	9.20
ATOM	301	O	GLU	117	15.122	10.891	28.899	1.00	8.75
ATOM	302	N	LEU	118	16.305	11.045	30.804	1.00	8.92
ATOM	303	CA	LEU	118	15.204	10.592	31.642	1.00	9.72
ATOM	304	CB	LEU	118	15.728	9.722	32.786	1.00	11.39
ATOM	305	CG	LEU	118	16.532	8.480	32.394	1.00	12.95
ATOM	306	CD1	LEU	118	16.853	7.667	33.643	1.00	14.11
ATOM	307	CD2	LEU	118	15.743	7.643	31.409	1.00	14.39
ATOM	308	C	LEU	118	14.508	11.826	32.200	1.00	10.39
ATOM	309	O	LEU	118	15.103	12.596	32.955	1.00	9.73
ATOM	310	N	SER	119	13.251	12.024	31.819	1.00	10.03
ATOM	311	CA	SER	119	12.510	13.187	32.284	1.00	10.69
ATOM	312	CB	SER	119	12.014	14.005	31.091	1.00	11.30
ATOM	313	OG	SER	119	13.080	14.335	30.217	1.00	11.39
ATOM	314	C	SER	119	11.321	12.770	33.132	1.00	10.86
ATOM	315	O	SER	119	10.503	11.959	32.706	1.00	10.01
ATOM	316	N	PRO	120	11.213	13.315	34.350	1.00	11.88
ATOM	317	CD	PRO	120	12.109	14.265	35.038	1.00	12.61
ATOM	318	CA	PRO	120	10.081	12.950	35.202	1.00	12.25
ATOM	319	CB	PRO	120	10.441	13.581	36.547	1.00	13.23
ATOM	320	CG	PRO	120	11.220	14.799	36.145	1.00	14.45
ATOM	321	C	PRO	120	8.782	13.505	34.623	1.00	12.69
ATOM	322	O	PRO	120	8.730	14.648	34.166	1.00	12.96
ATOM	323	N	VAL	121	7.742	12.682	34.627	1.00	12.46
ATOM	324	CA	VAL	121	6.440	13.088	34.118	1.00	12.87
ATOM	325	CB	VAL	121	5.797	11.948	33.306	1.00	12.58
ATOM	326	CG1	VAL	121	4.378	12.318	32.902	1.00	11.86
ATOM	327	CG2	VAL	121	6.642	11.666	32.074	1.00	13.10
ATOM	328	C	VAL	121	5.586	13.418	35.332	1.00	12.84
ATOM	329	O	VAL	121	4.902	14.440	35.378	1.00	13.81
ATOM	330	N	GLU	122	5.654	12.532	36.316	1.00	12.83
ATOM	331	CA	GLU	122	4.949	12.667	37.583	1.00	12.89
ATOM	332	CB	GLU	122	3.488	12.222	37.457	1.00	13.88
ATOM	333	CG	GLU	122	3.266	10.931	36.691	1.00	15.79
ATOM	334	CD	GLU	122	1.805	10.506	36.688	1.00	17.76
ATOM	335	OE1	GLU	122	1.385	9.812	35.741	1.00	18.31
ATOM	336	OE2	GLU	122	1.078	10.856	37.642	1.00	19.25
ATOM	337	C	GLU	122	5.705	11.759	38.538	1.00	12.49
ATOM	338	O	GLU	122	6.672	11.114	38.135	1.00	12.27
ATOM	339	N	ARG	123	5.288	11.701	39.795	1.00	11.54
ATOM	340	CA	ARG	123	5.998	10.854	40.739	1.00	10.92
ATOM	341	CB	ARG	123	5.344	10.896	42.120	1.00	11.82
ATOM	342	CG	ARG	123	5.972	9.885	43.059	1.00	12.96
ATOM	343	CD	ARG	123	5.602	10.112	44.501	1.00	14.90
ATOM	344	NE	ARG	123	6.104	9.014	45.318	1.00	14.69
ATOM	345	CZ	ARG	123	6.118	9.015	46.644	1.00	14.66
ATOM	346	NH1	ARG	123	5.658	10.067	47.309	1.00	15.83
ATOM	347	NH2	ARG	123	6.579	7.963	47.302	1.00	13.18
ATOM	348	C	ARG	123	6.109	9.399	40.288	1.00	9.45
ATOM	349	O	ARG	123	5.101	8.744	40.018	1.00	9.21
ATOM	350	N	GLY	124	7.345	8.909	40.217	1.00	8.93
ATOM	351	CA	GLY	124	7.601	7.530	39.828	1.00	8.60
ATOM	352	C	GLY	124	7.471	7.208	38.349	1.00	7.77
ATOM	353	O	GLY	124	7.656	6.060	37.947	1.00	7.28
ATOM	354	N	VAL	125	7.169	8.215	37.537	1.00	7.41
ATOM	355	CA	VAL	125	6.989	8.018	36.101	1.00	7.25
ATOM	356	CB	VAL	125	5.546	8.377	35.687	1.00	6.87
ATOM	357	CG1	VAL	125	5.339	8.101	34.212	1.00	7.15
ATOM	358	CG2	VAL	125	4.552	7.585	36.530	1.00	7.51
ATOM	359	C	VAL	125	7.955	8.874	35.291	1.00	7.01
ATOM	360	O	VAL	125	8.153	10.052	35.593	1.00	7.46
ATOM	361	N	VAL	126	8.539	8.286	34.249	1.00	6.88
ATOM	362	CA	VAL	126	9.493	8.999	33.403	1.00	6.56
ATOM	363	CB	VAL	126	10.942	8.553	33.703	1.00	6.83
ATOM	364	CG1	VAL	126	11.302	8.836	35.148	1.00	6.87
ATOM	365	CG2	VAL	126	11.087	7.064	33.401	1.00	7.21
ATOM	366	C	VAL	126	9.290	8.760	31.911	1.00	6.87
ATOM	367	O	VAL	126	8.579	7.842	31.505	1.00	7.11
ATOM	368	N	SER	127	9.911	9.612	31.099	1.00	5.95
ATOM	369	CA	SER	127	9.895	9.432	29.655	1.00	6.02
ATOM	370	CB	SER	127	9.447	10.696	28.908	1.00	6.80
ATOM	371	OG	SER	127	10.344	11.774	29.083	1.00	7.48
ATOM	372	C	SER	127	11.366	9.130	29.384	1.00	6.01
ATOM	373	O	SER	127	12.243	9.559	30.143	1.00	6.11
ATOM	374	N	ILE	128	11.636	8.375	28.330	1.00	5.10
ATOM	375	CA	ILE	128	13.003	7.997	27.999	1.00	5.69
ATOM	376	CB	ILE	128	13.170	6.472	28.121	1.00	5.40
ATOM	377	CG2	ILE	128	14.626	6.088	27.897	1.00	6.23
ATOM	378	CG1	ILE	128	12.698	6.014	29.511	1.00	5.86
ATOM	379	CD1	ILE	128	12.683	4.497	29.713	1.00	5.16
ATOM	380	C	ILE	128	13.268	8.455	26.572	1.00	5.76
ATOM	381	O	ILE	128	12.789	7.849	25.615	1.00	6.10
ATOM	382	N	PHE	129	14.041	9.530	26.442	1.00	5.77
ATOM	383	CA	PHE	129	14.324	10.126	25.141	1.00	6.64
ATOM	384	CB	PHE	129	14.092	11.638	25.237	1.00	7.28
ATOM	385	CG	PHE	129	14.167	12.355	23.920	1.00	7.76
ATOM	386	CD1	PHE	129	13.172	12.186	22.965	1.00	8.17
ATOM	387	CD2	PHE	129	15.227	13.214	23.643	1.00	8.14
ATOM	388	CE1	PHE	129	13.227	12.867	21.752	1.00	8.24
ATOM	389	CE2	PHE	129	15.293	13.897	22.435	1.00	8.09
ATOM	390	CZ	PHE	129	14.291	13.725	21.488	1.00	8.83
ATOM	391	C	PHE	129	15.722	9.859	24.588	1.00	7.07
ATOM	392	O	PHE	129	16.726	10.118	25.253	1.00	6.90
ATOM	393	N	GLY	130	15.773	9.344	23.363	1.00	7.82
ATOM	394	CA	GLY	130	17.048	9.073	22.720	1.00	7.44
ATOM	395	C	GLY	130	17.588	10.389	22.195	1.00	8.24
ATOM	396	O	GLY	130	17.134	10.892	21.170	1.00	7.64
ATOM	397	N	VAL	131	18.562	10.944	22.908	1.00	8.47
ATOM	398	CA	VAL	131	19.164	12.226	22.561	1.00	8.95
ATOM	399	CB	VAL	131	20.371	12.512	23.489	1.00	10.08
ATOM	400	CG1	VAL	131	20.979	13.857	23.157	1.00	12.10
ATOM	401	CG2	VAL	131	19.917	12.487	24.948	1.00	11.84
ATOM	402	C	VAL	131	19.606	12.383	21.107	1.00	8.99
ATOM	403	O	VAL	131	19.258	13.364	20.447	1.00	8.93
ATOM	404	N	ALA	132	20.367	11.416	20.606	1.00	7.92
ATOM	405	CA	ALA	132	20.880	11.473	19.241	1.00	8.44
ATOM	406	CB	ALA	132	22.082	10.529	19.103	1.00	8.07
ATOM	407	C	ALA	132	19.864	11.170	18.142	1.00	8.83
ATOM	408	O	ALA	132	19.806	11.876	17.132	1.00	9.23
ATOM	409	N	SER	133	19.066	10.123	18.337	1.00	8.54
ATOM	410	CA	SER	133	18.076	9.721	17.341	1.00	9.10
ATOM	411	CB	SER	133	17.626	8.279	17.592	1.00	9.66
ATOM	412	OG	SER	133	16.807	8.212	18.748	1.00	12.46
ATOM	413	C	SER	133	16.850	10.627	17.356	1.00	9.35
ATOM	414	O	SER	133	16.158	10.761	16.348	1.00	8.28
ATOM	415	N	ARG	134	16.585	11.234	18.510	1.00	9.32
ATOM	416	CA	ARG	134	15.436	12.113	18.696	1.00	10.27
ATOM	417	CB	ARG	134	15.380	13.170	17.588	1.00	12.72
ATOM	418	CG	ARG	134	16.623	14.046	17.501	1.00	16.89
ATOM	419	CD	ARG	134	16.855	14.803	18.799	1.00	19.81
ATOM	420	NE	ARG	134	18.015	15.691	18.743	1.00	23.62
ATOM	421	CZ	ARG	134	18.138	16.699	17.886	1.00	24.68
ATOM	422	NH1	ARG	134	17.172	16.945	17.012	1.00	25.92
ATOM	423	NH2	ARG	134	19.218	17.471	17.911	1.00	25.37
ATOM	424	C	ARG	134	14.121	11.331	18.745	1.00	9.51
ATOM	425	O	ARG	134	13.077	11.837	18.339	1.00	10.33
ATOM	426	N	PHE	135	14.178	10.097	19.237	1.00	8.65
ATOM	427	CA	PHE	135	12.980	9.269	19.374	1.00	8.38
ATOM	428	CB	PHE	135	13.106	7.940	18.621	1.00	8.83
ATOM	429	CG	PHE	135	12.898	8.035	17.139	1.00	8.66
ATOM	430	CD1	PHE	135	13.953	8.356	16.291	1.00	10.75
ATOM	431	CD2	PHE	135	11.649	7.765	16.584	1.00	8.52
ATOM	432	CE1	PHE	135	13.768	8.404	14.909	1.00	10.30
ATOM	433	CE2	PHE	135	11.450	7.811	15.205	1.00	8.67
ATOM	434	CZ	PHE	135	12.512	8.131	14.366	1.00	10.46
ATOM	435	C	PHE	135	12.768	8.924	20.842	1.00	7.92
ATOM	436	O	PHE	135	13.730	8.669	21.564	1.00	7.39
ATOM	437	N	PHE	136	11.512	8.920	21.279	1.00	7.93
ATOM	438	CA	PHE	136	11.184	8.529	22.642	1.00	7.08
ATOM	439	CB	PHE	136	9.824	9.077	23.091	1.00	7.49
ATOM	440	CG	PHE	136	9.838	10.515	23.482	1.00	6.98
ATOM	441	CD1	PHE	136	9.518	11.501	22.560	1.00	7.11
ATOM	442	CD2	PHE	136	10.155	10.884	24.785	1.00	7.49
ATOM	443	CE1	PHE	136	9.508	12.839	22.928	1.00	8.41
ATOM	444	CE2	PHE	136	10.150	12.218	25.166	1.00	8.09
ATOM	445	CZ	PHE	136	9.825	13.200	24.233	1.00	7.87
ATOM	446	C	PHE	136	11.033	7.019	22.576	1.00	7.61
ATOM	447	O	PHE	136	10.617	6.483	21.548	1.00	7.88
ATOM	448	N	VAL	137	11.378	6.331	23.654	1.00	6.58
ATOM	449	CA	VAL	137	11.189	4.890	23.691	1.00	6.26
ATOM	450	CB	VAL	137	12.017	4.231	24.805	1.00	6.19
ATOM	451	CG1	VAL	137	11.626	2.759	24.937	1.00	6.00
ATOM	452	CG2	VAL	137	13.503	4.351	24.488	1.00	6.61
ATOM	453	C	VAL	137	9.708	4.749	24.027	1.00	6.37
ATOM	454	O	VAL	137	9.213	5.417	24.937	1.00	6.96
ATOM	455	N	ALA	138	8.997	3.907	23.286	1.00	6.10
ATOM	456	CA	ALA	138	7.568	3.707	23.522	1.00	6.34
ATOM	457	CB	ALA	138	6.750	4.478	22.479	1.00	6.40
ATOM	458	C	ALA	138	7.220	2.226	23.461	1.00	6.19
ATOM	459	O	ALA	138	7.998	1.418	22.959	1.00	5.65
ATOM	460	N	MET	139	6.053	1.866	23.984	1.00	6.77
ATOM	461	CA	MET	139	5.622	0.472	23.949	1.00	6.76
ATOM	462	CB	MET	139	5.802	−0.193	25.316	1.00	7.13
ATOM	463	CG	MET	139	5.454	−1.675	25.304	1.00	8.40
ATOM	464	SD	MET	139	5.687	−2.479	26.897	1.00	9.30
ATOM	465	CE	MET	139	7.482	−2.496	27.008	1.00	8.92
ATOM	466	C	MET	139	4.161	0.391	23.529	1.00	7.49
ATOM	467	O	MET	139	3.318	1.120	24.055	1.00	6.81
ATOM	468	N	SER	140	3.873	−0.493	22.576	1.00	7.52
ATOM	469	CA	SER	140	2.515	−0.679	22.068	1.00	8.67
ATOM	470	CB	SER	140	2.557	−1.319	20.678	1.00	8.60
ATOM	471	OG	SER	140	2.937	−2.684	20.771	1.00	7.74
ATOM	472	C	SER	140	1.696	−1.573	22.996	1.00	9.77
ATOM	473	O	SER	140	2.226	−2.148	23.948	1.00	9.80
ATOM	474	N	SER	141	0.407	−1.702	22.701	1.00	9.71
ATOM	475	CA	SER	141	−0.483	−2.530	23.510	1.00	11.35
ATOM	476	CB	SER	141	−1.939	−2.307	23.091	1.00	11.04
ATOM	477	OG	SER	141	−2.161	−2.753	21.763	1.00	12.07
ATOM	478	C	SER	141	−0.130	−4.010	23.375	1.00	11.94
ATOM	479	O	SER	141	−0.628	−4.848	24.131	1.00	12.36
ATOM	480	N	LYS	142	0.724	−4.327	22.406	1.00	12.31
ATOM	481	CA	LYS	142	1.150	−5.705	22.186	1.00	13.17
ATOM	482	CB	LYS	142	1.258	−6.005	20.690	1.00	15.47
ATOM	483	CG	LYS	142	−0.063	−5.947	19.946	1.00	18.63
ATOM	484	CD	LYS	142	0.144	−6.189	18.463	1.00	21.02
ATOM	485	CE	LYS	142	−1.155	−6.052	17.690	1.00	23.01
ATOM	486	NZ	LYS	142	−0.927	−6.222	16.228	1.00	24.37
ATOM	487	C	LYS	142	2.498	−5.950	22.849	1.00	12.17
ATOM	488	O	LYS	142	3.071	−7.027	22.717	1.00	12.32
ATOM	489	N	GLY	143	3.000	−4.936	23.550	1.00	10.43
ATOM	490	CA	GLY	143	4.270	−5.061	24.245	1.00	9.42
ATOM	491	C	GLY	143	5.506	−4.804	23.404	1.00	8.75
ATOM	492	O	GLY	143	6.626	−5.029	23.862	1.00	8.31
ATOM	493	N	LYS	144	5.314	−4.321	22.181	1.00	7.56
ATOM	494	CA	LYS	144	6.437	−4.054	21.293	1.00	6.96
ATOM	495	CB	LYS	144	5.988	−4.152	19.833	1.00	7.62
ATOM	496	CG	LYS	144	7.102	−3.928	18.815	1.00	8.80
ATOM	497	CD	LYS	144	8.145	−5.037	18.867	1.00	8.64
ATOM	498	CE	LYS	144	9.230	−4.827	17.819	1.00	9.66
ATOM	499	NZ	LYS	144	10.256	−5.911	17.843	1.00	11.27
ATOM	500	C	LYS	144	7.067	−2.687	21.527	1.00	6.78
ATOM	501	O	LYS	144	6.392	−1.657	21.454	1.00	6.18
ATOM	502	N	LEU	145	8.365	−2.680	21.813	1.00	6.40
ATOM	503	CA	LEU	145	9.088	−1.428	22.014	1.00	6.12
ATOM	504	CB	LEU	145	10.437	−1.687	22.689	1.00	5.82
ATOM	505	CG	LEU	145	10.411	−1.994	24.186	1.00	6.37
ATOM	506	CD1	LEU	145	11.800	−2.423	24.650	1.00	7.01
ATOM	507	CD2	LEU	145	9.949	−0.756	24.944	1.00	7.23
ATOM	508	C	LEU	145	9.323	−0.795	20.646	1.00	6.17
ATOM	509	O	LEU	145	9.697	−1.483	19.695	1.00	6.13
ATOM	510	N	TYR	146	9.095	0.509	20.544	1.00	5.68
ATOM	511	CA	TYR	146	9.300	1.206	19.282	1.00	6.42
ATOM	512	CB	TYR	146	8.049	1.078	18.395	1.00	6.42
ATOM	513	CG	TYR	146	6.860	1.917	18.824	1.00	6.55
ATOM	514	CD1	TYR	146	6.665	3.197	18.304	1.00	6.78
ATOM	515	CE1	TYR	146	5.585	3.982	18.707	1.00	7.64
ATOM	516	CD2	TYR	146	5.943	1.438	19.758	1.00	7.13
ATOM	517	CE2	TYR	146	4.857	2.218	20.169	1.00	7.09
ATOM	518	CZ	TYR	146	4.687	3.487	19.640	1.00	7.80
ATOM	519	OH	TYR	146	3.627	4.269	20.044	1.00	7.00
ATOM	520	C	TYR	146	9.643	2.672	19.536	1.00	6.69
ATOM	521	O	TYR	146	9.400	3.201	20.625	1.00	6.62
ATOM	522	N	GLY	147	10.232	3.320	18.538	1.00	5.95
ATOM	523	CA	GLY	147	10.593	4.715	18.692	1.00	5.63
ATOM	524	C	GLY	147	9.469	5.638	18.268	1.00	5.37
ATOM	525	O	GLY	147	8.886	5.458	17.194	1.00	5.41
ATOM	526	N	SER	148	9.159	6.621	19.108	1.00	5.18
ATOM	527	CA	SER	148	8.104	7.588	18.810	1.00	5.87
ATOM	528	CB	SER	148	7.063	7.610	19.934	1.00	6.82
ATOM	529	OG	SER	148	5.963	8.438	19.586	1.00	5.87
ATOM	530	C	SER	148	8.744	8.965	18.662	1.00	6.42
ATOM	531	O	SER	148	9.319	9.496	19.612	1.00	7.70
ATOM	532	N	PRO	149	8.652	9.561	17.462	1.00	7.74
ATOM	533	CD	PRO	149	7.983	9.012	16.268	1.00	7.72
ATOM	534	CA	PRO	149	9.228	10.880	17.182	1.00	8.21
ATOM	535	CB	PRO	149	9.112	10.988	15.662	1.00	8.68
ATOM	536	CG	PRO	149	7.866	10.227	15.368	1.00	9.11
ATOM	537	C	PRO	149	8.561	12.038	17.924	1.00	8.51
ATOM	538	O	PRO	149	9.101	13.147	17.965	1.00	8.97
ATOM	539	N	PHE	150	7.385	11.782	18.493	1.00	8.22
ATOM	540	CA	PHE	150	6.674	12.791	19.274	1.00	8.18
ATOM	541	CB	PHE	150	5.525	13.429	18.466	1.00	9.01
ATOM	542	CG	PHE	150	4.593	12.444	17.820	1.00	10.81
ATOM	543	CD1	PHE	150	3.496	11.943	18.513	1.00	11.41
ATOM	544	CD2	PHE	150	4.798	12.037	16.506	1.00	11.41
ATOM	545	CE1	PHE	150	2.610	11.049	17.903	1.00	12.19
ATOM	546	CE2	PHE	150	3.922	11.144	15.886	1.00	12.69
ATOM	547	CZ	PHE	150	2.824	10.650	16.588	1.00	12.71
ATOM	548	C	PHE	150	6.173	12.145	20.567	1.00	7.43
ATOM	549	O	PHE	150	5.993	10.928	20.636	1.00	6.84
ATOM	550	N	PHE	151	5.967	12.965	21.593	1.00	6.95
ATOM	551	CA	PHE	151	5.550	12.482	22.908	1.00	7.33
ATOM	552	CB	PHE	151	5.862	13.567	23.945	1.00	8.43
ATOM	553	CG	PHE	151	5.952	13.062	25.355	1.00	8.59
ATOM	554	CD1	PHE	151	6.480	11.802	25.631	1.00	8.15
ATOM	555	CD2	PHE	151	5.558	13.870	26.418	1.00	10.06
ATOM	556	CE1	PHE	151	6.614	11.356	26.943	1.00	8.82
ATOM	557	CE2	PHE	151	5.690	13.433	27.738	1.00	9.79
ATOM	558	CZ	PHE	151	6.219	12.174	28.000	1.00	10.29
ATOM	559	C	PHE	151	4.086	12.059	22.994	1.00	7.03
ATOM	560	O	PHE	151	3.192	12.832	22.657	1.00	6.91
ATOM	561	N	THR	152	3.852	10.827	23.450	1.00	7.20
ATOM	562	CA	THR	152	2.495	10.286	23.581	1.00	6.32
ATOM	563	CB	THR	152	2.152	9.316	22.443	1.00	6.44
ATOM	564	OG1	THR	152	2.906	8.110	22.615	1.00	5.93
ATOM	565	CG2	THR	152	2.470	9.933	21.090	1.00	7.16
ATOM	566	C	THR	152	2.321	9.499	24.872	1.00	6.73
ATOM	567	O	THR	152	3.273	9.306	25.629	1.00	6.29
ATOM	568	N	ASP	153	1.102	9.013	25.097	1.00	7.13
ATOM	569	CA	ASP	153	0.801	8.240	26.292	1.00	7.68
ATOM	570	CB	ASP	153	−0.720	8.176	26.521	1.00	8.99
ATOM	571	CG	ASP	153	−1.477	7.518	25.379	1.00	9.77
ATOM	572	OD1	ASP	153	−0.949	7.437	24.252	1.00	9.30
ATOM	573	OD2	ASP	153	−2.631	7.095	25.614	1.00	10.99
ATOM	574	C	ASP	153	1.424	6.837	26.297	1.00	7.99
ATOM	575	O	ASP	153	1.304	6.110	27.282	1.00	8.34
ATOM	576	N	GLU	154	2.092	6.458	25.208	1.00	7.55
ATOM	577	CA	GLU	154	2.763	5.157	25.157	1.00	6.93
ATOM	578	CB	GLU	154	2.537	4.456	23.806	1.00	7.67
ATOM	579	CG	GLU	154	1.090	4.029	23.552	1.00	7.08
ATOM	580	CD	GLU	154	0.944	3.048	22.395	1.00	7.82
ATOM	581	OE1	GLU	154	1.765	3.091	21.456	1.00	6.93
ATOM	582	OE2	GLU	154	−0.010	2.237	22.417	1.00	7.76
ATOM	583	C	GLU	154	4.265	5.353	25.395	1.00	6.91
ATOM	584	O	GLU	154	5.048	4.409	25.276	1.00	6.34
ATOM	585	N	CYS	155	4.653	6.579	25.746	1.00	6.29
ATOM	586	CA	CYS	155	6.055	6.918	26.003	1.00	6.66
ATOM	587	CB	CYS	155	6.426	8.221	25.292	1.00	6.30
ATOM	588	SG	CYS	155	6.252	8.217	23.510	1.00	7.00
ATOM	589	C	CYS	155	6.352	7.099	27.492	1.00	7.01
ATOM	590	O	CYS	155	7.441	7.538	27.863	1.00	8.21
ATOM	591	N	THR	156	5.386	6.775	28.340	1.00	6.69
ATOM	592	CA	THR	156	5.560	6.931	29.780	1.00	5.87
ATOM	593	CB	THR	156	4.348	7.646	30.391	1.00	5.85
ATOM	594	OG1	THR	156	3.146	7.045	29.903	1.00	6.18
ATOM	595	CG2	THR	156	4.360	9.118	30.010	1.00	6.61
ATOM	596	C	THR	156	5.763	5.589	30.467	1.00	5.83
ATOM	597	O	THR	156	5.073	4.615	30.167	1.00	5.54
ATOM	598	N	PHE	157	6.710	5.553	31.401	1.00	5.70
ATOM	599	CA	PHE	157	7.043	4.327	32.113	1.00	5.65
ATOM	600	CB	PHE	157	8.341	3.739	31.545	1.00	6.27
ATOM	601	CG	PHE	157	8.240	3.314	30.111	1.00	5.92
ATOM	602	CD1	PHE	157	7.775	2.046	29.779	1.00	6.03
ATOM	603	CD2	PHE	157	8.582	4.192	29.089	1.00	5.50
ATOM	604	CE1	PHE	157	7.650	1.658	28.445	1.00	6.23
ATOM	605	CE2	PHE	157	8.459	3.814	27.758	1.00	5.35
ATOM	606	CZ	PHE	157	7.992	2.543	27.437	1.00	5.57
ATOM	607	C	PHE	157	7.244	4.545	33.604	1.00	6.10
ATOM	608	O	PHE	157	7.811	5.553	34.021	1.00	5.75
ATOM	609	N	LYS	158	6.784	3.590	34.403	1.00	6.36
ATOM	610	CA	LYS	158	6.976	3.661	35.843	1.00	6.76
ATOM	611	CB	LYS	158	5.964	2.772	36.567	1.00	7.54
ATOM	612	CG	LYS	158	4.531	3.239	36.431	1.00	8.41
ATOM	613	CD	LYS	158	3.599	2.436	37.322	1.00	10.05
ATOM	614	CE	LYS	158	2.186	2.986	37.247	1.00	11.40
ATOM	615	NZ	LYS	158	1.286	2.332	38.237	1.00	12.48
ATOM	616	C	LYS	158	8.389	3.153	36.111	1.00	6.88
ATOM	617	O	LYS	158	8.743	2.048	35.698	1.00	7.51
ATOM	618	N	GLU	159	9.195	3.966	36.785	1.00	6.50
ATOM	619	CA	GLU	159	10.568	3.598	37.117	1.00	7.08
ATOM	620	CB	GLU	159	11.446	4.847	37.124	1.00	8.45
ATOM	621	CG	GLU	159	12.930	4.578	37.312	1.00	10.72
ATOM	622	CD	GLU	159	13.754	5.830	37.101	1.00	12.99
ATOM	623	OE1	GLU	159	13.516	6.820	37.823	1.00	14.61
ATOM	624	OE2	GLU	159	14.632	5.829	36.212	1.00	14.45
ATOM	625	C	GLU	159	10.535	2.954	38.498	1.00	7.00
ATOM	626	O	GLU	159	10.389	3.639	39.515	1.00	6.45
ATOM	627	N	ILE	160	10.679	1.632	38.521	1.00	6.44
ATOM	628	CA	ILE	160	10.602	0.850	39.749	1.00	6.73
ATOM	629	CB	ILE	160	9.649	−0.348	39.534	1.00	7.25
ATOM	630	CG2	ILE	160	9.493	−1.142	40.827	1.00	8.05
ATOM	631	CG1	ILE	160	8.292	0.169	39.047	1.00	7.90
ATOM	632	CD1	ILE	160	7.370	−0.906	38.490	1.00	8.51
ATOM	633	C	ILE	160	11.946	0.336	40.245	1.00	7.21
ATOM	634	O	ILE	160	12.623	−0.426	39.553	1.00	6.33
ATOM	635	N	LEU	161	12.307	0.742	41.461	1.00	6.48
ATOM	636	CA	LEU	161	13.570	0.337	42.065	1.00	6.42
ATOM	637	CB	LEU	161	13.820	1.115	43.362	1.00	6.84
ATOM	638	CG	LEU	161	15.159	0.811	44.047	1.00	6.63
ATOM	639	CD1	LEU	161	16.301	1.238	43.135	1.00	7.13
ATOM	640	CD2	LEU	161	15.247	1.538	45.382	1.00	7.21
ATOM	641	C	LEU	161	13.618	−1.154	42.363	1.00	7.01
ATOM	642	O	LEU	161	12.632	−1.745	42.816	1.00	5.88
ATOM	643	N	LEU	162	14.785	−1.741	42.110	1.00	6.67
ATOM	644	CA	LEU	162	15.044	−3.160	42.333	1.00	7.48
ATOM	645	CB	LEU	162	15.362	−3.843	41.000	1.00	7.02
ATOM	646	CG	LEU	162	14.313	−3.734	39.893	1.00	7.49
ATOM	647	CD1	LEU	162	14.921	−4.162	38.563	1.00	7.87
ATOM	648	CD2	LEU	162	13.112	−4.591	40.254	1.00	8.37
ATOM	649	C	LEU	162	16.264	−3.270	43.243	1.00	7.49
ATOM	650	O	LEU	162	16.890	−2.264	43.570	1.00	7.10
ATOM	651	N	PRO	163	16.610	−4.493	43.677	1.00	8.02
ATOM	652	CD	PRO	163	15.853	−5.756	43.606	1.00	8.20
ATOM	653	CA	PRO	163	17.785	−4.635	44.542	1.00	8.81
ATOM	654	CB	PRO	163	17.822	−6.135	44.827	1.00	9.08
ATOM	655	CG	PRO	163	16.371	−6.514	44.808	1.00	8.96
ATOM	656	C	PRO	163	19.032	−4.175	43.784	1.00	9.09
ATOM	657	O	PRO	163	19.061	−4.205	42.554	1.00	8.86
ATOM	658	N	ASN	164	20.049	−3.743	44.522	1.00	9.58
ATOM	659	CA	ASN	164	21.314	−3.303	43.936	1.00	10.43
ATOM	660	CB	ASN	164	22.031	−4.499	43.304	1.00	11.05
ATOM	661	CG	ASN	164	22.486	−5.518	44.338	1.00	12.27
ATOM	662	OD1	ASN	164	22.849	−6.647	43.999	1.00	13.69
ATOM	663	ND2	ASN	164	22.478	−5.119	45.605	1.00	10.90
ATOM	664	C	ASN	164	21.246	−2.144	42.935	1.00	10.78
ATOM	665	O	ASN	164	22.006	−2.098	41.965	1.00	11.04
ATOM	666	N	ASN	165	20.332	−1.215	43.193	1.00	10.80
ATOM	667	CA	ASN	165	20.148	−0.009	42.390	1.00	11.74
ATOM	668	CB	ASN	165	21.426	0.836	42.423	1.00	14.07
ATOM	669	CG	ASN	165	21.142	2.328	42.341	1.00	17.00
ATOM	670	OD1	ASN	165	20.431	2.884	43.187	1.00	17.76
ATOM	671	ND2	ASN	165	21.691	2.983	41.322	1.00	18.46
ATOM	672	C	ASN	165	19.686	−0.178	40.944	1.00	10.73
ATOM	673	O	ASN	165	19.738	0.769	40.162	1.00	11.09
ATOM	674	N	TYR	166	19.245	−1.375	40.575	1.00	9.88
ATOM	675	CA	TYR	166	18.737	−1.586	39.221	1.00	9.07
ATOM	676	CB	TYR	166	18.721	−3.073	38.862	1.00	9.58
ATOM	677	CG	TYR	166	20.043	−3.623	38.390	1.00	11.15
ATOM	678	CD1	TYR	166	20.312	−3.767	37.028	1.00	11.97
ATOM	679	CE1	TYR	166	21.533	−4.271	36.582	1.00	13.55
ATOM	680	CD2	TYR	166	21.032	−3.991	39.300	1.00	13.15
ATOM	681	CE2	TYR	166	22.257	−4.493	38.866	1.00	14.56
ATOM	682	CZ	TYR	166	22.499	−4.630	37.506	1.00	14.88
ATOM	683	OH	TYR	166	23.707	−5.134	37.068	1.00	16.64
ATOM	684	C	TYR	166	17.302	−1.076	39.218	1.00	8.86
ATOM	685	O	TYR	166	16.690	−0.947	40.277	1.00	9.39
ATOM	686	N	ASN	167	16.772	−0.790	38.032	1.00	7.93
ATOM	687	CA	ASN	167	15.396	−0.332	37.895	1.00	8.47
ATOM	688	CB	ASN	167	15.332	1.143	37.480	1.00	9.78
ATOM	689	CG	ASN	167	15.806	2.084	38.561	1.00	11.84
ATOM	690	OD1	ASN	167	15.313	2.055	39.687	1.00	13.23
ATOM	691	ND2	ASN	167	16.760	2.939	38.218	1.00	13.91
ATOM	692	C	ASN	167	14.688	−1.136	36.818	1.00	7.74
ATOM	693	O	ASN	167	15.313	−1.607	35.863	1.00	8.53
ATOM	694	N	ALA	168	13.381	−1.302	36.989	1.00	7.42
ATOM	695	CA	ALA	168	12.559	−1.972	35.995	1.00	5.36
ATOM	696	CB	ALA	168	11.664	−3.014	36.640	1.00	5.62
ATOM	697	C	ALA	168	11.720	−0.827	35.451	1.00	5.33
ATOM	698	O	ALA	168	11.470	0.155	36.158	1.00	5.24
ATOM	699	N	TYR	169	11.299	−0.939	34.199	1.00	4.83
ATOM	700	CA	TYR	169	10.485	0.100	33.583	1.00	5.05
ATOM	701	CB	TYR	169	11.270	0.770	32.453	1.00	5.13
ATOM	702	CG	TYR	169	12.432	1.583	32.983	1.00	5.77
ATOM	703	CD1	TYR	169	12.254	2.900	33.403	1.00	6.00
ATOM	704	CE1	TYR	169	13.295	3.617	33.994	1.00	6.57
ATOM	705	CD2	TYR	169	13.686	1.001	33.157	1.00	5.97
ATOM	706	CE2	TYR	169	14.733	1.706	33.745	1.00	6.85
ATOM	707	CZ	TYR	169	14.533	3.009	34.164	1.00	7.84
ATOM	708	OH	TYR	169	15.568	3.690	34.774	1.00	8.35
ATOM	709	C	TYR	169	9.200	−0.524	33.071	1.00	5.40
ATOM	710	O	TYR	169	9.207	−1.297	32.114	1.00	5.61
ATOM	711	N	GLU	170	8.095	−0.185	33.723	1.00	5.44
ATOM	712	CA	GLU	170	6.794	−0.733	33.362	1.00	6.45
ATOM	713	CB	GLU	170	6.052	−1.154	34.635	1.00	7.61
ATOM	714	CG	GLU	170	4.608	−1.600	34.426	1.00	7.81
ATOM	715	CD	GLU	170	3.905	−1.897	35.740	1.00	8.83
ATOM	716	OE1	GLU	170	4.305	−1.316	36.772	1.00	9.65
ATOM	717	OE2	GLU	170	2.947	−2.698	35.742	1.00	9.00
ATOM	718	C	GLU	170	5.947	0.255	32.574	1.00	6.80
ATOM	719	O	GLU	170	5.883	1.441	32.906	1.00	6.00
ATOM	720	N	SER	171	5.304	−0.235	31.521	1.00	6.67
ATOM	721	CA	SER	171	4.445	0.615	30.709	1.00	7.04
ATOM	722	CB	SER	171	3.755	−0.201	29.615	1.00	6.44
ATOM	723	CG	SER	171	2.748	0.577	28.982	1.00	8.08
ATOM	724	C	SER	171	3.380	1.241	31.593	1.00	7.11
ATOM	725	O	SER	171	2.683	0.541	32.330	1.00	7.84
ATOM	726	N	TYR	172	3.247	2.560	31.516	1.00	7.12
ATOM	727	CA	TYR	172	2.246	3.243	32.314	1.00	8.72
ATOM	728	CB	TYR	172	2.507	4.748	32.327	1.00	9.00
ATOM	729	CG	TYR	172	1.543	5.499	33.207	1.00	11.05
ATOM	730	CD1	TYR	172	1.765	5.613	34.578	1.00	11.87
ATOM	731	CE1	TYR	172	0.849	6.260	35.403	1.00	13.42
ATOM	732	CD2	TYR	172	0.378	6.050	32.678	1.00	12.30
ATOM	733	CE2	TYR	172	−0.543	6.695	33.492	1.00	14.29
ATOM	734	CZ	TYR	172	−0.303	6.797	34.850	1.00	14.57
ATOM	735	OH	TYR	172	−1.220	7.438	35.652	1.00	17.42
ATOM	736	C	TYR	172	0.855	2.969	31.738	1.00	8.33
ATOM	737	O	TYR	172	−0.103	2.751	32.477	1.00	8.14
ATOM	738	N	LYS	173	0.748	2.975	30.414	1.00	8.55
ATOM	739	CA	LYS	173	−0.536	2.734	29.764	1.00	8.99
ATOM	740	CB	LYS	173	−0.496	3.209	28.306	1.00	9.71
ATOM	741	CG	LYS	173	−1.848	3.137	27.605	1.00	10.52
ATOM	742	CD	LYS	173	−1.861	3.932	26.300	1.00	10.91
ATOM	743	CE	LYS	173	−3.273	4.008	25.727	1.00	11.42
ATOM	744	NZ	LYS	173	−3.348	4.800	24.469	1.00	11.20
ATOM	745	C	LYS	173	−0.977	1.273	29.808	1.00	8.99
ATOM	746	O	LYS	173	−2.177	0.985	29.849	1.00	7.88
ATOM	747	N	TYR	174	−0.012	0.355	29.797	1.00	8.58
ATOM	748	CA	TYR	174	−0.302	−1.080	29.818	1.00	9.01
ATOM	749	CB	TYR	174	0.170	−1.724	28.510	1.00	8.57
ATOM	750	CG	TYR	174	−0.329	−0.998	27.285	1.00	8.58
ATOM	751	CD1	TYR	174	−1.691	−0.947	26.988	1.00	7.77
ATOM	752	CE1	TYR	174	−2.162	−0.224	25.894	1.00	7.99
ATOM	753	CD2	TYR	174	0.555	−0.314	26.450	1.00	7.31
ATOM	754	CE2	TYR	174	0.095	0.411	25.354	1.00	7.84
ATOM	755	CZ	TYR	174	−1.263	0.454	25.083	1.00	7.62
ATOM	756	OH	TYR	174	−1.721	1.185	24.014	1.00	7.92
ATOM	757	C	TYR	174	0.374	−1.771	31.002	1.00	9.15
ATOM	758	O	TYR	174	1.466	−2.332	30.873	1.00	8.77
ATOM	759	N	PRO	175	−0.277	−1.742	32.176	1.00	9.16
ATOM	760	CD	PRO	175	−1.589	−1.129	32.440	1.00	9.90
ATOM	761	CA	PRO	175	0.256	−2.361	33.391	1.00	9.10
ATOM	762	CB	PRO	175	−0.905	−2.227	34.375	1.00	10.40
ATOM	763	CG	PRO	175	−1.566	−0.966	33.941	1.00	10.31
ATOM	764	C	PRO	175	0.663	−3.813	33.180	1.00	9.23
ATOM	765	O	PRO	175	0.000	−4.556	32.455	1.00	9.54
ATOM	766	N	GLY	176	1.765	−4.207	33.806	1.00	9.48
ATOM	767	CA	GLY	176	2.226	−5.577	33.690	1.00	9.58
ATOM	768	C	GLY	176	3.192	−5.838	32.554	1.00	9.81
ATOM	769	O	GLY	176	3.677	−6.958	32.409	1.00	10.30
ATOM	770	N	MET	177	3.463	−4.823	31.739	1.00	8.82
ATOM	771	CA	MET	177	4.401	−4.975	30.629	1.00	8.52
ATOM	772	CB	MET	177	3.760	−4.515	29.320	1.00	10.08
ATOM	773	CG	MET	177	2.649	−5.439	28.859	1.00	11.46
ATOM	774	SD	MET	177	2.093	−5.117	27.179	1.00	12.85
ATOM	775	CE	MET	177	0.393	−5.651	27.292	1.00	13.23
ATOM	776	C	MET	177	5.660	−4.172	30.930	1.00	7.98
ATOM	777	O	MET	177	5.579	−3.010	31.326	1.00	7.40
ATOM	778	N	PHE	178	6.820	−4.797	30.739	1.00	7.68
ATOM	779	CA	PHE	178	8.094	−4.160	31.049	1.00	7.09
ATOM	780	CB	PHE	178	8.743	−4.864	32.250	1.00	8.40
ATOM	781	CG	PHE	178	7.874	−4.908	33.477	1.00	9.38
ATOM	782	CD1	PHE	178	6.767	−5.754	33.536	1.00	9.55
ATOM	783	CD2	PHE	178	8.150	−4.090	34.569	1.00	9.47
ATOM	784	CE1	PHE	178	5.947	−5.783	34.664	1.00	9.89
ATOM	785	CE2	PHE	178	7.336	−4.112	35.699	1.00	10.43
ATOM	786	CZ	PHE	178	6.232	−4.960	35.746	1.00	9.58
ATOM	787	C	PHE	178	9.107	−4.119	29.914	1.00	7.00
ATOM	788	O	PHE	178	9.128	−4.993	29.044	1.00	6.97
ATOM	789	N	ILE	179	9.955	−3.094	29.943	1.00	6.62
ATOM	790	CA	ILE	179	11.010	−2.925	28.949	1.00	6.43
ATOM	791	CB	ILE	179	11.736	−1.569	29.124	1.00	6.72
ATOM	792	CG2	ILE	179	12.947	−1.496	28.202	1.00	7.70
ATOM	793	CG1	ILE	179	10.767	−0.418	28.847	1.00	6.16
ATOM	794	CD1	ILE	179	11.411	0.966	28.933	1.00	6.64
ATOM	795	C	ILE	179	12.017	−4.038	29.195	1.00	6.58
ATOM	796	O	ILE	179	12.330	−4.344	30.342	1.00	6.50
ATOM	797	N	ALA	180	12.531	−4.639	28.129	1.00	6.56
ATOM	798	CA	ALA	180	13.496	−5.712	28.302	1.00	6.46
ATOM	799	CB	ALA	180	12.770	−7.005	28.677	1.00	6.63
ATOM	800	C	ALA	180	14.369	−5.953	27.082	1.00	6.85
ATOM	801	O	ALA	180	13.950	−5.731	25.945	1.00	6.24
ATOM	802	N	LEU	181	15.593	−6.405	27.344	1.00	6.67
ATOM	803	CA	LEU	181	16.561	−6.745	26.305	1.00	7.47
ATOM	804	CB	LEU	181	17.795	−5.839	26.379	1.00	7.48
ATOM	805	CG	LEU	181	17.654	−4.375	25.964	1.00	7.58
ATOM	806	CD1	LEU	181	18.985	−3.680	26.176	1.00	7.44
ATOM	807	CD2	LEU	181	17.230	−4.274	24.503	1.00	8.28
ATOM	808	C	LEU	181	16.987	−8.186	26.571	1.00	8.19
ATOM	809	O	LEU	181	17.144	−8.591	27.725	1.00	8.34
ATOM	810	N	ALA	182	17.176	−8.956	25.508	1.00	9.76
ATOM	811	CA	ALA	182	17.583	−10.346	25.648	1.00	9.78
ATOM	812	CB	ALA	182	16.934	−11.180	24.570	1.00	13.35
ATOM	813	C	ALA	182	19.095	−10.461	25.558	1.00	10.87
ATOM	814	O	ALA	182	19.778	−9.487	25.243	1.00	8.94
ATOM	815	N	LYS	183	19.616	−11.657	25.825	1.00	11.33
ATOM	816	CA	LYS	183	21.054	−11.872	25.779	1.00	12.77
ATOM	817	CB	LYS	183	21.402	−13.275	26.299	1.00	13.33
ATOM	818	CG	LYS	183	20.846	−14.427	25.487	1.00	14.58
ATOM	819	CD	LYS	183	21.231	−15.760	26.124	1.00	15.82
ATOM	820	CE	LYS	183	20.900	−16.937	25.222	1.00	17.00
ATOM	821	NZ	LYS	183	19.446	−17.024	24.917	1.00	18.11
ATOM	822	C	LYS	183	21.645	−11.650	24.389	1.00	13.15
ATOM	823	O	LYS	183	22.855	−11.458	24.254	1.00	14.24
ATOM	824	N	ASN	184	20.804	−11.668	23.356	1.00	13.74
ATOM	825	CA	ASN	184	21.291	−11.440	21.998	1.00	14.34
ATOM	826	CB	ASN	184	20.399	−12.141	20.960	1.00	15.53
ATOM	827	CG	ASN	184	18.962	−11.657	20.985	1.00	16.25
ATOM	828	OD1	ASN	184	18.628	−10.692	21.668	1.00	16.76
ATOM	829	ND2	ASN	184	18.102	−12.329	20.229	1.00	17.81
ATOM	830	C	ASN	184	21.369	−9.943	21.701	1.00	14.45
ATOM	831	O	ASN	184	21.711	−9.535	20.590	1.00	14.60
ATOM	832	N	GLY	185	21.051	−9.132	22.708	1.00	13.68
ATOM	833	CA	GLY	185	21.109	−7.688	22.557	1.00	13.05
ATOM	834	C	GLY	185	19.904	−7.049	21.894	1.00	12.79
ATOM	835	O	GLY	185	19.911	−5.848	21.631	1.00	13.80
ATOM	836	N	ALA	186	18.873	−7.841	21.619	1.00	10.99
ATOM	837	CA	ALA	186	17.670	−7.318	20.985	1.00	10.27
ATOM	838	CB	ALA	186	17.246	−8.225	19.828	1.00	10.89
ATOM	839	C	ALA	186	16.545	−7.197	22.004	1.00	8.96
ATOM	840	O	ALA	186	16.578	−7.820	23.063	1.00	8.77
ATOM	841	N	THR	187	15.545	−6.385	21.687	1.00	8.86
ATOM	842	CA	THR	187	14.427	−6.203	22.600	1.00	7.58
ATOM	843	CB	THR	187	13.518	−5.051	22.146	1.00	7.14
ATOM	844	OG1	THR	187	13.033	−5.325	20.825	1.00	7.19
ATOM	845	CG2	THR	187	14.281	−3.734	22.146	1.00	6.53
ATOM	846	C	THR	187	13.562	−7.452	22.700	1.00	8.41
ATOM	847	O	THR	187	13.527	−8.279	21.788	1.00	8.64
ATOM	848	N	LYS	188	12.876	−7.585	23.829	1.00	8.14
ATOM	849	CA	LYS	188	11.953	−8.684	24.056	1.00	9.20
ATOM	850	CB	LYS	188	12.271	−9.412	25.363	1.00	10.91
ATOM	851	CG	LYS	188	13.456	−10.356	25.287	1.00	12.37
ATOM	852	CD	LYS	188	13.577	−11.140	26.583	1.00	14.48
ATOM	853	CE	LYS	188	14.470	−12.345	26.416	1.00	16.86
ATOM	854	NZ	LYS	188	13.932	−13.324	25.422	1.00	15.93
ATOM	855	C	LYS	188	10.588	−8.020	24.167	1.00	9.15
ATOM	856	O	LYS	188	10.506	−6.823	24.443	1.00	8.81
ATOM	857	N	LYS	189	9.523	−8.783	23.944	1.00	8.51
ATOM	858	CA	LYS	189	8.170	−8.242	24.040	1.00	9.55
ATOM	859	CB	LYS	189	7.164	−9.237	23.452	1.00	11.24
ATOM	860	CG	LYS	189	7.433	−9.583	21.991	1.00	13.77
ATOM	861	CD	LYS	189	7.115	−8.412	21.070	1.00	15.40
ATOM	862	CE	LYS	189	5.666	−8.448	20.601	1.00	18.07
ATOM	863	NZ	LYS	189	4.706	−8.523	21.729	1.00	20.82
ATOM	864	C	LYS	189	7.842	−7.965	25.506	1.00	8.74
ATOM	865	O	LYS	189	8.100	−8.799	26.372	1.00	8.90
ATOM	866	N	GLY	190	7.273	−6.793	25.776	1.00	7.88
ATOM	867	CA	GLY	190	6.940	−6.421	27.140	1.00	8.10
ATOM	868	C	GLY	190	5.938	−7.314	27.851	1.00	8.54
ATOM	869	O	GLY	190	5.894	−7.339	29.086	1.00	7.39
ATOM	870	N	ASN	191	5.130	−8.045	27.090	1.00	8.98
ATOM	871	CA	ASN	191	4.135	−8.926	27.689	1.00	10.89
ATOM	872	CB	ASN	191	2.812	−8.829	26.922	1.00	12.28
ATOM	873	CG	ASN	191	2.937	−9.274	25.483	1.00	13.78
ATOM	874	OD1	ASN	191	4.035	−9.334	24.931	1.00	13.27
ATOM	875	ND2	ASN	191	1.803	−9.575	24.858	1.00	15.88
ATOM	876	C	ASN	191	4.607	−10.377	27.756	1.00	11.62
ATOM	877	O	ASN	191	3.802	−11.295	27.916	1.00	10.96
ATOM	878	N	ARG	192	5.917	−10.574	27.639	1.00	12.11
ATOM	879	CA	ARG	192	6.503	−11.910	27.715	1.00	13.33
ATOM	880	CB	ARG	192	7.076	−12.324	26.356	1.00	14.44
ATOM	881	CG	ARG	192	6.026	−12.464	25.263	1.00	16.19
ATOM	882	CD	ARG	192	6.663	−12.877	23.948	1.00	19.13
ATOM	883	NE	ARG	192	7.220	−14.224	24.013	1.00	22.54
ATOM	884	CZ	ARG	192	6.499	−15.337	23.905	1.00	24.09
ATOM	885	NH1	ARG	192	5.188	−15.266	23.725	1.00	24.60
ATOM	886	NH2	ARG	192	7.091	−16.521	23.977	1.00	25.22
ATOM	887	C	ARG	192	7.597	−11.957	28.780	1.00	13.36
ATOM	888	O	ARG	192	8.358	−12.919	28.861	1.00	13.48
ATOM	889	N	VAL	193	7.663	−10.908	29.596	1.00	13.03
ATOM	890	CA	VAL	193	8.654	−10.812	30.665	1.00	12.85
ATOM	891	CB	VAL	193	9.800	−9.841	30.284	1.00	11.28
ATOM	892	CG1	VAL	193	10.543	−10.360	29.064	1.00	11.22
ATOM	893	CG2	VAL	193	9.235	−8.457	30.003	1.00	11.65
ATOM	894	C	VAL	193	8.010	−10.305	31.957	1.00	13.63
ATOM	895	O	VAL	193	6.958	−9.660	31.927	1.00	14.01
ATOM	896	N	SER	194	8.647	−10.605	33.086	1.00	14.34
ATOM	897	CA	SER	194	8.166	−10.172	34.398	1.00	15.34
ATOM	898	CB	SER	194	7.728	−11.372	35.238	1.00	16.67
ATOM	899	OG	SER	194	8.833	−12.193	35.565	1.00	19.38
ATOM	900	C	SER	194	9.315	−9.453	35.095	1.00	14.47
ATOM	901	O	SER	194	10.480	−9.720	34.810	1.00	13.57
ATOM	902	N	PRO	195	9.003	−8.545	36.034	1.00	14.17
ATOM	903	CD	PRO	195	7.669	−8.211	36.562	1.00	14.38
ATOM	904	CA	PRO	195	10.048	−7.804	36.745	1.00	13.64
ATOM	905	CB	PRO	195	9.252	−6.826	37.607	1.00	14.45
ATOM	906	CG	PRO	195	8.002	−7.588	37.900	1.00	14.65
ATOM	907	C	PRO	195	11.033	−8.638	37.557	1.00	13.10
ATOM	908	O	PRO	195	12.046	−8.118	38.007	1.00	12.82
ATOM	909	N	THR	196	10.746	−9.921	37.753	1.00	12.72
ATOM	910	CA	THR	196	11.666	−10.771	38.509	1.00	12.30
ATOM	911	CB	THR	196	10.950	−11.988	39.121	1.00	12.60
ATOM	912	OG1	THR	196	10.332	−12.753	38.079	1.00	12.75
ATOM	913	CG2	THR	196	9.898	−11.540	40.121	1.00	13.99
ATOM	914	C	THR	196	12.785	−11.283	37.611	1.00	12.27
ATOM	915	O	THR	196	13.798	−11.786	38.094	1.00	12.60
ATOM	916	N	MET	197	12.593	−11.138	36.303	1.00	11.81
ATOM	917	CA	MET	197	13.561	−11.596	35.309	1.00	11.06
ATOM	918	CB	MET	197	12.832	−11.920	34.005	1.00	11.47
ATOM	919	CG	MET	197	11.814	−13.042	34.142	1.00	13.03
ATOM	920	SD	MET	197	10.747	−13.178	32.697	1.00	13.94
ATOM	921	CE	MET	197	11.878	−13.884	31.521	1.00	14.56
ATOM	922	C	MET	197	14.662	−10.574	35.050	1.00	10.53
ATOM	923	O	MET	197	14.399	−9.379	34.943	1.00	9.30
ATOM	924	N	ALA	198	15.896	−11.060	34.942	1.00	9.44
ATOM	925	CA	ALA	198	17.051	−10.205	34.708	1.00	8.58
ATOM	926	CB	ALA	198	18.327	−11.049	34.690	1.00	9.44
ATOM	927	C	ALA	198	16.958	−9.378	33.429	1.00	8.75
ATOM	928	O	ALA	198	17.544	−8.296	33.349	1.00	8.37
ATOM	929	N	VAL	199	16.234	−9.877	32.431	1.00	7.63
ATOM	930	CA	VAL	199	16.097	−9.152	31.170	1.00	6.86
ATOM	931	CB	VAL	199	15.388	−10.017	30.092	1.00	7.52
ATOM	932	CG1	VAL	199	16.272	−11.193	29.714	1.00	8.00
ATOM	933	CG2	VAL	199	14.043	−10.511	30.608	1.00	7.54
ATOM	934	C	VAL	199	15.353	−7.819	31.325	1.00	6.93
ATOM	935	O	VAL	199	15.382	−6.981	30.421	1.00	5.85
ATOM	936	N	THR	200	14.704	−7.615	32.470	1.00	6.77
ATOM	937	CA	THR	200	13.972	−6.369	32.721	1.00	7.57
ATOM	938	CB	THR	200	12.578	−6.631	33.340	1.00	8.45
ATOM	939	OG1	THR	200	12.736	−7.158	34.664	1.00	8.56
ATOM	940	CG2	THR	200	11.790	−7.622	32.500	1.00	8.63
ATOM	941	C	THR	200	14.725	−5.457	33.692	1.00	7.78
ATOM	942	O	THR	200	14.247	−4.370	34.028	1.00	7.71
ATOM	943	N	HIS	201	15.899	−5.896	34.137	1.00	7.34
ATOM	944	CA	HIS	201	16.695	−5.125	35.095	1.00	7.59
ATOM	945	CB	HIS	201	17.452	−6.073	36.028	1.00	8.17
ATOM	946	CG	HIS	201	16.568	−6.922	36.887	1.00	9.32
ATOM	947	CD2	HIS	201	16.861	−7.736	37.930	1.00	9.55
ATOM	948	ND1	HIS	201	15.205	−7.007	36.709	1.00	10.97
ATOM	949	CE1	HIS	201	14.695	−7.834	37.604	1.00	9.67
ATOM	950	NE2	HIS	201	15.681	−8.290	38.358	1.00	11.80
ATOM	951	C	HIS	201	17.704	−4.199	34.426	1.00	7.38
ATOM	952	O	HIS	201	18.618	−4.663	33.742	1.00	7.78
ATOM	953	N	PHE	202	17.553	−2.894	34.640	1.00	7.48
ATOM	954	CA	PHE	202	18.466	−1.922	34.048	1.00	7.69
ATOM	955	CB	PHE	202	17.716	−0.988	33.091	1.00	7.39
ATOM	956	CG	PHE	202	17.187	−1.673	31.866	1.00	7.17
ATOM	957	CD1	PHE	202	15.966	−2.334	31.894	1.00	7.73
ATOM	958	CD2	PHE	202	17.922	−1.674	30.686	1.00	6.55
ATOM	959	CE1	PHE	202	15.480	−2.988	30.765	1.00	6.46
ATOM	960	CE2	PHE	202	17.447	−2.327	29.545	1.00	6.72
ATOM	961	CZ	PHE	202	16.220	−2.986	29.587	1.00	6.52
ATOM	962	C	PHE	202	19.188	−1.080	35.092	1.00	8.95
ATOM	963	O	PHE	202	18.561	−0.494	35.980	1.00	9.11
ATOM	964	N	LEU	203	20.511	−1.019	34.972	1.00	9.21
ATOM	965	CA	LEU	203	21.334	−0.237	35.890	1.00	10.82
ATOM	966	CB	LEU	203	22.627	−0.989	36.224	1.00	10.86
ATOM	967	CG	LEU	203	23.571	−0.292	37.213	1.00	11.00
ATOM	968	CD1	LEU	203	22.887	−0.188	38.568	1.00	12.23
ATOM	969	CD2	LEU	203	24.871	−1.069	37.341	1.00	11.91
ATOM	970	C	LEU	203	21.689	1.103	35.256	1.00	12.02
ATOM	971	O	LEU	203	22.303	1.149	34.191	1.00	11.12
ATOM	972	N	PRO	204	21.288	2.214	35.892	1.00	13.88
ATOM	973	CD	PRO	204	20.358	2.318	37.029	1.00	14.72
ATOM	974	CA	PRO	204	21.596	3.540	35.351	1.00	16.16
ATOM	975	CB	PRO	204	20.832	4.478	36.280	1.00	16.31
ATOM	976	CG	PRO	204	19.679	3.632	36.748	1.00	15.39
ATOM	977	C	PRO	204	23.101	3.786	35.409	1.00	18.73
ATOM	978	O	PRO	204	23.716	3.625	36.466	1.00	18.38
ATOM	979	N	ARG	205	23.688	4.165	34.278	1.00	21.09
ATOM	980	CA	ARG	205	25.123	4.432	34.213	1.00	24.19
ATOM	981	CB	ARG	205	25.867	3.219	33.650	1.00	25.55
ATOM	982	CG	ARG	205	25.752	1.972	34.509	1.00	28.06
ATOM	983	CD	ARG	205	27.109	1.329	34.774	1.00	30.17
ATOM	984	NE	ARG	205	28.040	2.244	35.432	1.00	31.85
ATOM	985	CZ	ARG	205	28.888	3.045	34.792	1.00	33.10
ATOM	986	NH1	ARG	205	28.933	3.050	33.464	1.00	33.73
ATOM	987	NH2	ARG	205	29.690	3.849	35.480	1.00	33.12
ATOM	988	C	ARG	205	25.442	5.659	33.366	1.00	25.09
ATOM	989	O	ARG	205	24.577	6.174	32.659	1.00	24.94
ATOM	990	N	ALA	206	26.690	6.117	33.459	1.00	26.79
ATOM	991	CA	ALA	206	27.188	7.281	32.722	1.00	28.01
ATOM	992	CB	ALA	206	28.274	6.843	31.745	1.00	28.80
ATOM	993	C	ALA	206	26.107	8.065	31.983	1.00	28.96
ATOM	994	O	ALA	206	26.376	8.463	30.829	1.00	29.66
ATOM	995	O1	HOH	1000	12.829	−2.036	17.116	1.00	7.18
ATOM	996	O1	HOH	1001	20.982	8.993	22.203	1.00	7.57
ATOM	997	O1	HOH	1002	12.768	12.229	28.379	1.00	10.08
ATOM	998	O1	HOH	1003	21.297	3.228	27.387	1.00	6.83
ATOM	999	O1	HOH	1004	2.864	4.158	28.500	1.00	7.52
ATOM	1000	O1	HOH	1005	10.142	−1.516	16.908	1.00	7.61
ATOM	1001	O1	HOH	1006	11.860	−3.327	19.508	1.00	5.26
ATOM	1002	O1	HOH	1007	9.727	7.593	26.490	1.00	7.03
ATOM	1003	O1	HOH	1008	10.236	−4.757	26.340	1.00	7.00
ATOM	1004	O1	HOH	1009	10.856	2.908	42.825	1.00	6.81
ATOM	1005	O1	HOH	1010	6.747	4.115	39.695	1.00	8.95
ATOM	1006	O1	HOH	1011	12.036	−3.230	32.828	1.00	8.27
ATOM	1007	O1	HOH	1012	4.734	16.208	33.512	1.00	12.05
ATOM	1008	O1	HOH	1013	10.093	0.852	15.744	1.00	8.23
ATOM	1009	O1	HOH	1014	0.631	2.544	19.025	1.00	10.23
ATOM	1010	O1	HOH	1015	18.399	−0.846	45.247	1.00	8.81
ATOM	1011	O1	HOH	1016	3.640	7.004	20.379	1.00	5.67
ATOM	1012	O1	HOH	1017	19.978	−6.895	33.952	1.00	10.33
ATOM	1013	O1	HOH	1018	3.469	1.860	26.778	1.00	10.74
ATOM	1014	O1	HOH	1019	19.254	1.746	45.513	1.00	11.57
ATOM	1015	O1	HOH	1020	9.787	−5.206	22.223	1.00	9.48
ATOM	1016	O1	HOH	1021	9.165	4.101	14.820	1.00	9.74
ATOM	1017	O1	HOH	1022	19.029	7.930	20.394	1.00	7.35
ATOM	1018	O1	HOH	1023	11.450	−7.648	40.765	1.00	14.67
ATOM	1019	O1	HOH	1024	10.751	−7.011	20.455	1.00	9.99
ATOM	1020	O1	HOH	1025	−0.784	0.040	20.721	1.00	11.72
ATOM	1021	O1	HOH	1026	−2.374	3.652	22.178	1.00	16.42
ATOM	1022	O1	HOH	1027	16.329	18.885	28.375	1.00	19.76
ATOM	1023	O1	HOH	1028	18.501	−6.259	40.698	1.00	14.77
ATOM	1024	O1	HOH	1029	18.291	−13.454	23.318	1.00	15.78
ATOM	1025	O1	HOH	1030	10.174	5.278	12.637	1.00	13.08
ATOM	1026	O1	HOH	1031	1.243	0.722	34.708	1.00	17.19
ATOM	1027	O1	HOH	1032	2.523	−3.821	18.445	1.00	16.48
ATOM	1028	O1	HOH	1033	15.902	−4.895	19.077	1.00	10.14
ATOM	1029	O1	HOH	1034	16.085	18.258	38.411	1.00	20.04
ATOM	1030	O1	HOH	1035	8.277	3.225	41.854	1.00	12.99
ATOM	1031	O1	HOH	1036	16.439	13.001	35.214	1.00	16.54
ATOM	1032	O1	HOH	1037	28.131	5.663	18.293	1.00	20.92
ATOM	1033	O1	HOH	1038	14.371	−11.433	40.776	1.00	17.19
ATOM	1034	O1	HOH	1039	6.487	15.695	21.119	1.00	14.88
ATOM	1035	O1	HOH	1040	2.321	−12.928	26.470	1.00	11.64
ATOM	1036	O1	HOH	1041	10.693	5.971	40.782	1.00	17.64
ATOM	1037	O1	HOH	1042	12.532	16.814	29.258	1.00	17.91
ATOM	1038	O1	HOH	1043	15.955	−13.066	32.485	1.00	14.81
ATOM	1039	O1	HOH	1044	18.984	3.255	12.162	1.00	21.70
ATOM	1040	O1	HOH	1045	10.025	10.429	37.944	1.00	16.17
ATOM	1041	O1	HOH	1046	10.284	−14.324	27.916	1.00	18.92
ATOM	1042	O1	HOH	1047	14.189	5.045	17.917	1.00	14.25
ATOM	1043	O1	HOH	1048	1.288	−3.987	37.554	1.00	20.26
ATOM	1044	O1	HOH	1049	10.779	−7.111	14.686	1.00	24.75
ATOM	1045	O1	HOH	1050	11.650	13.767	17.828	1.00	19.59
ATOM	1046	O1	HOH	1051	14.534	9.188	36.954	1.00	19.75
ATOM	1047	O1	HOH	1052	19.606	−7.904	38.613	1.00	20.20
ATOM	1048	O1	HOH	1053	9.427	9.736	48.436	1.00	15.68
ATOM	1049	O1	HOH	1054	−2.792	−4.538	26.019	1.00	26.55
ATOM	1050	O1	HOH	1055	−0.725	−9.213	26.754	1.00	18.89
ATOM	1051	O1	HOH	1056	−0.278	6.480	29.432	1.00	23.19
ATOM	1052	O1	HOH	1057	25.587	−11.455	24.178	1.00	14.80
ATOM	1053	O1	HOH	1058	16.900	10.379	36.579	1.00	17.89
ATOM	1054	O1	HOH	1059	3.126	15.011	20.955	1.00	13.94
ATOM	1055	O1	HOH	1060	24.968	5.423	18.006	1.00	15.86
ATOM	1056	O1	HOH	1061	18.376	−0.133	12.322	1.00	22.38
ATOM	1057	O1	HOH	1062	15.661	−10.620	22.135	1.00	20.09
ATOM	1058	O1	HOH	1063	14.444	−8.692	41.510	1.00	17.78
ATOM	1059	O1	HOH	1064	−2.099	2.685	19.269	1.00	18.56
ATOM	1060	O1	HOH	1065	15.065	−14.513	30.168	1.00	20.78
ATOM	1061	O1	HOH	1066	−0.478	−12.428	26.525	1.00	19.85
ATOM	1062	O1	HOH	1067	24.619	−2.274	41.474	1.00	20.99
ATOM	1063	O1	HOH	1068	14.937	4.314	41.154	1.00	23.31
ATOM	1064	O1	HOH	1069	18.536	3.179	40.506	1.00	20.39
ATOM	1065	O1	HOH	1070	12.859	4.828	42.895	1.00	23.36
ATOM	1066	O1	HOH	1071	21.098	−7.930	36.367	1.00	22.36
ATOM	1067	O1	HOH	1072	−4.171	−0.822	29.589	1.00	31.08
ATOM	1068	O1	HOH	1073	5.760	16.825	18.804	1.00	26.85
ATOM	1069	O1	HOH	1074	14.347	16.052	36.421	1.00	26.89
ATOM	1070	O1	HOH	1075	26.493	−4.497	20.667	1.00	19.77
ATOM	1071	O1	HOH	1076	2.436	9.335	39.933	1.00	22.58
ATOM	1072	O1	HOH	1077	28.166	−2.047	20.549	1.00	17.57
ATOM	1073	O1	HOH	1078	26.366	−10.689	21.612	1.00	24.57
ATOM	1074	O1	HOH	1079	24.829	−14.257	24.782	1.00	24.91
ATOM	1075	O1	HOH	1080	13.266	−13.588	42.052	1.00	24.33
ATOM	1076	O1	HOH	1081	24.443	2.764	17.515	1.00	24.61
ATOM	1077	O1	HOH	1082	4.269	−9.664	30.981	1.00	24.44
ATOM	1078	O1	HOH	1083	4.143	−10.890	22.698	1.00	22.21
ATOM	1079	O1	HOH	1084	18.040	4.749	42.785	1.00	25.14
ATOM	1080	O1	HOH	1085	1.136	−9.675	22.102	1.00	27.39
ATOM	1081	O1	HOH	1086	9.192	14.318	28.336	1.00	22.60
ATOM	1082	O1	HOH	1087	19.935	−4.720	19.071	1.00	29.60
ATOM	1083	O1	HOH	1088	4.049	−5.334	16.898	1.00	21.65
ATOM	1084	O1	HOH	1089	27.148	9.654	24.125	1.00	24.37
ATOM	1085	O1	HOH	1090	16.984	11.029	13.815	1.00	27.26
ATOM	1086	O1	HOH	1091	20.544	−2.126	18.367	1.00	22.99
ATOM	1087	O1	HOH	1092	23.707	3.151	39.432	1.00	28.88
ATOM	1088	O1	HOH	1093	−1.920	−4.147	30.571	1.00	27.39
ATOM	1089	O1	HOH	1094	11.899	−14.917	37.385	1.00	32.81
ATOM	1090	O1	HOH	1095	20.795	14.376	16.474	1.00	29.46
ATOM	1091	S	SO4	300	17.194	−14.451	26.775	1.00	23.76
ATOM	1092	O1	SO4	300	17.779	−15.800	26.674	1.00	25.07
ATOM	1093	O2	SO4	300	16.796	−13.993	25.435	1.00	24.21
ATOM	1094	O3	SO4	300	16.011	−14.509	27.652	1.00	24.52
ATOM	1095	O4	SO4	300	18.190	−13.524	27.343	1.00	23.52
ATOM	1096	S	SO4	301	13.826	−5.555	16.116	1.00	19.81
ATOM	1097	O1	SO4	301	13.393	−6.524	15.095	1.00	21.58
ATOM	1098	O2	SO4	301	13.720	−4.187	15.584	1.00	19.29
ATOM	1099	O3	SO4	301	12.996	−5.693	17.324	1.00	20.41
ATOM	1100	O4	SO4	301	15.233	−5.831	16.467	1.00	22.09
ATOM	1101	S	SO4	302	13.249	23.091	32.541	1.00	76.58
ATOM	1102	O1	SO4	302	13.950	22.101	31.704	1.00	76.66
ATOM	1103	O2	SO4	302	12.256	22.409	33.391	1.00	76.74
ATOM	1104	O3	SO4	302	14.229	23.787	33.395	1.00	76.89
ATOM	1105	O4	SO4	302	12.559	24.071	31.680	1.00	76.61
END

[0121]

Claims

1. An isolated FGF4 polypeptide having the amino acid sequence set forth for FGF4 in FIG. 2 (top line) with substitution of an alanine amino acid residue for another amino acid residue selected from the group consisting of: (a) tyrosine at amino acid residue 87; (b) phenylalanine at amino acid residue 129; (c) phenylalanine at amino acid residue 151, (d) glutamic acid at amino acid residue 159; (e) phenylalanine at amino acid residue 166; (f) leucine at amino acid residue 203; (g) arginine at amino acid residue 205; (h) asparagine at amino acid residue 89; (i) lysine at amino acid residue 198; (j) asparagine at amino acid residue 89; (k) lysine at amino acid residue 183; (l) lysine at amino acid residue 188; (m) lysine at amino acid residue 183; (n) arginine at amino acid residue 103; (o) lysine at amino acid residue 144; (p) arginine at amino acid residue 103; (q) glutamic acid at amino acid residue 117; (r) leucine at amino acid residue 162; (s) histidine at amino acid residue 201; (t) proline at amino acid residue 163; (u) proline at amino acid residue 195; (v) threonine at amino acid residue 196; and of the FGF4 polypeptide sequence set forth in FIG. 2.

2. An isolated FGF4 polypeptide according to claim 1(O), wherein there is a further substitution of an alanine residue for arginine at amino acid residue 103.

3. An isolated FGF4 polypeptide according to claim 1(H), wherein there are further substitutions of alanine residues for lysine at amino acid residue 198, lysine at amino acid residue 183, and lysine at amino acid residue 188.

4. A crystal belonging to the orthorhombic space group P212121 with unit cell dimensions a=40.37, b=53.3 and c=56.23.

5. A crystal comprising a polypeptide having the amino acid sequence set forth in FIG. 2 (top line).

6. A mutant FGF4 polypeptide, wherein at least one amino acid residue in a region of the FGF4 module selected from the primary binding site, the secondary binding site and the heparin binding site is different from the wild-type FGF4 molecule.

7. A method for producing an FGF4 mutant protein comprising the steps of; a) providing a nucleic acid encoding FGF4 protein; b) changing at least one codon encoding an amino acid present in said FGF4 protein; c) expressing said nucleic acid; d) isolating said FGF4 protein; and e) testing said protein of step (d) for biologic activity.

8. The method of claim 7, wherein the codon of step (b) is in a region of said FGF4 protein selected from the primary binding site, the secondary binding site, and the heparin binding site.

9. The method of claim 8, wherein said mutant FGF4 protein is an agonist.

10. The method of claim 8, wherein said mutant FGF4 protein is an antagonist.

11. A crystalline form of FGF4.

12. The crystalline form of FGF4 of claim 11, belonging to the orthorhombic space P212121 with unit cell dimensions a=40.37 Å, b=53.3 Å, and c=56.23 Å.

13. The crystalline form of claim 11, having the coordinates set forth in Appendix B.