| Crystal structure of the complex of hepatocyte growth factor beta chain with met receptor and methods of use -> Monitor Keywords |
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Crystal structure of the complex of hepatocyte growth factor beta chain with met receptor and methods of useRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai, Cyclopeptides, 25 Or More Peptide Repeating Units In Known Peptide Chain StructureCrystal structure of the complex of hepatocyte growth factor beta chain with met receptor and methods of use description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060069019, Crystal structure of the complex of hepatocyte growth factor beta chain with met receptor and methods of use. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of 35 U.S.C. .sctn. 119(e) to U.S. Ser. No. 60/568,865 filed May 6, 2004, which application is hereby incorporated by reference. BACKGROUND [0002] The receptor tyrosine kinase Met, and its ligand, hepatocyte growth factor (HGF, also called scatter factor), have been implicated in promoting invasive growth of many tumor types due to inappropriate activation of Met function (Jankowski et al., 2003; Nardone et al., 2003; Trusolino and Comoglio, 2002; Birchmeier et al., 2003). This activation can arise from a variety of sources, but in each case the Met receptor activates signaling cascades that normally function to organize groups of cells into branching, tubular structures that are present in a variety of organs (Montesano et al., 1992; Sonnenberg et al., 1993; Rosen et al., 1994; Trusolino and Comoglio, 2002; Zhang and Vande Woude, 2003). The Met receptor plays a unique role during development as a master switch, which can stimulate proliferation and motility necessary for the full program of growth and scattering of cells. Its role in the invasiveness of many cancers makes it an attractive target for therapeutics (Ma et al., 2003). However, many questions remain about how the ligand, HGF, binds to Met and induces its tyrosine kinase cascade and thus leads to a biological response. [0003] The Met receptor is part of a larger family of growth factor receptors with identical domain architecture that includes the Ron and Sea receptors (Monsin et al., 1992, Huff et al., 1993). The extracellular portions of Met family members are composed of three domain types. The N-terminal 500 residues fold into a Sema domain, which shares sequence homology with domains found in the Semaphorin and plexin families of neural development proteins (Winburg et al., 1998). As reported recently, Sema domains form a 7-bladed .beta.-propeller structure (Antipenkov et al., 2003, Love et al., 2003). Met undergoes proteolytic cleavage within the Sema domain during normal processing, although the role for this remains unclear since cells that are unable to cleave Met show normal levels of Met activation upon ligand binding (Komada et al., 1993). A PSI domain, a small domain spanning about 50 residues and containing 4 disulfide bonds, follows the Sema domain. In addition to the Met receptor family, PSI domains are also found in the plexins, Semaphorins and integrins, hence its name (Bork et al., 1999). In Met, the PSI domain is connected via 4 IPT domains to the transmembrane helix and the kinase domain in the intracellular portion of the receptor. IPT domains are related to immunoglobulin-like domains and are named after their presence in plexins and transcription factors (Takagi et al., 1995). [0004] HGF is a large growth factor of 728 residues that is produced as an inactive single-chain precursor which is proteolytically processed to form the biologically active disulfide-linked .alpha./.beta.-heterodimer (Nakamura et al., 1989; Hartmann et al., 1992; Kataoka et al., 2003). The .alpha.-chain folds into an N-terminal domain (N-domain) followed by 4 Kringle domains. The .beta.-chain starts with residue Val495 and is homologous to the protease domain of chymotrypsin like serine proteases, which, like HGF, are activated by a proteolytic cleavage event (Perona and Craig, 1995; Hedstrom 2002). However, no protease activity has been demonstrated for HGF .beta.-chain (Lokker et al., 1992) consistent with the absence of the key serine and histidine residues that are part of the `catalytic triad` Asp[c102]-His[c57]-Ser[c195] ([chymotrypsinogen numbering]) required for catalytic activity in serine proteases. [0005] Comparisons of the biologically active, two-chain HGF, and the inactive single-chain HGF precursor on the Met receptor have shown that both forms of HGF bind Met with similar affinity, but only the cleaved, mature form of HGF is able to activate Met (Lokker et al., 1992). In addition, various C-terminally truncated fragments of the .alpha.-chain (termed NK1, NK2, or NK4 depending on the number of Kringle domains retained) bind Met; in many cases they act as potent antagonists of Met receptor function (Cioce et al., 1996; Chan et al., 1991; Date et al., 1997). Studies involving the cross-linking of Met receptors by a variety of specific antibodies to its extracellular domain have demonstrated that simple dimerization of Met is sufficient for activation (Prat et al., 1998). Based on these characteristics, the fundamental mechanism for Met dimerization remains unclear. [0006] Currently, there is no detailed structural information about HGF .beta.-chain complexed with Met receptor. A completely solved crystal structure of the HGF .beta.-chain complexed with Met receptor is needed, for example, for assays for Met-ligand (e.g., HGF .beta.-chain) interaction and function, modeling the structure-function relationship of Met and other molecules, diagnostic assays for mutation-induced pathologies, and rational design of agents useful in modulating Met or HGF activity or activation. SUMMARY [0007] In some embodiments, the present disclosure provides a crystalline form of hepatocyte growth factor beta chain (HGF .beta.) complexed with Met receptor, and the structural coordinates of the crystal. Coordinates of a crystal structure solved by molecular replacement are listed in Table 2. In some embodiments, HGF .beta. comprises an amino acid sequence of SEQ ID NO: 1 or conservative substitutions thereof and the Met receptor comprises an amino acid sequence of SEQ ID NO:3 or conservative substitutions thereof. [0008] In some embodiments, the disclosure provides a crystal structure of HGF .beta. complexed with Met receptor, as well as use of the crystal structure to model Met receptor activity when complexed with HGF .beta.. This use of the structure includes: modeling the interaction of ligands with the Met receptor; activation and inhibition of Met receptor; and the rational design of modulators of Met receptor activity. For example, these modulators include ligands that interact with Met receptor and modulate Met receptor activities, such as cell migration, HGF .beta. binding to Met, and Met phosphorylation and signaling. [0009] In other embodiments, the amino acid residues that form the binding site for the Met receptor on HGF .beta. are identified and are useful, for example, in methods to model the structure of HGF binding site and to identify agents that can bind or fit into the binding site. In addition, the amino acid positions that form the binding site for HGF .beta. on Met have been identified and are useful, for example, in methods to model the structure of the Met ligand binding site and to identify other agents that can bind or fit into the binding site. BRIEF DESCRIPTION OF FIGURES [0010] FIG. 1A shows a superposition representation of HGF .beta.-chain (grey) and plasmin. The plasmin .alpha. chain is shown as a thin dark line. Selected side-chains of HGF .beta. and plasmin are shown as sticks. They include the residues of the catalytic triad in serine proteases (His[c57], Asp[c102], and Ser[c195]) and the respective residues in the catalytically inactive HGF .beta.-chain (Gln534, Asp578 and Tyr673), the N-terminal Val495 (V495; Val16 in plasmin) and Asp672 (D672; Asp194 in plasmin). After maturation, the N-terminus of Val495 of HGF is inserted into the core of the protein. The N-terminal amine forms a salt bridge with the side-chain of Asp672 and thus rearranges the loops that carry the catalytic triad. The numbering system with a lower case c is that of the chymotyrpsinogen numbering system. [0011] FIG. 1B shows the same superposition of FIG. 1A rotated 180.degree. around the y-axis. The surface of HGF .beta.-chain is grey. Cysteines 561 (C561) and 604 (C604) in HGF .beta.-chain and the Asp598 (D598) are shown. The .alpha.-chain of plasmin follows a groove that is also present on the HGF surface. The distance requirements for the formation of the disulfide bond between the .alpha.- and the .beta.-chain analogous to plasmin and MSP are not satisfied. The two cysteines on the plasmin .alpha.-chain are shown as stick stubs. [0012] FIG. 1C shows the sequence alignment of selected regions of HGF, MSP, and plasmin. (SEQ ID NOs:7-9) The alignment shows the Cys residues that are present in the a and .beta. chain of HGF. The Cys at position 487 in the .alpha. chain of HGF is conserved when compared with MSP and plasmin. However, the Cys residues in the .beta. chain of HGF are not at conserved positions, but are found at positions 561 and 604. A disulfide bond between amino acid residues at 487 in the .alpha. chain and the cysteine residue at 604 in the .beta. chain may be formed. However, given the location of cysteine 561 in the three-dimensional structure, this residue could also form a disulfide bond with amino acid residue 487 in the .alpha. chain. Asterisks indicate amino acid residues that are conserved when the three sequences are compared and dots indicate amino acids that are conservative substitutions. [0013] FIGS. 2A-C show representations of the complex of Met and HGF .beta.-chain. FIGS. 2A and 2B show ribbon representations with HGF .beta.-chain. [0014] FIG. 2A provides a view onto the `top` side of the propeller. The numbers in the center refer to the blades. The .beta.-strands in blade 1 are labeled A, B, C, and D. Disordered residues in the represented model are indicated with dotted lines and the dotted line associated numbers refer to the last and first amino acid residues present in the model. [0015] FIG. 2B provides a side view of the same complex of FIG. 2A. Note that the loops on the top face of the propeller are longer than the ones on the bottom face. All figures were made using Pymol (DeLano, 2002). [0016] FIG. 2C provides a surface representation of the Met Sema domain and an associated HGF .beta.-chain represented as a gray ribbon. The left panel of FIG. 2C captures the complex in the same view as FIG. 2A and shows approximate molecular dimensions. The right panel of FIG. 2C is a view towards the bottom of the propeller and indicates a proteolysis site. [0017] FIG. 3 shows sequence alignment of the Sema domains of human Met receptor (Met_h) (SEQ ID NO:10), human Sema4D (hSema4d) (SEQ ID NO:11), and mouse Sema3A (mSema3a) (SEQ ID NO:12). The secondary structure elements depicted refer to the Met structure. The structural elements identified as A1, B1, C1, etc. refer to .beta. sheets that form the blades 1-7 of the propeller of the human Met receptor. For example, A1, B1, C1 and D1 identify the amino acids that form propellor blade 1 of the Met Sema domain. The amino acids forming other blades of the propeller are also identified. The boxes indicate structural equivalent positions between Met and Sema4D. The coordinates of the Sema3A structure were unavailable. Dots above the amino acid residues indicate these residues contact HGF .beta.. Residues in the dimer interface of Sema4D are shaded. Cysteines engaged in disulfide bonds are marked with letters A to G and those with same letter form a disulfide bond. Residues that are disordered in the represented structure are shown in italics. [0018] FIG. 4A shows a superposition representation of Met and Sema4D. Note the structural similarities within the .beta.-propellers and the differences in the insertions. The topology of the PSI domains in both structures is identical, but the relative orientation in comparison to the Sema domains is rather different. [0019] FIGS. 4B and 4C shows two different views of a model of a potential Met-dimer based on the dimer of Sema4D and the superposition shown in FIG. 4A. The interface between the two molecules forming the Sema4D dimer is large and buries approximately 2,500 .ANG..sup.2. If this dimerization interface were present on Met, the respective interface in Met would be much smaller due to the different conformation of the loops that correspond to the loops forming the dimer interface in Sema4D. Also shown are two HGF .beta. molecules. Residues shown as spheres represent Cys604 (C604) and Cys561 (C561), which are potential disulfide partners of Cys487 in the .alpha.-chain of HGF. [0020] FIG. 5A shows an open-book surface representation view of the Met-HGF complex interface. Atoms of an amino acid residues of HGF .beta. (shown on the left) that are closer than about 4.7 .ANG. to an atom of an amino acid of Met include Y513, K516, R533, Q534, P537, Y673, E670, Y619, D578, R647, P693, C669, V692, C697, E656, G694, G696, R695, I699, K649, and R702. Atoms of an amino acid residues of Met (shown on the right) closer than about 4.7 .ANG. to an atom of an amino acid residues of HGF .beta. include R218, K220, E221, T222, L229, T230, E167, D190, R191, F192, K223, Y126, D127, D128, H148, S286, and Y125. Contact residues are labeled. HGF is on the left side and Met is on the right side. The three underlined amino acid residue numbers indicate the residues that form the catalytic triad in serine proteases. 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