Crystal structure of the complex of hepatocyte growth factor beta chain with Met receptor and methods of use

Abstract

The disclosure provides a crystal structure of a complex of the HGF β-chain with am extracellular fragment of the Met receptor, as well as use of the crystal structure in the design, identification, and selection of ligands that modulate the Met Receptor and the interaction of HGF with the Met receptor.

1 . A crystal of HGF β complexed with Met comprising a human hepatocyte growth factor beta chain comprising an amino acid sequence of SEQ ID NO:1 or conservative substitutions thereof complexed with an extracellular fragment of a Met Receptor comprising an amino acid sequence of SEQ ID NO:3 or conservative substitutions thereof.  
     
     
         2 . Crystalline form of a complex of HGF β-chain with an extracellular fragment of the Met Receptor.  
     
     
         3 . A crystal of a 1:1 complex of the HGF β-chain with an extracellular fragment of the Met Receptor having a space group symmetry of P2 1 2 1 2 and comprising a unit cell having the dimensions of a, b, and c, where a is about 137.1 Å, b is about 186.4 Å, and c is about 66.7 Å.  
     
     
         4 . The cocrystal of  claim 3 , wherein the HGF β-chain with an extracellular fragment of the Met Receptor has the three-dimensional coordinates of Table 2.  
     
     
         5 . The crystal of  claim 1 , wherein the crystal diffracts X-rays for the determination of atomic coordinates to a resolution of 5 Å or better.  
     
     
         6 . A composition comprising a crystal of  claim 1 , and a carrier.  
     
     
         7 . A molecule or molecular complex comprising at least a portion of the Met binding site for HGF β of a polypeptide comprising an amino acid sequence of SEQ ID NO:3 or conservative substitution thereof, wherein the binding site comprises at least one amino acid residue corresponding to residues 124 to 128, 148, 167, 190 to 192, 218, 220 to 224, 229, 230, 286, 414 or mixtures thereof, the binding site defined by a set of points having a root mean square deviation of less than about 0.70 Å from points representing the backbone atoms of the amino acids as represented by the structure coordinates listed in Table 2.  
     
     
         8 . (canceled)  
     
     
         9 . (canceled)  
     
     
         10 . A three-dimensional configuration of points, wherein at least a portion of the points are derived from structure coordinates representing locations of at least the backbone atoms of amino acids defining the Met binding site for HGF β.  
     
     
         11 . (canceled)  
     
     
         12 . The three-dimensional configuration of points of  claim 10  displayed as a holographic image, a stereodiagram, a model, or a computer-displayed image.  
     
     
         13 . A machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein a machine programmed with instructions for using such data displays a graphical three-dimensional representation of at least one molecule or molecular complex comprising at least a portion of a Met binding site for HGF β, the binding site defined by a set of points having a root mean square deviation of less than about 0.05 Å from points representing the atoms of the amino acids as represented by the structure coordinates listed in Table 2.  
     
     
         14 . (canceled)  
     
     
         15 . A method for obtaining structural information about a molecule or molecular complex comprising applying of at least a portion of the HGF β:Met structure coordinates of a crystal of  claim 5  to an X-ray diffraction pattern of the molecule or molecular complex's crystal structure to generate a three-dimensional electron density map of at least a portion of the molecule or molecular complex.  
     
     
         16 - 22 . (canceled)  
     
     
         23 . A method of identifying a molecule that binds Met and blocks binding of HGF β to Met comprising: 
 (a) searching a molecular structure database with the structural coordinates of a crystal of  claim 5;  and    (b) selecting a molecule from the database that mimics the structural coordinates of bound HGF in HGF β:Met.    
     
     
         24 . A method of assessing agents that are antagonists or agonists of HGF and/or Met comprising: 
 applying at least a portion of the crystallography coordinates of a crystal of  claim 5  to a computer algorithm that generates a 3 dimensional model of HGF β:Met or of the Met binding site for HGF βsuitable for designing molecules that are antagonists or agonists; and    searching a molecular structure database to identify potential antagonists or agonists of Met or HGF β:Met.    
     
     
         25 . The method of  claim 24 , further comprising: 
 (a) synthesizing or obtaining the antagonist or agonist;    (b) contacting the antagonist or agonist with Met and selecting the antagonist or agonist that binds to Met.    
     
     
         26 . The method of any of  claim 24 , further comprising selecting an agonist or antagonist that can associate with at least one or more or all amino acid residues on the Met receptor that comprise amino acids corresponding to residues Y125, Y126, D127, H148, E168, R191, F192, R218, E221, K223, D224 or mixtures thereof of a polypeptide comprising an amino acid sequence of SEQ ID NO:3.  
     
     
         27 . The method of  claim 24 , further comprising selecting an agonist or antagonist that can associate with at least one or more or all amino acid residues on HGF β that comprise amino acids corresponding to residues Y513, K516, R533, D578, Y619, R647, K649, E656, E670, Y673, R695, R702 or mixtures thereof of a polypeptide comprising an amino acid sequence of SEQ ID NO:1.  
     
     
         28 . (canceled)  
     
     
         29 . A molecule or molecular complex comprising at least a portion of the HGF β binding site for Met of a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 or conservative substitution thereof, wherein the binding site comprises at least one amino acid residue corresponding to amino acid residues 513, 516, 533, 534, 537, 578, 619, 647, 649, 656, 669, 670, 673, 692, 693, 694, 695, 696, 697, 699, 702 or mixtures thereof, the binding site defined by a set of points having a root mean square deviation of less than about 0.70 Å from points representing the backbone atoms of the amino acids as represented by the structure coordinates listed in Table 2.  
     
     
         30 . The three-dimensional configuration of points of  claim 29 , wherein at least a portion of the points are derived from structure coordinates representing locations of at least the backbone atoms of amino acids defining the Met binding site for HGF β.  
     
     
         31 . The three-dimensional configuration of points of  claim 29  displayed as a holographic image, a stereodiagram, a model, or a computer-displayed image.  
     
     
         32 . A machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein a machine programmed with instructions for using such data displays a graphical three-dimensional representation of at least one molecule or molecular complex comprising at least a portion of a Met binding site on HGF β, the binding site defined by a set of points having a root mean square deviation of less than about 0.05 Å from points representing the atoms of the amino acids as represented by the structure coordinates listed in Table 2.  
     
     
         33 . (canceled)