Crystal structures and methods using same

Abstract

The present invention relates generally to the fields of molecular biology and growth factor regulation. More specifically, the invention concerns modulators of FGFR3 function, and the identification and uses of said modulators.

1 . A crystal of FGFR3 complexed with an anti-FGFR3 antibody comprising a human FGFR3 comprising sequence of SEQ ID NO:272 or conservative substitutions thereof complexed with an anti-FGFR3 antibody comprising (a) a light chain variable domain comprising the amino acid sequence of SEQ ID NO:274 or conservative substitutions thereof, and (b) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:275 or conservative substitutions thereof. 
     
     
         2 . Crystalline form of a complex of FGFR3 and an anti-FGFR3 antibody. space group P2 1 2 1 2 1  with cell parameters of a=58.5 Å, b=99.3 Å and c=143.7 Å. 
     
     
         3 . A crystal of a 1:1 complex of FGFR3 and an anti-FGFR3 antibody having a space group having a space group symmetry of P2 1 2 1 2 1  and comprising a unit cell having the dimensions of a, b, and c, wherein a=58.5 Å, b=99.3 Å and c=143.7 Å. 
     
     
         4 . A cocrystal of FGFR3 with an anti-FGFR3 antibody having the three-dimensional coordinates of Table 6. 
     
     
         5 . The crystal of  claim 4 , 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 any of  claims 1 - 5 , and a carrier. 
     
     
         7 . A molecule or molecular complex comprising at least a portion of the binding site of FGFR3 or conservative substitution thereof, wherein the binding site comprises at least one amino acid residue corresponding to residues 158, 170, 171, 173, 175, and/or 315 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 6. 
     
     
         8 . The molecule or molecular complex of  claim 7 , wherein the binding site comprises at least one amino acid residue corresponding to residues 158, 159, 169, 170, 171, 173, 175, 205, 207, and/or 315 or mixtures thereof. 
     
     
         9 . The molecule or molecular complex of  claim 7 , wherein the binding site comprises at least one amino acid residue corresponding to residues 154, 155, 158, 159, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 177, 202, 205, 207, 210, 212, 214, 216, 217, 241, 246, 247, 248, 278, 279, 280, 281, 282, 283, 314, 315, 316, 317 and/or 318 or mixtures thereof. 
     
     
         10 . A computer-implemented method for causing a display of a graphical three-dimensional representation of the structure of a portion of a crystal of FGFR3 complexed with an anti-FGFR3 antibody, or structural homologs thereof, wherein the method comprises:
 causing said display of said graphical three-dimensional representation by a computer system programmed with instructions for transforming structure coordinates into said graphical three-dimensional representation of said structure and for displaying said graphical three-dimensional representation,   wherein said graphical three-dimensional representation is generated by transforming said structure coordinates into said graphical three-dimensional representation of said structure,   wherein said structure coordinates comprise structure coordinates of the backbone atoms of the portion of the crystal,   wherein the portion of the crystal comprises an FGFR3 binding site, and   wherein the crystal has the space group symmetry P2 1 2 1 2 1 .   
     
     
         11 . The computer-implemented method of  claim 10 , wherein the FGFR3:anti-FGFR3 antibody crystal comprises a polypeptide comprising an amino acid sequence shown in Table 6 or conservative substitution thereof, and further comprises an antibody comprising (a) a light chain variable domain comprising the amino acid sequence of SEQ ID NO:274 or conservative substitutions thereof, and (b) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:275 or conservative substitutions thereof. 
     
     
         12 . The computer-implemented method of  claim 10 , wherein the structure coordinates are defined in Table 6. 
     
     
         13 . The computer-implemented method of  claim 10 , wherein the structure coordinates comprise the structure coordinates of the backbone atoms of the amino acid residues corresponding to residues 158, 170, 171, 173, 175, and/or 315 of FGFR3. 
     
     
         14 . The computer-implemented method of  claim 10 , wherein the structure coordinates comprise the structure coordinates of the backbone atoms of the amino acid residues corresponding to residues 154, 155, 158, 159, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 177, 202, 205, 207, 210, 212, 214, 216, 217, 241, 246, 247, 248, 278, 279, 280, 281, 282, 283, 314, 315, 316, 317, 318 of FGFR3. 
     
     
         15 . The computer-implemented method of  claim 10 , wherein the structure coordinates are determined by homology modeling. 
     
     
         16 . A machine-readable data storage medium comprising a data storage material encoded with machine-readable instructions for:
 (a) transforming data into a graphical three-dimensional representation for the structure of a portion of a crystal of FGFR3 complexed with an anti-FGFR3 antibody, or structural homologs thereof; and   (b) causing the display of said graphical three-dimensional representation;   wherein said data comprise structure coordinates of the backbone atoms of the amino acids defining an FGFR3 binding site; and wherein the crystal or structural homolog has the space group symmetry P2 1 2 1 2 1 .   
     
     
         17 . A computer system for displaying a three-dimensional graphical representation for the structure of a portion of a crystal of FGFR3 complexed with an anti-FGFR3 antibody, or structural homologs thereof, comprising:
 (a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprise structure coordinates of the backbone atoms of the amino acids defining an FGFR3 binding site, wherein the crystal or structural homolog has the space group symmetry P2 1 2 1 2 1 ;   (b) a working memory;   (c) a central processing unit coupled to said working memory and to said machine-readable data storage medium for processing said machine-readable data into sad three-dimensional graphical representation; and   (d) a display coupled to said central processing unit for displaying said three-dimensional graphical representation.   
     
     
         18 . A method for obtaining structural information about a molecule or molecular complex comprising applying at least a portion of the structure coordinates of a FGFR3 complexed with an anti-FGFR3 antibody to an X-ray diffraction pattern of the molecule or molecular complex's crystal structure to cause the generation of a three-dimensional electron density map of at least a portion of the molecule or molecular complex;
 wherein the FGFR3:anti-FGFR3 antibody crystal comprises a polypeptide comprising an amino acid sequence of SEQ ID NO:272 or conservative substitution thereof, and further comprises an antibody comprising (a) a light chain variable domain comprising the amino acid sequence of SEQ ID NO:274 or conservative substitutions thereof, and (b) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:275 or conservative substitutions thereof;   wherein the FGFR3:anti-FGFR3 antibody crystal diffracts x-rays for the determination of atomic coordinates to a resolution of 5 Å or better.   
     
     
         19 . A method of screening for molecules that may be antagonists or agonists of FGFR3 comprising:
 (a) computationally screening agents against a three-dimensional model to identify potential antagonists or agonists of FGFR3;   wherein the three-dimensional model comprises a three-dimensional model of at least a portion of a crystal of a FGFR3 complexed with an anti-FGFR3 antibody;   wherein the three dimensional model is generated from at least a portion of the structure coordinates of the crystal by a computer algorithm for generating a three-dimensional model of the crystal useful for identifying agents that are potential antagonists or agonists of FGFR3;   wherein the FGFR3: anti-FGFR3 antibody crystal comprises a polypeptide comprising an amino acid sequence SEQ ID NO:272 or conservative substitution thereof, and further comprises an antibody comprising (a) a light chain variable domain comprising the amino acid sequence of SEQ ID NO:274 or conservative substitutions thereof, and (b) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:275 or conservative substitutions thereof; and   wherein the FGFR3:anti-FGFR3 antibody crystal diffracts x-rays for the determination of atomic coordinates to a resolution of 5 Å or better.