Crystals of the tyrosine kinase domain of non-insulin receptor tyrosine kinases

Abstract

Crystals of the tyrosine kinase domain of cytoplasmic tyrosine kinases and receptor tyrosine kinases that undergo ligand-mediated receptor dimerization are provided. In particular, crystals of a mutant of the tyrosine kinase domain of fibroblast growth factor receptor 1 (FLGK), alone and in complex with a non-hydrolyzable adenosine triphosphate analogue, are provided. Also provided are the high resolution three dimensional structures of crystalline FLGK, both alone and in co-complex with the adenosine triphosphate analogue, as determined by X-ray diffraction.

What is claimed is:  
     
         1 . A crystalline form of a polypeptide corresponding to the catalytic domain of a non-insulin receptor tyrosine kinase.  
     
     
         2 . The crystalline polypeptide of  claim 1  in which the non-insulin receptor tyrosine kinase is a receptor tyrosine kinase.  
     
     
         3 . The crystalline polypeptide of  claim 1  in which the non-insulin receptor tyrosine kinase is a cytoplasmic tyrosine kinase.  
     
     
         4 . The crystalline polypeptide of  claim 2  in which the non-insulin receptor tyrosine kinase is FGF-R, PDGF-R, KDR, CCK4, MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, ROR1, or MUSK.  
     
     
         5 . The crystalline polypeptide of  claim 3  in which the cytoplasmic tyrosine kinase is SRC, BRK, BTK; CSK, ABL, ZAP70, FES, FAK, JAK, or ACK.  
     
     
         6 . A derivative crystal comprising the crystalline form of the polypeptide of  claim 1 ,  2 ,  3 ,  4  or  5  in a covalent association with a heavy metal atom.  
     
     
         7 . A co-crystal comprising the crystalline form of the polypeptide of  claim 1 ,  2 ,  3 ,  4  or  5  in association with a compound such as a cofactor, substrate, substrate analog, inhibitor, or allosteric effector.  
     
     
         8 . The co-crystal of  claim 7  in which the compound is a non-hydrolyzable analog of ATP.  
     
     
         9 . A crystal of an FGF receptor tyrosine kinase domain protein, wherein the crystal is characterized by having monoclinic unit cells and space group symmetry C2.  
     
     
         10 . The crystal of  claim 9 , wherein the FGF receptor tyrosine kinase domain protein is FLGK.  
     
     
         11 . The FLGK crystal of  claim 10 , wherein the monoclinic unit cells have dimensions of about a=208.3±0.2 Å, b=57.8±0.2 Å, c=65.5±0.2 Å and β=107.2°±0.2°.  
     
     
         12 . The FLGK crystal of  claim 10 , wherein the monoclinic unit cells have dimensions of about a=211.6±0.2 Å, b=51.3±0.2 Å, c=66.1±0.2 Å and β=107.7°±0.2°.  
     
     
         13 . The FLGK crystal of  claim 10 , wherein the crystal is a native crystal.  
     
     
         14 . The native FLGK crystal of  claim 13 , wherein the FLGK has a three-dimensional structure characterized by the atomic structure coordinates of Table 3.  
     
     
         15 . The FLGK crystal of  claim 10 , wherein the crystal is a heavy atom derivative crystal.  
     
     
         16 . The FLGK crystal of  claim 10 , wherein the crystal is a co-crystal.  
     
     
         17 . The co-crystal of  claim 16 , wherein the FLGK protein has a three-dimensional structure characterized by the atomic structure coordinates of Table 4.  
     
     
         18 . A polypeptide corresponding to the catalytic domain of a non-insulin receptor tyrosine kinase, containing at least about 20 amino acid residues upstream of the first glycine in the conserved glycine-rich region of the catalytic domain, and at least about 17 amino acid residues downstream of the conserved arginine located at the C-terminal boundary of the catalytic domain.  
     
     
         19 . The polypeptide of  claim 18  in which the non-insulin receptor tyrosine kinase is a receptor tyrosine kinase.  
     
     
         20 . The polypeptide of  claim 18  in which the non-insulin receptor tyrosine kinase is a cytoplasmic tyrosine kinase.  
     
     
         21 . The polypeptide of  claim 18  in which the non-insulin receptor tyrosine kinase is FGF-R, PDGF-R, KDR, CCK4, MET, TRKA, AXL, TIE, EPH, RYK, DDR, ROS, RET, LTK, ROR1, or MUSK.  
     
     
         22 . The polypeptide of  claim 20  in which the cytoplasmic kinase is SRC, BRK, BTK, CSK, ABL, ZAP70, FES, FAK, JAK, or ACK.  
     
     
         23 . The polypeptide of  claim 21  or  22  having the amino acid sequence shown in FIGS. 6A or  6 B.  
     
     
         24 . A method of-using the polypeptide of  claim 18 ,  19 ,  20 ,  21  or  22  to form a crystal, comprising: 
 (a) mixing a volume of polypeptide solution with a reservoir solution; and  
 (b) incubating the mixture obtained in step (a) over the reservoir solution in a closed container, under conditions suitable for crystallization.  
 
     
     
         25 . A method of obtaining FGF receptor tyrosine kinase domain polypeptide in crystalline form, the method comprising the steps of: 
 (a) mixing a volume of polypeptide solution with an equal volume of reservoir solution, wherein the polypeptide solution comprises 1 mg/mL to 60 mg/mL FGF-type tyrosine kinase domain protein, 10 mM to 200 mM buffering agent, 0 mM to 20 mM dithiothreitol and has a pH of about 5.5 to about 7.5, and wherein the reservoir solution comprises 10% to 30% (w/v) polyethylene glycol, 0.1 M to 0.5 M ammonium sulfate, 0% to 20% (w/v) ethylene glycol or glycerol, 10 mM to 200 mM buffering agent and has a pH of about 5.5 to about 7.5; and    (b) incubating the mixture obtained in step (a) over said reservoir solution in a closed container at a temperature between 0° and 25° until crystals form.    
     
     
         26 . The method of  claim 25 , wherein the polypeptide solution comprises about 10 mg/mL FGF receptor tyrosine kinase domain, about 10 mM sodium chloride, about 2 mM dithiothreitol, about 10 mM Tris-HCl and has a pH of about 8; the reservoir buffer comprises about 16% (w/v) polyethylene glycol (MW 10000), about 0.3 M ammonium sulfate, about 5%. ethylene glycol or glycerol, about 100 mM bis-Tris and has a pH of about 6.5; and the temperature is about 4° C.  
     
     
         27 . The method of  claim 25 , wherein the polypeptide solution includes a compound such as a cofactor, substrate, substrate analog, inhibitor or allosteric effector.  
     
     
         28 . The method of  claim 25 , wherein the compound is a non-hydrolyzable analog of ATP.  
     
     
         29 . A cDNA encoding an FGF receptor tyrosine kinase domain protein, wherein a coding strand of the cDNA has the nucleotide sequence of SEQ ID NO:5.