Methods for crystallizing erk2 polypeptides

Abstract

The present invention relates to crystals of the ERK2 polypeptide and complexes thereof which are useful, inter alia, for structure assisted drug design.

1 . A crystal comprising mouse Ah 6 -ERK2 polypeptide optionally phosphorylated or thiophosphorylated on one or more amino acids. 
     
     
         2 . A computer for producing a three-dimensional representation of
 (i) unphosphorylated mouse Ah 6 -ERK2 or a homologue thereof, (ii) unphosphorylated mouse ERK2 or a homologue thereof, (iii) diphosphorylated mouse Ah 6 -ERK2 or a homologue thereof, (iv) diphosphorylated mouse ERK2 or a homologue thereof, (v) dithiophosphorylated mouse Ah 6 -ERK2 or a homologue thereof, (vi) dithiophosphorylated mouse ERK2 or a homologue thereof, (vii) unphosphorylate mouse Ah 6 -ERK2 complexed with olomoucine   
       
         
           
           
               
               
           
         
          or a homologue thereof; or (viii) unphosphorylate mouse ERK2 complexed with olomoucine 
       
       
         
           
           
               
               
           
         
          or a homologue thereof; wherein said computer comprises:
 (a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises the structure coordinates of Table 1, 2, 3, or 4; 
 (b) a working memory for storing instructions for processing said machine-readable data; 
 (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 said three-dimensional representation; and 
 (d) a display unit coupled to said central-processing unit for displaying said three-dimensional representation. 
 
       
     
     
         3 . The computer of  claim 2  wherein the root mean square deviation between said homologue and the structure coordinates set forth in Table 1, 2, 3, or 4 is less than about 1 Å. 
     
     
         4 . The computer of  claim 3  wherein the root mean square deviation between the homologue and the structure coordinates set forth in Table 1, 2, 3, or 4 is less than about 0.5 Å. 
     
     
         5 . The computer of  claim 4  wherein the root mean square deviation between the homologue and the structure coordinates set forth in Table 1, 2, 3, or 4 is less than about 0.1 Å. 
     
     
         6 . The computer of  claim 2  wherein the display unit is displaying the three dimensional representation. 
     
     
         7 . A method for making a crystal comprising placing a mouse ERK2 polypeptide in an aqueous buffered solution comprising from about 1% to about 8% PEG 400 (v/v), about 2.4M ammonium sulfate and a pH of from about 5.5 to about 6.7. 
     
     
         8 . The method of  claim 7  wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-6. 
     
     
         9 . The method of  claim 7  wherein the polypeptide is crystallized by the hanging-drop vapor diffusion method. 
     
     
         10 . A method for making a crystal comprising placing a mouse ERK2 polypeptide in an aqueous buffered solution of having a pH of from about 5 to about 6.2 and a concentration of from about 5% to about 30% isopropanol (v/v). 
     
     
         11 . The method of  claim 10  wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-6. 
     
     
         12 . The method of  claim 10  wherein the polypeptide is crystallized by the hanging-drop vapor diffusion method. 
     
     
         13 . A method for making a crystal comprising placing a mouse ERK2 polypeptide in an aqueous buffered solution of having a pH of from about 5 to about 6.2 and a concentration of from about 5% to about 30% isopropanol (v/v). 
     
     
         14 . The method of  claim 13  wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-6. 
     
     
         15 . The method of  claim 13  wherein the polypeptide is crystallized by the hanging-drop vapor diffusion method. 
     
     
         16 . A method for making a crystal comprising placing a mouse ERK2 polypeptide in an aqueous buffered solution comprising from about 1%, to about 8% PEG 400 (v/v), about 2.4M ammonium sulfate and a pH of from about 5.5 to about 6.7, crystallizing the polypeptide by the hanging drop vapor diffusion method and soaking said crystal in a solution comprising olomoucine. 
     
     
         17 . The method of  claim 16  wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-6.