Compositions of hepatitis C virus NS5B polymerase and methods for crystallizing same

Abstract

The invention relates to the purification, crystallization of and structure of hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. Also, crystallization conditions for NS5B are provided. Further, the atomic coordinates for the NS5B protein are disclosed. Examples of its use for the determination of the three-dimensional atomic structures of HCV NS5B or HCV NS5B in complex with substrates or substrate analogs or inhibitors are also provided.

We claim:  
     
         1 . A crystalline composition comprising a HCV NS5B polypeptide.  
     
     
         2 . The composition of  claim 1  wherein the HCV NS5B polypeptide comprises tNS5B.  
     
     
         3 . A composition comprising a NS5B protein having at least one amino acid substitution at position 335, 344 or 550 of SEQ ID NO: 1.  
     
     
         4 . The composition according to  claim 3  wherein the amino acid substitution at position 335 is a threonine to a valine substitution.  
     
     
         5 . The composition according to  claim 3 , wherein the amino acid substitution at position 344 is a valine to an alanine substitution.  
     
     
         6 . The composition according to  claim 3 , wherein the amino acid substitution at position 550 is an arginine to a glutamine substitution.  
     
     
         7 . A composition comprising a NS5B protein having at least one amino acid substitution at position 335, 344 or 550 of a histidine-tagged NS5B protein.  
     
     
         8 . The composition according to  claim 7 , wherein the NS5B protein comprises SEQ ID NO: 1.  
     
     
         9 . A method of preparing purified, crystalline HCV NS5B comprising the steps of: 
 (a) stabilizing a NS5B protein preparation in a solution containing a protein stabilizing agent;    (b) subjecting the NS5B protein preparation to cation exchange chromatography in a buffer containing glycine; and    (c) allowing crystals to form in a precipitant containing a protein stabilizing agent, a salt and polyethylene glycol under conditions in which crystallization occurs.    
     
     
         10 . The method of  claim 9  wherein the NS5B protein preparation comprises tNS5B.  
     
     
         11 . The method of  claim 9  wherein the glycine is removed from the NS5B protein preparation after cation exchange chromatography.  
     
     
         12 . The method of  claim 11 , wherein the glycine is removed by dialysis.  
     
     
         13 . The method of  claim 9  wherein the crystals are allowed to form using a microbatch method.  
     
     
         14 . The method of  claim 9 , wherein the crystals are allowed to form using vapor diffusion.  
     
     
         15 . The method of  claim 9 , wherein the protein stabilizing agent is glycerol.  
     
     
         16 . The method of  claim 15 , wherein the glycerol is provided at a concentration of 10% (volume/volume).  
     
     
         17 . The method of  claim 9 , wherein glycine is provided at a concentration of one molar.  
     
     
         18 . The method of  claim 9 , wherein the polyethylene glycol has a molecular weight of 4000-5000 daltons.  
     
     
         19 . A machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the structure coordinates of a NS5B polypeptide according to Table 1, or a homologue thereof wherein said homologue comprises alpha carbon atoms that have a root mean square deviation between equivalent alpha carbon atoms of the polypeptide of not more than 3.0 Å.  
     
     
         20 . A method of obtaining structural information concerning a molecule of unknown structure by using the structure coordinates set forth in Table 1, comprising the steps of: 
 (a) generating x-ray diffraction data from said crystallized molecule; and    (b) applying crystallographic phases derived from at least a portion of the structure coordinates set forth in Table 1 to said x-ray diffraction pattern to generate a three-dimensional electron density map of at least a portion of the molecule.    
     
     
         21 . The method of  claim 20 , wherein the molecule is a polymerase.  
     
     
         22 . A method of obtaining structural information concerning a molecular complex of unknown structure by using the structure coordinates set forth in Table 1, comprising the steps of: 
 (a) generating x-ray diffraction data from said crystallized molecule; and    (b) applying crystallographic phases derived from at least a portion of the structure coordinates set forth in Table 1 to said x-ray diffraction pattern to generate a three-dimensional electron density map of at least a portion of the molecule.    
     
     
         23 . The method of  claim 22 , wherein the molecular complex contains a NS5B polypeptide.