Crystalline TNF-alpha-converting enzyme and uses thereof

Abstract

A tumor necrosis factor-α converting enzyme (TACE) is produced, purified, and crystallized. The three-dimensional coordinates of the crystal are obtained by X-ray diffraction. The coordinates can be recorded on a computer readable medium, or are part of a video memory, where they can be used as part of a system for studying for studying TACE. The coordinates are also used in designing, screening, and developing compounds that associate with TACE.

We claim:  
     
         1 . A composition comprising a polypeptide in crystalline form, wherein the polypeptide is a TNF-α-converting enzyme (TACE) polypeptide.  
     
     
         2 . A composition according to  claim 1 , wherein the TACE polypeptide comprises the TACE catalytic domain (TCD).  
     
     
         3 . A composition according to  claim 1 , wherein the TACE polypeptide is the expression product of a polynucleotide encoding the pro and catalytic domains of TACE.  
     
     
         4 . A composition according to  claim 1 , wherein the TACE polypeptide is the expression product of a polynucleotide encoding the amino acid residues 1-477 of TACE.  
     
     
         5 . A composition according to  claim 4 , wherein the polynucleotide is substituted such that amino acid residue Ser266 is changed to Ala and amino acid residue Asn542 is changed to Gln, and wherein a second polynucleotide encoding the sequence Gly-Ser-(His) 6  is fused to the C-terminus.  
     
     
         6 . A composition according to  claim 1 , further comprising a binding partner suitable for co-crystallization with the TACE polypeptide.  
     
     
         7 . A composition according to  claim 6 , wherein the binding partner is a hydroxamate-based binding partner.  
     
     
         8 . A composition according to  claim 6 , wherein the binding partner is N-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide.  
     
     
         9 . A composition according to  claim 1 , wherein the crystal has a crystal structure diffracting to 2.0 Å.  
     
     
         10 . A composition according to  claim 1 , wherein the crystal is monoclinic.  
     
     
         11 . A composition according to  claim 1 , wherein the unit cell of the crystal comprises four crystallographically independent TCD molecules.  
     
     
         12 . A composition according to  claim 11 , wherein the TCD molecules are in an asymmetric unit.  
     
     
         13 . A composition according to  claim 1 , wherein the crystal is of monoclinic space group P2 1  and the cell has the constants a=61.38 A, b=126.27 A, c=81.27 A, and β=107.41°.  
     
     
         14 . A composition according to  claim 1 , wherein the polypeptide is characterized by the structure coordinates according to Table 1, or a substantial part thereof.  
     
     
         15 . A method for crystallizing a TACE polypeptide, comprising: 
 (A) mixing a solution comprising a TACE polypeptide and a binding partner with a crystallization buffer; and    (B) crystallizing the mixture of step (A) by drop vapor diffusion to form a crystalline precipitate.    
     
     
         16 . The method according to  claim 15 , further comprising: 
 (C) transferring seeds from the crystalline precipitate formed by the drop vapor diffusion, along with a crystallization promoter, into a mixture of a concentrated solution comprising a TACE polypeptide and binding partner substrate, and a crystallization buffer; and    (D) crystallizing the mixture of step (C) by drop vapor diffusion to form a crystal.    
     
     
         17 . The method of  claim 15 , wherein said crystallization buffer is 0.1M Na Citrate pH 5.4, 20% w/v PEG 4000, and 20% v/v isopropanol.  
     
     
         18 . The method of  claim 15 , wherein the binding partner is N-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine, 2-(amino)ethyl amide.  
     
     
         19 . The method of  claim 15 , wherein crystallization is at a temperature ranging from 4 to 20 degrees Celsius.  
     
     
         20 . The method of  claim 15 , wherein the solution comprising the TACE polypeptide and the binding partner is at a concentration of about 5 mg/mL to about 12 mg/mL in a buffer.  
     
     
         21 . The method of  claim 20 , wherein the solution is mixed with the crystallization buffer in a 1:1 ratio.  
     
     
         22 . A TACE crystal made by co-crystallizing a TACE polypeptide with a co-crystallization substrate.  
     
     
         23 . A method of identifying a compound that associates with TACE, comprising (A) designing a compound that associates with a catalytic domain of a TACE polypeptide, using atomic coordinates from a region selected from the group consisting of the S1′ region, the S1′S3′ pocket and atoms which bind a catalytic zinc, (B) synthesizing said compound, and (C) determining in vitro whether said compound associates with said catalytic domain, 
 wherein said atomic coordinates are selected from Table 1.  
 
     
     
         24 . The method of  claim 23 , wherein said region comprises the atomic coordinates for the S1′ region.  
     
     
         25 . The method of  claim 23 , wherein said region comprises the atomic coordinates for the S1′S3′ pocket.  
     
     
         26 . The method of  claim 23 , wherein said region comprises the atomic coordinates for atoms which bind a catalytic zinc.  
     
     
         27 . The method of  claim 26 , wherein said atoms comprise atoms His405, His409 and His415.