Method of using a crystal of the N-terminal domain of a signal transducer and activator of transcription

Abstract

The present invention provides a crystal containing the N-terminal domain of a STAT protein that is of sufficient quality to perform X-ray crystallographic studies. Methods of preparing the crystals are include in the invention. The present invention further discloses the three-dimensional structure of the crystal. The present invention also provides methods of using the structural information in drug discovery and drug development.

What is claimed is:  
     
         1 . A crystal of an N-terminal domain of a STAT protein, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the N-terminal domain of the STAT protein to a resolution of greater than 5.0 Angstroms.  
     
     
         2 . The crystal of  claim 1  wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the N-terminus to a resolution of greater than 3.0 Angstroms.  
     
     
         3 . The crystal of  claim 1  wherein the N-terminal domain comprises the amino acid sequence: 
 Arg Xaa  H Xaa Leu Xaa Xaa Trp  H Xaa Glu Xaa Gln Xaa Trp (SEQ ID NO: 1) wherein:  H Xaa is either Ile, Leu, Val, Phe, or Tyr.  
 
     
     
         4 . The crystal of  claim 3  wherein the N-terminal domain of the STAT protein is contained in a peptide fragment that consists of 100 to 150 amino acids.  
     
     
         5 . The crystal of  claim 4  wherein the N-terminal domain of the STAT protein comprises amino acids 4-112 of SEQ ID NO:2.  
     
     
         6 . The crystal of  claim 5  wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the N-terminus to a resolution of 1.45 Angstroms.  
     
     
         7 . The crystal of  claim 5  having a space group of P6 5 22 and a unit cell of dimensions a=79.5 1 Å, b=79.51 Å, and c=84.68 Å.  
     
     
         8 . The crystal of  claim 1  having secondary structural elements comprising eight helices (α1-α8) that are assembled into a hook-like structure having an inner and outer surface, wherein: 
 (a) the first four helices (α1-α4) form a ring-shaped element having a proximal and a distal surface;  
 (b) helices six (α6) and seven (α7) form an anti-parallel coiled-coil having a proximal and a distal surface;  
 (c) helix five (α5) connects the ring-shaped element to the anti-parallel coiled-coil; and  
 (d) helix eight (α8) is wrapped around the distal surface of the ring-shaped element; wherein the inner surface of the hook-like structure is formed by the intersection of the proximal surface of the ring-shaped element with the proximal surface of the antiparallel coiled-coil.  
 
     
     
         9 . A method of using the crystal of  claim 1  in a drug screening assay comprising: 
 (a) selecting a potential drug by performing rational drug design with the three-dimensional structure determined for the crystal, wherein said selecting is performed in conjunction with computer modeling;  
 (b) contacting the potential drug with a dimeric STAT protein N-terminal domain; and  
 (c) detecting the binding of the potential drug with the N-terminal domain; wherein a drug is selected that binds to the N-terminal domain.  
 
     
     
         10 . The method of  claim 9  wherein contacting the potential drug with a dimeric STAT protein N-terminal domain is performed with a STAT protein fragment containing a STAT protein N-terminal domain comprising SEQ ID NO: 1.  
     
     
         11 . The method of  claim 10  wherein the STAT protein fragment is labeled.  
     
     
         12 . The method of  claim 10  wherein the STAT protein fragment is bound to a solid support.  
     
     
         13 . A method of using the crystal of  claim 1  in a drug screening assay comprising: 
 (a) selecting a potential drug by performing rational drug design with the three-dimensional structure determined for the crystal, wherein said selecting is performed in conjunction with computer modeling;  
 (b) contacting the potential drug with two or more dimeric STAT proteins in the presence of a nucleic acid containing at least two adjacent weak binding sites for STAT protein dimers; and  
 (c) detecting the effect of the potential drug on the binding of the dimeric STAT proteins to each other and/or to the nucleic acid; wherein a potential drug is selected as a candidate drug when it either enhances or diminishes the binding of the dimeric STAT proteins to each other and/or the nucleic acid.  
 
     
     
         14 . The method of  claim 13  further comprising: 
 (d) growing a supplemental crystal containing a protein-drug complex formed between the dimeric N-terminal domain and the candidate drug, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the protein-ligand complex to a resolution of greater than 5.0 Angstroms;  
 (e) determining the three-dimensional structure of the supplemental crystal with molecular replacement analysis; and  
 (f) selecting a drug by performing rational drug design with the three-dimensional structure determined for the supplemental crystal, wherein said selecting is performed in conjunction with computer modeling.  
 
     
     
         15 . A method for identifying a drug that enhances or diminishes the ability of STAT protein dimers to induce the expression of a gene operably under the control of a promoter containing at least two adjacent weak binding sites for STAT protein dimers comprising: 
 (a) selecting a potential drug by performing rational drug design with the three-dimensional structure determined for the crystal of  claim 1 , wherein said selecting is performed in conjunction with computer modeling;    (b) measuring the level of expression of a first reporter gene and a second reporter gene contained by a host cell in the presence and absence of the potential drug; wherein the first reporter gene is operably linked to a first promoter containing at least two adjacent weak binding sites for STAT protein dimers, and the second reporter gene is operably linked to a second promoter comprising at least one strong binding site for a STAT protein dimer; wherein the binding of STAT protein dimers to the two adjacent weak binding sites induces the expression of the first reporter gene, and wherein the binding of the STAT protein dimer to the strong binding site induces the expression of the second reporter gene; and wherein the host cell contains STAT protein dimers; and    (c) comparing the level of expression of the first reporter gene with that of the second reporter gene in the presence and absence of the potential drug, wherein when the presence of the potential drug results in an increase in the level of expression of the first reporter gene but not that of the second reporter gene, the potential drug is identified as a drug that enhances the ability of STAT protein dimers to induce the expression of a gene operably under the control of a promoter containing at least two adjacent weak binding sites for STAT protein dimers; and when the presence of a potential drug results in a decrease in the level of expression of the first reporter gene but not that of the second reporter gene the potential drug is identified as a drug that inhibits the ability of STAT protein dimers to induce the expression of a gene operably under the control of a promoter containing at least two adjacent weak binding sites for STAT protein dimers.    
     
     
         16 . The method of  claim 15  wherein the host cell is a mammalian cell.  
     
     
         17 . The method of  claim 15  wherein the first reporter gene is contained by a first host cell, and the second reporter gene is contained by a second host cell; and wherein the first host cell and second host cell both contain STAT protein dimers.  
     
     
         18 . The method of  claim 15  wherein the weak STAT binding sites are selected from the group consisting of sites present in the regulatory regions of the MIG gene, the c-fos gene and the interferon-γ gene.  
     
     
         19 . The method of  claim 15  further comprising: 
 (d) growing a supplemental crystal containing a protein-drug complex formed between the dimeric N-terminal domain and the drug, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the protein-ligand complex to a resolution of greater than 5.0 Angstroms;  
 (e) determining the three-dimensional structure of the supplemental crystal with molecular replacement analysis; and  
 (f) selecting a drug by performing rational drug design with the three-dimensional structure determined for the supplemental crystal, wherein said selecting is performed in conjunction with computer modeling.  
 
     
     
         20 . A method for identifying a drug that modulates the ability of adjacent STAT protein dimers to interact and bind to adjacent DNA binding sites comprising: 
 (a) selecting a potential drug by performing rational drug design with the three-dimensional structure determined for the crystal of  claim 1 , wherein said selecting is performed in conjunction with computer modeling;    (b) measuring the binding affinity of the STAT protein, or a fragment thereof that comprises the N-terminal domain, to a nucleic acid comprising 2 adjacent weak STAT DNA binding sites in the presence and absence of the potential drug;    (c) measuring the binding affinity of the STAT protein, or the fragment, to a nucleic acid comprising a single strong STAT binding site in the presence and absence of the potential drug; and    (d) comparing the binding affinity measured in step (b) in the presence and absence of the potential drug with the binding affinity measured in step (c) in the presence and absence of the potential drug, wherein a potential drug which causes an increase in the binding affinity measured in step (b) but not in the binding affinity measured in step (c) is identified as a drug that enhances the interaction between adjacent activated STAT dimers, and a potential drug which causes a decrease in the binding affinity measured in step (b) but not in the binding affinity measured in step (c) is identified as a drug that inhibits the interaction between adjacent activated STAT dimers.    
     
     
         21 . A method for identifying a drug that modulates the ability of adjacent STAT protein dimers to interact and bind to adjacent DNA binding sites comprising: 
 (a) measuring the binding affinity of the STAT protein comprising the N-terminal domain, to a nucleic acid comprising two adjacent weak STAT DNA binding sites in the presence and absence of a potential drug;    (b) measuring the binding affinity of a modified form of the STAT protein lacking the α4-tryptophan of N-terminal domain with the nucleic acid in the presence and absence of the potential drug; and    (c) comparing the binding affinity measured in step (a) in the presence and absence of the potential drug with the binding affinity measured in step (b) in the presence and absence of the potential drug, wherein a potential drug which causes an increase in the binding affinity measured in step (a) but not in the binding affinity measured in step (b) is identified as a drug that enhances the interaction between adjacent activated STAT dimers, and a potential drug which causes a decrease in the binding affinity measured in step (a) but not in the binding affinity measured in step (b) is identified as a drug that inhibits the interaction between adjacent activated STAT dimers.    
     
     
         22 . A method of making the crystal of  claim 1  comprising placing an aliquot of a solution containing the STAT N-terminal domain fragment on a cover slip as a hanging drop above a well containing a reservoir buffer that comprises 0.2 M Na + CH 3 COO − , 0.1 M Tris/HCL pH 8.0, 17% PEG4000.  
     
     
         23 . The method of  claim 22  wherein said solution is prepared by combining a preparation of the STAT N-terminal domain fragment that contains 20 mg/ml of the STAT N-terminal domain fragment in 50 mM Hepes/HCl pH 8.0, 150 mM KCl, 2.5 mM CaCl 2 , and 5 mM DTT with an equal volume of the reservoir buffer; and wherein the aliquot comprises approximately 1 μl of said solution.  
     
     
         24 . The method of  claim 23  wherein the STAT N-terminal domain fragment is the STAT-4 N-terminal domain fragment.