Crystallization and structure determination of staphylococcus aureus thymidylate kinase

Abstract

An unliganded form of Staphylococcus aureus thymidylate kinase ( S. aureus TMK) has been crystallized, and the three dimensional x-ray crystal structure has been solved to 2.3 Å resolution. The x-ray crystal structure is useful for solving the structure of other molecules or molecular complexes, and designing inhibitors of S. aureus TMK activity.

What is claimed is:  
     
         1 . A molecule or molecular complex comprising at least a portion of an  S. aureus  thymidylate kinase or thymidylate kinase-like TMP binding pocket, wherein the TMP binding pocket comprises the amino acids listed in Table 1, the TMP binding pocket being defined by a set of points having a root mean square deviation of less than about 2.1 Å from points representing the backbone atoms of said amino acids as represented by the structure coordinates listed in FIG. 2.  
     
     
         2 . The molecule or molecular complex of  claim 1 , wherein the TMP binding pocket comprises the amino acids listed in Table 2.  
     
     
         3 . The molecule or molecular complex of  claim 1 , wherein the TMP binding pocket comprises the amino acids listed in Table 3.  
     
     
         4 . A molecule or molecular complex comprising at least a portion of an  S. aureus  thymidylate kinase TMP/ATP substrate binding pocket, wherein the TMP substrate binding pocket comprises the amino acids listed in Table 4, the substrate binding pocket being defined by a set of points having a root mean square deviation of less than about 2.1 Å from points representing the backbone atoms of said amino acids as represented by the structure coordinates listed in FIG. 2.  
     
     
         5 . The molecule or molecular complex of  claim 4 , wherein the TMP/ATP binding pocket comprises the amino acids listed in Table 5.  
     
     
         6 . The molecule or molecular complex of  claim 4 , wherein the TMP/ATP binding pocket comprises the amino acids listed in Table 6.  
     
     
         7 . A molecule or molecular complex that is structurally homologous to an  S. aureus  thymidylate kinase molecule or molecular complex, wherein the  S. aureus  thymidylate kinase molecule or molecular complex is represented by at least a portion of the structure coordinates listed in FIG. 2.  
     
     
         8 . A scalable three dimensional configuration of points, at least a portion of said points derived from structure coordinates of at least a portion of an  S. aureus  thymidylate kinase molecule or molecular complex listed in FIG. 2 comprising at least one of an  S. aureus  thymidylate kinase or thymidylate kinase-like TMP or TMP/ATP binding pocket.  
     
     
         9 . The scalable three dimensional configuration of points of  claim 8 , wherein substantially all of said points are derived from structure coordinates of an  S. aureus  thymidylate kinase molecule or molecular complex listed in FIG. 2.  
     
     
         10 . The scalable three dimensional configuration of points of  claim 8  wherein at least a portion of the points derived from the  S. aureus  thymidylate kinase structure coordinates are derived from structure coordinates representing the locations of at least the backbone atoms of amino acids defining an  S. aureus  thymidylate kinase TMP binding pocket, the TMP binding pocket comprising the amino acids listed in Table 1.  
     
     
         11 . The scalable three dimensional configuration of points of  claim 10 , wherein the TMP binding pocket comprises the amino acids listed in Table 2.  
     
     
         12 . The scalable three dimensional configuration of points of  claim 10 , wherein the TMP binding pocket comprises the amino acids listed in Table 3.  
     
     
         13 . The scalable three dimensional configuration of points of  claim 8  wherein at least a portion of the points derived from the  S. aureus  thymidylate kinase structure coordinates are derived from structure coordinates representing the locations of at least the backbone atoms of amino acids defining an  S. aureus  thymidylate kinase TMP/ATP binding pocket, the TMP/ATP substrate binding pocket comprising the amino acids listed in Table 4.  
     
     
         14 . The scalable three dimensional configuration of points of  claim 13 , wherein the TMP/ATP binding pocket comprises the amino acids listed in Table 5.  
     
     
         15 . The scalable three dimensional configuration of points of  claim 13 , wherein the TMP/ATP binding pocket comprises the amino acids listed in Table 6.  
     
     
         16 . The scalable three dimensional configuration of points of  claim 8  displayed as a holographic image, a stereodiagram, a model or a computer-displayed image.  
     
     
         17 . A scalable three dimensional configuration of points, at least a portion of the points derived from structure coordinates of at least a portion of a molecule or a molecular complex that is structurally homologous to an  S. aureus  thymidylate kinase molecule or molecular complex and comprises at least one of an  S. aureus  thymidylate kinase or thymidylate kinase-like TMP or TMP/ATP binding pocket.  
     
     
         18 . The scalable three-dimensional configuration of points of  claim 17  displayed as a holographic image, a stereodiagram, a model or a computer-displayed image  
     
     
         19 . A machine-readable data storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of at least one molecule or molecular complex selected from the group consisting of: 
 (i) a molecule or molecular complex comprising at least a portion of an  S. aureus  thymidylate kinase or thymidylate kinase-like TMP binding pocket comprising the amino acids listed in Table 1, the TMP binding pocket defined by a set of points having a root mean square deviation of less than about 2.1 Å from points representing the backbone atoms of said amino acids as represented by structure coordinates listed in FIG. 2;    (ii) a molecule or molecular complex comprising at least a portion of an  S. aureus  thymidylate kinase or thymidylate kinase-like TMP/ATP binding pocket comprising the amino acids listed in Table 4, the TMP/ATP binding pocket defined by a set of points having a root mean square deviation of less than about 2.1 Å from points representing the backbone atoms of said amino acids as represented by structure coordinates listed in FIG. 2; and    (iii) a molecule or molecular complex that is structurally homologous to an  S. aureus  thymidylate kinase molecule or molecular complex, wherein the  S. aureus  thymidylate kinase molecule or molecular complex is represented by at least a portion of the structure coordinates listed in FIG. 2.    
     
     
         20 . A machine-readable data storage medium comprising a data storage material encoded with a first set of machine readable data which, when combined with a second set of machine readable data, using a machine programmed with instructions for using said first set of data and said second set of data, can determine at least a portion of the structure coordinates corresponding to the second set of machine readable data, wherein said first set of data comprises a Fourier transform of at least a portion of the structural coordinates for  S. aureus  thymidylate kinase listed in FIG. 2; and said second set of data comprises an x-ray diffraction pattern of a molecule or molecular complex of unknown structure.  
     
     
         21 . A method for obtaining structural information about a molecule or a molecular complex of unknown structure comprising: 
 crystallizing the molecule or molecular complex;    generating an x-ray diffraction pattern from the crystallized molecule or molecular complex;    applying at least a portion of the structure coordinates set forth FIG. 2 to the x-ray diffraction pattern to generate a three-dimensional electron density map of at least a portion of the molecule or molecular complex whose structure is unknown.    
     
     
         22 . A method for homology modeling an  S. aureus  thymidylate kinase homolog comprising: 
 aligning the amino acid sequence of an  S. aureus  thymidylate kinase homolog with an amino acid sequence of  S. aureus  thymidylate kinase and incorporating the sequence of the  S. aureus  thymidylate kinase homolog into a model of  S. aureus  thymidylate kinase derived from structure coordinates set forth in FIG. 2 to yield a preliminary model of the  S. aureus  thymidylate kinase homolog;    subjecting the preliminary model to energy minimization to yield an energy minimized model;    remodeling regions of the energy minimized model where stereochemistry restraints are violated to yield a final model of the  S. aureus  thymidylate kinase homolog.    
     
     
         23 . A computer-assisted method for identifying an inhibitor of  S. aureus  thymidylate kinase activity comprising: 
 supplying a computer modeling application with a set of structure coordinates of a molecule or molecular complex, the molecule or molecular complex comprising at least a portion of an  S. aureus  thymidylate kinase or thymidylate kinase-like TMP binding pocket, the TMP binding pocket comprising the amino acids listed in Table 1;    supplying the computer modeling application with a set of structure coordinates of a chemical entity; and    determining whether the chemical entity is an inhibitor expected to bind to or interfere with the molecule or molecular complex, wherein binding to or interfering with the molecule or molecular complex is indicative of potential inhibition of  S. aureus  thymidylate kinase activity.    
     
     
         24 . A computer-assisted method for identifying an inhibitor of  S. aureus  thymidylate kinase activity comprising: 
 supplying a computer modeling application with a set of structure coordinates of a molecule or molecular complex, the molecule or molecular complex comprising at least a portion of an  S. aureus  thymidylate kinase or thymidylate kinase-like TMP/ATP binding pocket, the TMP/ATP binding pocket comprising the amino acids listed in Table 4;    supplying the computer modeling application with a set of structure coordinates of a chemical entity; and    determining whether the chemical entity is an inhibitor expected to bind to or interfere with the molecule or molecular complex, wherein binding to or interfering with the molecule or molecular complex is indicative of potential inhibition of  S. aureus  thymidylate kinase activity.    
     
     
         25 . The method of  claim 23  wherein the TMP binding pocket comprises the amino acids listed in Table 1, the TMP binding pocket being defined by a set of points having a root mean square deviation of less than about 2.1 Å from points representing the backbone atoms of said amino acids as represented by structure coordinates listed in FIG. 2.  
     
     
         26 . The method of  claim 24  wherein the TMP/ATP binding pocket comprises the amino acids listed in Table 4, the TMP/ATP binding pocket being defined by a set of points having a root mean square deviation of less than about 2.1 Å from points representing the backbone atoms of said amino acids as represented by structure coordinates listed in FIG. 2.  
     
     
         27 . The method of  claim 23  or  24  wherein determining whether the chemical entity is an inhibitor expected to bind to or interfere with the molecule or molecular complex comprises performing a fitting operation between the chemical entity and a binding pocket of the molecule or molecular complex, followed by computationally analyzing the results of the fitting operation to quantify the association between the chemical entity and the binding pocket.  
     
     
         28 . The method of  claim 23  or  24  further comprising screening a library of chemical entities.  
     
     
         29 . A computer-assisted method for designing an inhibitor of  S. aureus  thymidylate kinase activity comprising: 
 supplying a computer modeling application with a set of structure coordinates of a molecule or molecular complex, the molecule or molecular complex comprising at least a portion of an  S. aureus  thymidylate kinase or thymidylate kinase-like TMP binding pocket, the TMP binding pocket comprising the amino acids listed in Table 1;    supplying the computer modeling application with a set of structure coordinates for a chemical entity;    evaluating the potential binding interactions between the chemical entity and substrate binding pocket of the molecule or molecular complex;    structurally modifying the chemical entity to yield a set of structure coordinates for a modified chemical entity; and    determining whether the modified chemical entity is an inhibitor expected to bind to or interfere with the molecule or molecular complex, wherein binding to or interfering with the molecule or molecular complex is indicative of potential inhibition of  S. aureus  thymidylate kinase activity.    
     
     
         30 . A computer-assisted method for designing an inhibitor of  S. aureus  thymidylate kinase activity comprising: 
 supplying a computer modeling application with a set of structure coordinates of a molecule or molecular complex, the molecule or molecular complex comprising at least a portion of an  S. aureus  thymidylate kinase or thymidylate kinase-like TMP/ATP binding pocket, the TMP/ATP binding pocket comprising the amino acids listed in Table 4;    supplying the computer modeling application with a set of structure coordinates for a chemical entity;    evaluating the potential binding interactions between the chemical entity and substrate binding pocket of the molecule or molecular complex;    structurally modifying the chemical entity to yield a set of structure coordinates for a modified chemical entity; and    determining whether the modified chemical entity is an inhibitor expected to bind to or interfere with the molecule or molecular complex, wherein binding to or interfering with the molecule or molecular complex is indicative of potential inhibition of  S. aureus  thymidylate kinase activity.    
     
     
         31 . The method of  claim 29  wherein the TMP binding pocket comprises the amino acids listed in Table 1, the TMP binding pocket being defined by a set of points having a root mean square deviation of less than about 2.1 Å from points representing the backbone atoms of said amino acids as represented by structure coordinates listed in FIG. 2.  
     
     
         32 . The method of  claim 30  wherein the TMP/ATP binding pocket comprises the amino acids listed in Table 1, the TMP/ATP binding pocket being defined by a set of points having a root mean square deviation of less than about 2.1 Å from points representing the backbone atoms of said amino acids as represented by structure coordinates listed in FIG. 2.  
     
     
         33 . The method of  claim 29  or  30  wherein determining whether the modified chemical entity is an inhibitor expected to bind to or interfere with the molecule or molecular complex comprises performing a fitting operation between the chemical entity and a binding pocket of the molecule or molecular complex, followed by computationally analyzing the results of the fitting operation to quantify the association between the chemical entity and the binding pocket.  
     
     
         34 . The method of  claim 29  or  30  wherein the set of structure coordinates for the chemical entity is obtained from a chemical fragment library  
     
     
         35 . A computer-assisted method for designing an inhibitor of  S. aureus  thymidylate kinase activity de novo comprising: 
 supplying a computer modeling application with a set of structure coordinates of a molecule or molecular complex, the molecule or molecular complex comprising at least a portion of an  S. aureus  thymidylate kinase or thymidylate kinase-like TMP binding pocket, wherein the TMP substrate binding pocket comprises the amino acids listed in Table 1;    computationally building a chemical entity represented by set of structure coordinates; and    determining whether the chemical entity is an inhibitor expected to bind to or interfere with the molecule or molecular complex, wherein binding to or interfering with the molecule or molecular complex is indicative of potential inhibition of  S. aureus  thymidylate kinase activity.    
     
     
         36 . A computer-assisted method for designing an inhibitor of  S. aureus  thymidylate kinase activity de novo comprising: 
 supplying a computer modeling application with a set of structure coordinates of a molecule or molecular complex, the molecule or molecular complex comprising at least a portion of an  S. aureus  thymidylate kinase or thymidylate kinase-like TMP/ATP binding pocket, wherein the TMP/ATP binding pocket comprises the amino acids listed in Table 4;    computationally building a chemical entity represented by set of structure coordinates; and    determining whether the chemical entity is an inhibitor expected to bind to or interfere with the molecule or molecular complex, wherein binding to or interfering with the molecule or molecular complex is indicative of potential inhibition of  S. aureus  thymidylate kinase activity.    
     
     
         37 . The method of  claim 35  wherein the TMP binding pocket comprises the amino acids listed in Table 1, the TMP binding pocket being defined by a set of points having a root mean square deviation of less than about 2.1 Å from points representing the backbone atoms of said amino acids as represented by structure coordinates listed in FIG. 2.  
     
     
         38 . The method of  claim 36  wherein the TMP/ATP binding pocket comprises the amino acids listed in Table 4, the TMP/ATP binding pocket being defined by a set of points having a root mean square deviation of less than about 2.1 Å from points representing the backbone atoms of said amino acids as represented by structure coordinates listed in FIG. 2.  
     
     
         39 . The method of  claim 35  or  36  wherein determining whether the chemical entity is an inhibitor expected to bind to or interfere with the molecule or molecular complex comprises performing a fitting operation between the chemical entity and a binding pocket of the molecule or molecular complex, followed by computationally analyzing the results of the fitting operation to quantify the association between the chemical entity and the binding pocket.  
     
     
         40 . The method of any of claims  23 ,  24 ,  29 ,  30 ,  35 , or  36  further comprising supplying or synthesizing the potential inhibitor, then assaying the potential inhibitor to determine whether it inhibits  S. aureus  TMK activity.  
     
     
         41 . A method for making an inhibitor of  S. aureus  TMK activity, the method comprising chemically or enzymatically synthesizing a chemical entity to yield an inhibitor of  S. aureus  TMK activity, the chemical entity having been identified during a computer-assisted process comprising supplying a computer modeling application with a set of structure coordinates of a molecule or molecular complex, the molecule or molecular complex comprising at least a portion of at least one of a  S. aureus  thymidylate kinase or thymidylate kinase-like TMP or TMP/AT binding pocket; supplying the computer modeling application with a set of structure coordinates of a chemical entity; and determining whether the chemical entity is expected to bind to or interfere with the molecule or molecular complex at a binding pocket, wherein binding to or interfering with the molecule or molecular complex is indicative of potential inhibition of  S. aureus  TMK activity.  
     
     
         42 . A method for making an inhibitor of  S. aureus  TMK activity, the method comprising chemically or enzymatically synthesizing a chemical entity to yield an inhibitor of  S. aureus  TMK activity, the chemical entity having been designed during a computer-assisted process comprising supplying a computer modeling application with a set of structure coordinates of a molecule or molecular complex, the molecule or molecular complex comprising at least a portion of at least one of a  S. aureus  thymidylate kinase or thymidylate kinase-like TMP or TMP/ATP binding pocket; supplying the computer modeling application with a set of structure coordinates for a chemical entity; evaluating the potential binding interactions between the chemical entity and a binding pocket of the molecule or molecular complex; structurally modifying the chemical entity to yield a set of structure coordinates for a modified chemical entity; and determining whether the chemical entity is expected to bind to or interfere with the molecule or molecular complex at the binding pocket, wherein binding to or interfering with the molecule or molecular complex is indicative of potential inhibition of  S. aureus  TMK activity.  
     
     
         43 . A method for making an inhibitor of  S. aureus  TMK activity, the method comprising chemically or enzymatically synthesizing a chemical entity to yield an inhibitor of  S. aureus  TMK activity, the chemical entity having been designed during a computer-assisted process comprising supplying a computer modeling application with a set of structure coordinates of a molecule or molecular complex, the molecule or molecular complex comprising at least a portion of at least one of a  S. aureus  thymidylate kinase or thymidylate kinase-like TMP or TMP/ATP binding pocket; computationally building a chemical entity represented by set of structure coordinates; and determining whether the chemical entity is expected to bind to or interfere with the molecule or molecular complex at a binding pocket, wherein binding to or interfering with the molecule or molecular complex is indicative of potential inhibition of  S. aureus  TMK activity.  
     
     
         44 . An inhibitor of  S. aureus  thymidylate kinase activity identified, designed or made according to the method of any of the claims  23 ,  24 ,  29 ,  30 ,  35 ,  36 ,  41 ,  42 , or  43 .  
     
     
         45 . A composition comprising an inhibitor of  S. aureus  thymidylate kinase activity identified or designed according to the method of any of the claims  23 ,  24 ,  29 ,  30 ,  35 ,  36 ,  41 ,  42 , or  43 .  
     
     
         46 . A pharmaceutical composition comprising an inhibitor of  S. aureus  thymidylate kinase activity identified or designed according to the method of any of the claims  23 ,  24 ,  29 ,  30 ,  35 ,  36 ,  41 ,  42 , or  43  or a salt thereof, and pharmaceutically acceptable carrier.  
     
     
         47 . A method for crystallizing an  S. aureus  thymidylate kinase molecule or molecular complex comprising: 
 preparing purified  S. aureus  thymidylate kinase at a concentration of about 1 mg/ml to about 50 mg/ml; and    crystallizing  S. aureus  thymidylate kinase from a solution comprising about 5 wt. % to about 50 wt. % PEG, about 0.05 M to about 0.5 M MgCl 2 , and about 0 wt. % to about 20 wt. % DMSO, wherein the solution is buffered to a pH of about 6 to about 7.    
     
     
         48 . A method for crystallizing an  S. aureus  thymidylate kinase molecule or molecular complex comprising: 
 preparing purified  S. aureus  thymidylate kinase at a concentration of about 1 mg/ml to about 50 mg/ml; and    crystallizing  S. aureus  thymidylate kinase from a solution comprising about 2 mM to about 20 mM β,γ-difluoromethylene-bisphosphonate adenosine monophosphate and about 0 wt. % to about 20 wt. % DMSO, wherein the solution is buffered to a pH of about 6 to about 7.    
     
     
         49 . A crystal of  S. aureus  thymidylate kinase.  
     
     
         50 . The crystal of  claim 49  having the trigonal space group symmetry P2 1 .  
     
     
         51 . The crystal of  claim 49  comprising a unit cell having dimensions of a, b, and c; wherein a is about 40 Å to about 60 Å, b is about 80 Å to about 100 Å, and c is about 40 Å to about 60 Å; and wherein α=γ=90° and β is about 80° to about 120°.  
     
     
         52 . The crystal of  claim 49  comprising atoms arranged in a spatial relationship represented by the structure coordinates listed in FIG. 2.  
     
     
         53 . The crystal of  claim 49  having amino acid sequence SEQ ID NO:1.  
     
     
         54 . The crystal of  claim 49  having amino acid sequence SEQ ID NO:1, with the proviso that at least one methionine is replaced with selenomethionine.