Crystals of MarR polypeptides, regulators of multiple antibiotic resistance

Abstract

The crystal structure of the product, crystals of the MarR protein, a regulator of multiple antibiotic resistance in Escherichia coli , and methods of crystallization of the MarR protein are described.

1 . A crystallized MarR family polypeptide, wherein said crystallized MarR family polypeptide is crystallized under appropriate conditions such that the three dimensional structure of said MarR family polypeptide can be determined.  
     
     
         2 . The crystallized MarR family polypeptide of  claim 1 , wherein the crystallized MarR family polypeptide is a structural homolog of crystallized MarR.  
     
     
         3 . The crystallized MarR family polypeptide of  claim 1 , wherein said MarR family polypeptide is selected from the group consisting of MarR, SlyA, EmrR (MprA), PapX, PrsX, HpcR, Ec17kD of  Escherichia coli ; MarR, SlyA, EmrR of  Salmonella typhimurium ; MexR of  Pseudomonas aeruginosa ; PecS of  Erwinia chrysanthemi ; BadR of  Rhodopseudomonas palustris ; OrfE of  Burkholderia pseudomallei ; YdcH, YhbI, YkmA, YkoM, Orf7, YfiV, YetL, YdgJ, YwoH, YwaE, YwhA, Hpr, YybA, YxaD, YsmB, YusO, YpoP, YkvE of  Bacillus subtilus ; Orf7 of  Bacillus firmus ; Orf145, Orf141 of  Staphylococcus sciuri ; CinR of  Butyrivibrio fibrisolvens ; Orf158 of  Sphingomonas aromaticivorans ; NhhD of  Rhodococcus rhodochrous ; Orf1 of  Streptomyces peucetius;  14.7kD, Rv1404, Rv0737, Rv0042c, Yz08 (15.6kD) of  Mycobacterium tuberculosis ; Yz08 (15.6kD) of  Mycobacterium leprae ; MTH313 of  Methanobacterium thermoautotrophicum ; Lrs14 of  Sulfolobus solfataricus ; CinR of  Archaeoglobus fulgidus ; PetP of  Rhodobacter capsulatus ; and SlyA (E293909) of  Sinorhizobium meliloti.    
     
     
         4 . The crystallized MarR family polypeptide of  claim 1 , wherein said MarR family polypeptide is MarR.  
     
     
         5 . The crystallized MarR family polypeptide of  claim 4 , wherein the peptide sequence of MarR comprises the amino acid sequence shown in SEQ ID NO:2.  
     
     
         6 . The crystallized MarR family polypeptide of  claim 4 , wherein said three dimensional structure has the space group of I4 I 22.  
     
     
         7 . The crystallized MarR family polypeptide of  claim 1 , wherein said appropriate conditions include the presence of a MarR family polypeptide modulator.  
     
     
         8 . The crystallized MarR family polypeptide of  claim 7 , wherein said MarR family polypeptide inhibitor is salicylate.  
     
     
         9 . The crystallized MarR family polypeptide of  claim 1 , wherein said three dimensional structure is determined to a resolution of 5 Å or better.  
     
     
         10 . The crystallized MarR family polypeptide of  claim 9 , wherein said three dimensional structure is determined to a resolution of 3 Å or better.  
     
     
         11 . The crystallized MarR family polypeptide of  claim 10 , wherein said three dimensional structure is determined to a resolution of 2.5 Å or better.  
     
     
         12 . The crystallized MarR family polypeptide of  claim 11 , wherein said three dimensional structure is determined to a resolution of 2.3 Å or better.  
     
     
         13 . The crystallized MarR family polypeptide of  claim 4 , wherein said crystallized MarR family polypeptide has a structure defined by the atomic coordinates given in  FIG. 7 .  
     
     
         14 . The crystallized MarR family polypeptide of  claim 4 , wherein said crystallized MarR family polypeptide has a structure defined by the atomic coordinates given in  FIG. 8 .  
     
     
         15 . The crystallized MarR family polypeptide of  claim 1 , wherein said MarR family polypeptide has a winged-helix structure.  
     
     
         16 . A crystallized MarR family polypeptide defined by the atomic coordinates given in  FIG. 7 .  
     
     
         17 . A crystallized MarR family polypeptide defined by the atomic coordinates given in  FIG. 8 .  
     
     
         18 . A method for determining the three dimensional structure of MarR family polypeptide, comprising 
 crystallizing the MarR family polypeptide under appropriate conditions such that crystals are formed; and    analyzing the crystallized MarR family polypeptide, such that the three dimensional structure of the MarR family polypeptide is determined.    
     
     
         19 . The method of  claim 18 , wherein said crystallized MarR family polypeptide is analyzed by x-ray diffraction techniques.  
     
     
         20 . The method of  claim 18 , wherein said MarR family polypeptide is MarR.  
     
     
         21 . The method of  claim 20 , wherein MarR comprises the amino acid sequence set forth in SEQ ID NO:2.  
     
     
         22 . The method of  claim 18 , wherein said appropriate conditions comprise the presence of a MarR family polypeptide modulator.  
     
     
         23 . The method of  claim 22 , wherein said MarR family polypeptide modulator is salicylate.  
     
     
         24 . The method of  claim 18 , wherein said three dimensional structure is determined to a resolution of 5 Å or better.  
     
     
         25 . The method of  claim 24 , wherein said three dimensional structure is determined to a resolution of 3 Å or better.  
     
     
         26 . The method of  claim 25 , wherein said three dimensional structure is determined to a resolution of 2.5 Å or better.  
     
     
         27 . The method of  claim 26 , wherein said three dimensional structure is determined to a resolution of 2.3 Å or better.  
     
     
         28 . The method of  claim 18 , wherein said MarR family polypeptide is selected from the group consisting of: MarR, SlyA, EmrR (MprA), PapX, PrsX, HpcR, Ec17kD of  Escherichia coli ; MarR, SlyA, EmrR of  Salmonella typhimurium ; MexR of  Pseudomonas aeruginosa ; PecS of  Erwinia chrysanthemi ; BadR of  Rhodopseudomonas palustris ; OrfE of  Burkholderia pseudomallei ; YdcH, YhbI, YkmA, YkoM, Orf7, YfiV, YetL, YdgJ, YwoH, YwaE, YwhA, Hpr, YybA, YxaD, YsmB, YusO, YpoP, YkvE of  Bacillus subtilus ; Orf7 of  Bacillus firmus ; Orf145, Orf141 of  Staphylococcus sciuri ; CinR of  Butyrivibrio fibrisolvens ; Orf158 of  Sphingomonas aromaticivorans ; NhhD of  Rhodococcus rhodochrous ; Orf1 of  Streptomyces peucetius;  14.7kD, Rv1404, Rv0737, Rv0042c, Yz08 (15.6kD) of  Mycobacterium tuberculosis ; Yz08 (15.6kD) of  Mycobacterium leprae ; MTH313. of  Methanobacterium thermoautotrophicum ; Lrs14 of  Sulfolobus solfataricus ; CinR of  Archaeoglobus fulgidus ; PetP of  Rhodobacter capsulatus ; and SlyA (E293909) of  Sinorhizobium meliloti.    
     
     
         29 . A method of making a crystal of a MarR family polypeptide comprising: contacting MarR with a modulator to form a complex and allowing crystals of the complex to grow.