title: The FxRxHrS motif: a conserved region essential for DNA binding of the VirR response regulator from Clostridium perfringens.
abstract: The VirSR two-component signal transduction pathway regulates virulence and toxin production in Clostridium perfringens, the causative agent of gas gangrene. The response regulator, VirR, binds to repeat sequences located upstream of the promoter and is directly responsible for the transcriptional activation of pfoA, the structural gene for the cholesterol-dependent cytolysin, perfringolysin O. Comparative sequence analysis of the 236 amino acid residue VirR protein revealed a two-domain structure: a typical N-terminal response regulator domain and an uncharacterised C-terminal domain. Database searching revealed that over 40 other proteins, many of which appeared to be response regulators or transcriptional activators, had homology with the VirR C-terminal domain (VirRc). Multiple sequence alignment of this VirRc family revealed a highly conserved region that was designated the FxRxHrS motif. By deletion analysis this motif was shown to be essential for the functional integrity of the VirR protein. Alanine scanning mutagenesis and subsequent phenotypic analysis indicated that conserved residues located within the motif were required for activity. These residues extended from L179 to N194. More detailed site-directed mutagenesis showed that amino acid residues R186, H188 and S190 were essential for activity since even conservative substitutions in these positions resulted in non-functional proteins. Three of the mutant proteins, R186K, S190A and S190C, were purified and shown by in vitro gel shift analysis to be unable to bind to the specific target DNA with the same efficiency as the wild-type protein. These data reveal for the first time that VirRc functions as a DNA binding domain in which the highly conserved FxRxHrS motif has a functional role. These studies have important implications for this new family of transcriptional factors since they imply that the conserved FxRxHrS motif may be involved in DNA binding in all of these proteins, irrespective of their biological role.
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics; D003016:Clostridium perfringens / Q000235:genetics* / Q000378:metabolism; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D030561:Databases, Nucleic Acid; D005796:Genes; D017930:Genes, Reporter; D006460:Hemolysin Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods; D017433:Protein Structure, Secondary; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D006460:Hemolysin Proteins; D014157:Transcription Factors; C068267:mry protein, Streptococcus pyogenes; C020452:Clostridium perfringens theta-toxin