Role of cysteinyl residues in sensing Pb(II), Cd(II), and Zn(II) by the plasmid pI258 CadC repressor.
The cadCA operon of Staphylococcus aureus plasmid pI258 confers resistance to salts of the soft metals lead, cadmium, and zinc. The operon is regulated by CadC, a member of the ArsR family of metal-responsive transcriptional repressors. In this study the role of the five cysteine residues of CadC in soft metal ion sensing was investigated. Cys-7, Cys-11, Cys-52, Cys-58, and Cys-60 were changed individually to glycine or serine residues. The effect of the cadC mutations was examined in Escherichia coli using a green fluorescent protein reporter system. None of the mutations affected the ability of CadC to repress gfp expression. Neither Cys-11 nor Cys-52 was required for in vivo response to Pb(II), Zn(II), or Cd(II). Cys-7, Cys-58, or Cys-60 mutations each reduced or eliminated soft metal sensing. Wild-type and mutant CadC proteins were purified, and the effect of the substitutions on DNA binding was determined using a restriction enzyme protection assay. Binding of wild-type CadC protected cad operator DNA from digestion at the single SspI site, and the addition of Pb(II), Zn(II), or Cd(II) resulted in deprotection. Chemical modification of the cysteine residues in CadC had no effect on protection but eliminated deprotection. C11G and C52G proteins exhibited wild-type properties in vitro. C7G, C58S, and C60G proteins were able to be protected from SspI digestion but had reduced responses to soft metal ions. The results indicate that Cys-7, Cys-58, and Cys-60 are involved in sensing those soft metals and suggest that they are ligands to Pb(II), Zn(II), and Cd(II).
Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA.
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Sun
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Wong
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Rosen
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002104:Cadmium / Q000032:analysis*; D003545:Cysteine / Q000378:metabolism*; D007854:Lead / Q000032:analysis*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000032:analysis*
D001426:Bacterial Proteins; C080050:CadC protein, Bacteria; D012097:Repressor Proteins; D002104:Cadmium; D007854:Lead; D015032:Zinc; D003545:Cysteine