title: Structural and functional characterization of Mycobacterium tuberculosis CmtR, a PbII/CdII-sensing SmtB/ArsR metalloregulatory repressor.
abstract: The SmtB/ArsR family of prokaryotic metalloregulators are winged-helix transcriptional repressors that collectively provide resistance to a wide range of both biologically required and toxic heavy-metal ions. CmtR is a recently described Cd(II)/Pb(II) regulator expressed in Mycobacterium tuberculosis that is structurally distinct from the well-characterized SmtB/ArsR Cd(II)/Pb(II) sensor, Staphylococcus aureus plasmid pI258-encoded CadC. From functional analyses and a multiple sequence alignment of CmtR paralogs, M. tuberculosis CmtR is proposed to bind Pb(II) and Cd(II) via coordination by Cys57, Cys61, and Cys102 [Cavet et al. (2003) J. Biol. Chem. 278, 44560-44566]. We establish here that both wild-type and C102S CmtR are homodimers and bind Cd(II) and Pb(II) via formation of cysteine thiolate-rich coordination bonds. UV-vis optical spectroscopy, (113)Cd NMR spectroscopy (delta = 480 ppm), and (111m)Cd perturbed angular correlation (PAC) spectroscopy suggest two or three thiolate donors in the wild-type protein. Cys57 and Cys61 anchor the coordination complex, while Cys102 plays only an accessory role in stabilizing the metal chelate in the free protein because C102S CmtR binds Cd(II) and Zn(II) with only approximately 10-20-fold lower affinity relative to wild-type CmtR but approximately 100-1000-fold lower for Pb(II). Quantitative investigation of CmtR-cmt O/P binding equilibria using fluorescence anisotropy, however, reveals that Cys102 functions as a key allosteric metal ligand, because substitution of Cys102 abrogates disassembly of oligomeric CmtR-cmt O/P oligomeric complexes. The implications of these findings on the evolution of distinct metal-sensing sites in a family of homologous proteins are discussed.
authors.affiliation: Department of Biochemistry and Biophysics, 2128 TAMU, Texas A&M University, College Station, Texas 77843-2128, USA.
authors.forename: Yun
authors.initials: Y
authors.lastname: Wang
authors.forename: Lars
authors.initials: L
authors.lastname: Hemmingsen
authors.forename: David P
authors.initials: DP
authors.lastname: Giedroc
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002104:Cadmium / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D007854:Lead / Q000378:metabolism*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000378:metabolism; D020836:Protein Structure, Quaternary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013053:Spectrophotometry; D040681:Structural Homology, Protein; D017186:Titrimetry
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D002104:Cadmium; D007854:Lead; D003545:Cysteine