Characterization of Mycobacterium tuberculosis CmtR_Mtb, a Pb(ii)/Cd(ii)-sensing SmtB/ArsR metalloregulatory repressor, and a homolog from S. coelicolor A3(2)
MetadataShow full item record
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. CmtRMtb is a recently described CdII/PbII regulator expressed in M. tuberculosis that is structurally distinct from the wellcharacterized SmtB/ArsR CdII/PbII sensor, S. aureus plasmid pI258-encoded CadC. From functional analyses and a multiple sequence alignment of CmtR homologs, CmtRMtb is proposed to bind PbII and CdII via coordination by Cys57, Cys61 and Cys102 [Cavet et al. (2003) J. Biol. Chem. 278, 44560-44566]. To better understand the mechanism how CmtRMtb utilizes specific metal ions to perform transcriptional repressor function, both CmtRMtb and its homolog in S. coelicolor A3(2) (CmtRSc) were studied. We establish here that both wild-type and C102S CmtRMtb are homodimers and bind CdII and PbII via formation of cysteine thiolate-rich coordination bonds. UV-Vis optical spectroscopy and 113Cd NMR spectroscopy (ÃÂ´=480 ppm) suggest two or three thiolate donors, while 111mCd perturbed angular correlation (PAC) spectroscopy establish an unusual trigonal pyramidal coordination eometry. C102S CmtRMtb binds CdII and ZnII with only Ã¢ÂÂ 10-20 fold lower affinity relative to wild-type CmtRMtb, but Ã¢ÂÂ 100-1000-fold lower for PbII. Quantitative investigation of CmtR-cmt O/P binding equilibria using fluorescence anisotropy reveals that Cys57 and Cys61 anchor the coordination complex with Cys102 functioning as a key allosteric ligand, while playing only an accessory role in stabilizing the metal complex in the free protein. Similar metal titration experiments were carried out with a putative CmtR homolog from S. coelicolor A3(2) (CmtRSc) and a double cysteine substitution mutant C110G/C111S CmtRSc. The implications of these findings on the evolution of distinct metal sensing sites in a family of homologous proteins are discussed.
Wang, Yun (2005). Characterization of Mycobacterium tuberculosis CmtR_Mtb, a Pb(ii)/Cd(ii)-sensing SmtB/ArsR metalloregulatory repressor, and a homolog from S. coelicolor A3(2). Master's thesis, Texas A&M University. Texas A&M University. Available electronically from