Characterization of Mycobacterium tuberculosis CmtRMtb, a Pb(II)/Cd(II)-sensing SmtB/ArsR metalloregulatory repressor, and a homolog from S. coelicolor A3(2) [electronic resource] /
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 well characterized 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 etal. (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 Cd II and Pb II via formation of cysteine thiolate-rich coordination bonds. UV-Vis optical spectroscopy and 113 Cd NMR spectroscopy ([delta]=480 ppm) suggest two or three thiolate donors, while 111m Cd perturbed angular correlation (PAC) spectroscopy establish an unusual trigonal pyramidal coordination eometry. C102S CmtRMtb binds Cd II and Zn II with only [asymp]10-20 fold lower affinity relative to wild-type CmtRMtb, but [asymp] 100-1000-fold lower for Pb II. 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.
School:Texas A&M International University
School Location:USA - Texas
Source Type:Master's Thesis
Keywords:major biochemistry smtb arsr cmtrm tb metalloregulatory repressor
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