Structural and functional characterization of Mycobacterium tuberculosis CmtR, a PbII/CdII-sensing SmtB/ArsR metalloregulatory repressor

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, Cd-113 NMR spectroscopy (delta = 480 ppm), and Cd-111m 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 approximate to 10-20-fold lower affinity relative to wild-type CmtR but approximate to 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.