The SmtB/ArsR family of prokaryotic metalloregulatory transcriptional repressors represses the expression of operons linked to stressinducing concentrations of di-and multivalent heavy metal ions. Derepression results from direct binding of metal ions by these homodimeric 'metal sensor' proteins. An evolutionary analysis, coupled with comparative structural and spectroscopic studies of six SmtB/ArsR family members, suggests a unifying 'theme and variations' model, in which individual members have evolved distinct metal selectivity profiles by alteration of one or both of two structurally distinct metal coordination sites. These two metal sites are designated K3N (or K3) and K5 (or K5C), named for the location of the metal binding ligands within the known or predicted secondary structure of individual family members. The K3N/K3 sensors, represented by Staphylococcus aureus pI258 CadC, Listeria monocytogenes CadC and Escherichia coli ArsR, form cysteine thiolate-rich coordination complexes (S 3 or S 4 ) with thiophilic heavy metal pollutants including Cd(II), Pb(II), Bi(III) and As(III) via inter-subunit coordination by ligands derived from the K3 helix and the N-terminal 'arm' (CadCs) or from the K3 helix only (ArsRs). The K5/K5C sensors Synechococcus SmtB, Synechocystis ZiaR, S. aureus CzrA, and Mycobacterium tuberculosis NmtR form metal complexes with biologically required metal ions Zn(II), Co(II) and Ni(II) characterized by four or more coordination bonds to a mixture of histidine and carboxylate ligands derived from the C-terminal K5 helices on opposite subunits. Direct binding of metal ions to either the K3N or K5 sites leads to strong, negative allosteric regulation of repressor operator/promoter binding affinity, consistent with a simple model for derepression. We hypothesize that distinct allosteric pathways for metal sensing have coevolved with metal specificities of distinct K3N and K5 coordination complexes.

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Staphylococcus aureus pI258 CadC is an extrachromosomally encoded metalloregulatory repressor protein from the ArsR superfamily which negatively regulates the expression of the cad operon in a metal-dependent fashion. The metalloregulatory hypothesis holds that direct binding of thiophilic divalent cations including Cd(II), Pb(II), and Zn(II) by CadC allosterically regulates the DNA binding activity of CadC to the cad operator/promoter (O/P). This report presents a detailed characterization of the metal binding and DNA binding properties of wild-type CadC. The results of analytical ultracentrifugation experiments suggest that both apo- and Cd(1)-CadC are stable or weakly dissociable homodimers characterized by a K(dimer) = 3.0 x 10(6) M(-1) (pH 7.0, 0.20 M NaCl, 25.0 degrees C) with little detectable effect of Cd(II) on the dimerization equilibrium. As determined by optical spectroscopy, the stoichiometry of Cd(II) and Pb(II) binding is approximately 0.7-0.8 mol/mol of wild-type CadC monomer. Chelator (EDTA) competition binding isotherms reveal that Cd(II) binds very tightly, with K(Cd) = 4.3 (+/-1.8) x 10(12) M(-1). The results of UV-Vis and X-ray absorption spectroscopy of the Cd(1) complex are consistent with a tetrathiolate (S(4)) complex formed by four cysteine ligands. The (113)Cd NMR spectrum reveals a single resonance of delta = 622 ppm, consistent with an S(3)(N,O) or unusual upfield-shifted S(4) complex. The Pb(II) complex reveals two prominent absorption bands at 350 nm (epsilon = 4000 M(-1) cm(-1)) and 250 nm (epsilon = 41 000 M(-1) cm(-1)), spectral properties consistent with three or four thiolate ligands to the Pb(II) ion. The change in the anisotropy of a fluorescein-labeled oligonucleotide containing the cad O/P upon binding CadC and analyzed using a dissociable CadC dimer binding model reveals that apo-CadC forms a high-affinity complex [K(a) = (1.1 +/- 0.3) x 10(9) M(-1); pH 7.0, 0.40 M NaCl, 25 degrees C], the affinity of which is reduced approximately 300-fold upon the binding of a single molar equivalent of Cd(II) or Pb(II). The implications of these findings on the mechanism of metalloregulation are discussed.