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.

No abstract

Staphylococcus aureusA bout one-third of all proteins exploit specific metal ions to assist in macromolecular folding and͞or function at the active site of metalloenzymes (1). All cells restrict the number of bioavailable metal atoms to avoid any excess that would otherwise compete with native metal ion sites that do not support biological activity (2). Essentially all cell types contain intracellular metal sensors that detect surplus metal ions and control the expression of genes encoding proteins that expel or sequester the extra ions (3). For some metals and some cell types, a complementary set of sensors detect deficiency and regulate genes encoding proteins that acquire more of the required ions (4, 5). It is currently poorly understood how such metal-sensing metalloregulators accurately discriminate between various metal ions.SmtB͞ArsR-family regulators are ubiquitous in bacterial genomes and bind to the operator͞promoter (O͞P) regions of gene(s) encoding proteins involved in metal export or sequestration, repressing transcription (for a review, see ref. 6). As the concentration of metal ion increases, the effector-binding sites of the regulators become occupied eliciting a conformational change that weakens the affinity for the O͞P region, allowing transcription to proceed. Members of the SmtB͞ArsR family include: As(III), Sb(III), Bi(III)-responsive ArsR (7), Zn(II)-responsive SmtB (8), Cd(II), Pb(II), Bi(III)-responsive CadC (9-11), Zn(II)-responsive ZiaR (12), Co(II), Zn(II)-responsive CzrA (13,14), and, most recently, Ni(II), Co(II)-responsive NmtR (15).Comparative structural and spectroscopic studies of six SmtB͞ ArsR family members reveal that individual members are characterized by one or both of two structurally distinct metal coordination sites (6, 11, 15-20). These two metal sites are designated ␣3N (or ␣3) and ␣5 (or ␣5C), named for the location of the metal-binding ligands within the known or predicted secondary structure of individual family members. The coordination environment and precise ligand set of the ␣3, ␣3N, and͞or ␣5, ␣5C sites in the different SmtB͞ArsR proteins differ and are presumed to contribute toward metal selectivity. A sequence comparison for proteins discussed herein is shown in Fig. 1 and highlights these sites.Here we report insights gained from the study of two additional family members, Staphylococcus aureus CzrA and Mycobacterium tuberculosis NmtR. CzrA and NmtR share 30% sequence identity and a high degree of similarity (60%) yet respond to distinct but partially overlapping metal profiles in vivo. S. aureus CzrA is a Co(II)͞Zn(II)-specific sensor that regulates the expression of the czr operon, which encodes a Co(II)͞ Zn(II)-facilitated pump, CzrB, that effluxes metal out of the cell (13, 14). Electromobility-shift assays and in vivo expression studies indicate that Zn(II) is the strongest inducer of CzrA regulation, with Co(II) also capable of regulation but only at higher concentrations than Zn(II). Other metals, including Ni(II), have little to no effect on derep...

NmtR from Mycobacterium tuberculosis is a new member of the ArsR-SmtB family of metal sensor transcriptional repressors. NmtR binds to the operator-promoter of a gene encoding a P 1 type ATPase (NmtA), repressing transcription in vivo except in medium supplemented with nickel or, to some extent, cobalt. In a cyanobacterial host, Synechococcus PCC 7942 strain R2-PIM8(smt), NmtR-mediated repression is alleviated by cobalt but not nickel or zinc addition, while the related sensor SmtB responds exclusively to zinc. Quantification of the number of atoms of nickel per cell shows that NmtR nickel sensitivity correlates with cytosolic nickel contents. Differential metal discrimination in a common cytosol by SmtB (zinc) and NmtR (cobalt) is not simply explained by affinities at equilibrium; although NmtR does bind nickel substantially more tightly than SmtB, it has a higher affinity for zinc than for cobalt and binds cobalt more weakly than SmtB. SmtB is known to bind and sense zinc at interhelical four-coordinate, tetrahedral sites across the C-terminal ␣5 helices, while absorption spectroscopy of Co(II)-and Ni(II)-substituted NmtR reveals five-and six-coordinate metal complexes. Sitedirected mutagenesis identifies six potential cobalt/ nickel ligands that are obligatory for inducer recognition but not repression by NmtR, four of which (Asp 91 , His 93 , His 104 , His 107 ) align with ␣5 ligands of SmtB with two additional His provided by a carboxyl-terminal "extension" (designated ␣5C). Gel retardation assays reveal that zinc does not allosterically regulate NmtR-DNA binding at concentrations where lower affinity cobalt does. These data suggest that two additional ligands form hexacoordinate metal complexes and are crucial for driving allosteric regulation of DNA binding by NmtR, thereby allowing NmtR to preferentially sense metals that favor higher coordination numbers relative to SmtB.Cells contain regulatory proteins to detect and respond to deficiency or excess of essential metals to maintain sufficient atoms to satisfy the requirements of metalloproteins while avoiding toxicity (1). The ArsR-SmtB family of transcriptional repressors associate with the promoters of genes encoding proteins involved in the efflux and/or sequestration of excess metal (2). De-repression occurs when the repressors bind metal effectors coincident with the number of atoms exceeding an optimal cell quota. SmtB-mediated repression is alleviated by Zn(II) (3), ZiaR by Zn(II) (4), ArsR by As(III), Sb(III), and Bi(III) (5), CadC by Cd(II), Pb(II) and Bi(III) (6 -8), and CzrA by Co(II) and Zn(II) (9, 10). Clearly these sensors discriminate between different metals in vivo, but the factors dictating which inorganic elements elicit responses remain to be defined.There is rich literature describing metal coordination by numerous small molecules in vitro and established theories cataloguing the factors likely to influence metal selectivity in vivo (11). The ligand environments of metal ions are also known in a vast array of metalloenzymes (12). The...

Metal ion homeostasis in prokaryotes is maintained by metal-responsive transcriptional regulatory proteins that regulate the transcription of genes encoding proteins responsible for metal detoxification, sequestration, efflux and uptake. These metalloregulatory, or metal sensor proteins, bind a wide range of specific metal ions directly; this in turn, allosterically regulates (enhances or decreases) operator/promoter binding affinity or promoter structure. Recent structural studies reveal five distinct families of metal sensor proteins. The MerR and ArsR/SmtB families regulate the expression of genes required for metal ion detoxification, efflux and sequestration; here, metal binding leads to activation (MerR) or derepression (ArsR/SmtB) of the resistance operon. In contrast, the DtxR, Fur, and NikR families regulate genes encoding proteins involved in metal ion uptake; in these cases, the metal ion functions as a co-repressor in turning off uptake genes under metal-replete conditions. Inspection of the structures of representative members from each metal sensor family reveals several common characteristics: (1) they function as homo-oligomers (either dimers or tetramers); (2) metal-binding ligands are found at subunit interfaces, with ligands derived from more than one protomer; this likely helps drive quaternary structural changes that mediate allosteric coupling between the metal and DNA binding sites; and (3) the primary determinant of metal ion selectivity within each protein family is dictated by the coordination geometry of the metal chelate, with trends consistent with expectations from fundamental inorganic chemistry. This review highlights recent efforts to elucidate the structure of metal sensing chelates and the molecular mechanisms of allosteric coupling in metal sensor proteins.