Spectroscopic Properties of the Metalloregulatory Cd(II) and Pb(II) Sites of <i>S. aureus</i> pI258 CadC

Busenlehner, Laura S.; Cosper, Nathaniel J.; Scott, Robert A.; Rosen, Barry P.; Wong, Marco D.; Giedroc, David P.

doi:10.1021/bi010006g

Cited by 89 publications

(199 citation statements)

References 41 publications

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“…The data reveal that both CzrA and NmtR are well described as dissociable homodimers under these conditions, with the magnitude of K dimer generally in the 10 5 M Ϫ1 range for both apo-CzrA and -NmtR. This value is comparable to that previously found for the apo-zinc sensor, SmtB, but Ϸ10-fold less than that for apo-CadC (19). The magnitude of K dimer in the presence of bound metal is consistently larger than that determined for the apoproteins, but the effect is relatively small (within a factor of two to three).…”

Section: Resultssupporting

confidence: 80%

“…NmtR, which discriminates toward Ni(II) and Co(II), also uses imidazoles. CadC and ArsR possess ␣3N and ␣3 metal sites that have all-cysteinyl coordination spheres that prefer softer Lewis acids, such as Cd(II), Pb(II), and Bi(III) (11,19,20). Interestingly, S. aureus pI258 CadC contains an intact ␣5 metal site that has been shown to bind Zn(II) and Co(II) directly; however, the binding of metal here is not regulatory for DNA binding in vitro (11) or in vivo (33).…”

Section: Discussionmentioning

confidence: 99%

“…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)(16)(17)(18)(19)(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.…”

mentioning

confidence: 99%

“…The partial specific volumes ( ) of CzrA and NmtR were calculated to be 0.740 ml͞g Ϫ1 and 0.736 ml͞g Ϫ1 , respectively, using the SEDNTERP program (22). Sedimentation equilibrium data were fit by using ORIGIN software (Microcal, Northampton, MA) to a single ideal species model and a self-association model of a single ideal species, assuming a monomer-dimer equilibrium characterized by the association constant K dimer , as described (19). The association constant was converted from absorbance to units of M Ϫ1 by using the calculated extinction coefficients 235 ϭ 22,543 M Ϫ1 ⅐cm Ϫ1 and 235 ϭ 25,246 M Ϫ1 ⅐cm Ϫ1 for CzrA and NmtR, respectively, and a path length l ϭ 1.2 cm.…”

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confidence: 99%

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Structural elements of metal selectivity in metal sensor proteins

Pennella

Shokes

Cosper

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultssupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Structural elements of metal selectivity in metal sensor proteins

Pennella

Shokes

Cosper

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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108

270

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“…Two distinct types of metal binding sites in a CadC dimer have been proposed (4). The type 1 metal binding site, also designated ␣3N, is a thiolate-rich site composed of Cys-7 and Cys-11 in the N terminus and Cys-58 and Cys-60 in the first helix of the DNA binding site (2,30). The type 2 metal binding site, also designated ␣5, is proposed to be an oxygen/nitrogen site at the dimer interface.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of the Staphylococcus aureus pI258 CadC Cd(II)/Pb(II)/Zn(II)-Responsive Repressor

et al. 2005

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The Staphylococcus aureus plasmid pI258 cadCA operon encodes a P-type ATPase, CadA, that confers resistance to the heavy metals Cd(II), Zn(II), and Pb(II). Expression of this heavy-metal efflux pump is regulated by CadC, a homodimeric repressor that dissociates from the cad operator/promoter upon binding of Cd(II), Pb(II), or Zn(II). CadC is a member of the ArsR/SmtB family of metalloregulatory proteins. Here we report the X-ray crystal structure of CadC at 1.9 Å resolution. The dimensions of the protein dimer are approximately 30 Å by 40 Å by 70 Å. Each monomer contains six ␣-helices and a three-stranded ␤-sheet. Helices 4 and 5 form a classic helix-turn-helix motif that is the putative DNA binding region. The ␣1 helix of one monomer crosses the dimer to approach the ␣4 helix of the other monomer, consistent with the previous proposal that these two regulatory metal binding sites for the inducer cadmium or lead are each formed by Cys-7 and Cys-11 from the N terminus of one monomer and Cys-58 and Cys-60 of the other monomer. Two nonregulatory metal binding sites containing zinc are formed between the two antiparallel ␣6 helices at the dimerization interface. This is the first reported three-dimensional structure of a member of the ArsR/SmtB family with regulatory metal binding sites at the DNA binding domain and the first structure of a transcription repressor that responds to the heavy metals Cd(II) and Pb(II).

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