Structural elements of metal selectivity in metal sensor proteins

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...

Solution structure of a paradigm ArsR family zinc sensor in the DNA-bound state

“…As a result, metal binding drives the DNA-bound CzrA off the operator while inhibiting subsequent binding of Zn(II)-bound CzrA to the DNA, with both scenarios leading to reduced operator-promoter occupancy and transcriptional derepression in vivo (Fig. S5) These quaternary structural transitions may be facilitated by the relatively weak dimer interface (31), which would allow the protomers to readily reorient themselves to maximize the complementarity of the protein-ligand [Zn(II) or DNA] interface in each conformational allosteric ''end'' state, P⅐D and P⅐Zn 2 .…”

“…We have focused much of our efforts on the Zn(II)/Co(II) sensor S. aureus CzrA as a paradigm system for understanding allosteric regulation of ArsR/SmtB family repressors by metal ions (10). Previous work with CzrA and the homologous Zn(II) sensor from Synechococcus, SmtB, has investigated in detail the free repressor dimer (denoted P) and the allosterically inhibited metal-bound form (P⅐Zn 2 ) (8,9,20,31). Here, we present the solution structure of the DNA operator-bound form of S. aureus CzrA and define key determinants of the protein-DNA interface.…”

“…Recombinant CzrA was expressed in E. coli BL21(DE3) cells at 37°C and purified as described previously (31) . ) were added to the culture 1 h before induction of protein expression with IPTG (at an A600 of ϳ0.6) (36).…”

“…4B using a fluorescein-labeled 28-bp CzrO DNA duplex (see Methods). The parameters reflect the results from two or three independent titrations fitted to a dimer-linkage model with K dimer fixed to the wild-type CzrA value of 1.7 ϫ 10 5 M Ϫ1 under these solution conditions (31). *The numbers in parentheses are the standard error on the fitted r i value(s) around the last significant figure; if not shown, this number is Ͻ1.…”

“…*The numbers in parentheses are the standard error on the fitted r i value(s) around the last significant figure; if not shown, this number is Ͻ1. Conditions: 10 mM Hepes, 0.4 M NaCl, pH 7.0, 25.0 (Ϯ 0.1)°C (9,31). † Fixed to the value that reflects the ⌬r (0.018) from the wild-type CzrA titration because saturation was not obtained for these mutant CzrAs.…”