The SmtB/ArsR family of metalloregulatory transcriptional repressors: structural insights into prokaryotic metal resistance

Abstract

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.

Metal Binding Studies and EPR Spectroscopy of the Manganese Transport Regulator MntR

“…Fura-2 was selected for initial experiments because its affinities for Mn 2+ , Fe 2+ , Co 2+ , and Cd 2+ ions are known and are in a range (picomolar to nanomolar) observed for many metalloregulatory proteins (40). Surprisingly, in our competition experiments between Fura-2 and MntR, where MntR was present in a 10-fold excess over Fura-2, MntR proved unable to compete for the metal ions Mn 2+ , Co 2+ , and Cd 2+ .…”

“…Both dyes are frequently employed as ratiometric Ca 2+ /Mg 2+ chelators but can also bind transition metal ions with related spectral changes. The affinities of Fura-2 for several transition metal ions has been determined (20,32-34), while for Mag-fura-2 only the binding constant with Zn 2+ has been reported (35).Fura-2 was selected for initial experiments because its affinities for Mn 2+ , Fe 2+ , Co 2+ , and Cd 2+ ions are known and are in a range (picomolar to nanomolar) observed for many metalloregulatory proteins (40). Surprisingly, in our competition experiments between Fura-2 and MntR, where MntR was present in a 10-fold excess over Fura-2, MntR proved unable to compete for the metal ions Mn 2+ , Co 2+ , and Cd 2+ .…”

“…Ni 2+ may also fail to form a dinuclear site, or the geometry of such a site with this metal ion may be significantly different, which again fails to produce the requisite allosteric change in MntR. Overall, we would concur with the hypothesis of Glasfeld et al (15) that the geometry and stoichiometry (dinuclear) of metal binding to MntR is more significant to the mechanism of activation than metal ion affinity.It is interesting that Fur and DtxR family members exhibit metal affinities generally much weaker than members of MerR and ArsR/SmtB families, the latter of which bind cognate ions in the 10 -9 to 10 -21 M range (8,37,40,(52)(53)(54)(55)(56)(57) (1 mM) and 40 times higher than that of Cd 2+ (0.5 mM) (63). Furthermore, in several Bacillus species, the minimal Mn 2+ concentration required for normal vegetative growth is ∼10 -7 M, 10 -6 -10 -3 M for production of secondary metabolites, and 10 -4 -10 -3 M for cultural longevity (64).…”

Transcription factors Rv0081 and Rv3334 connect the early and the enduring hypoxic response of Mycobacterium tuberculosis

“…Members of this family typically function as transcriptional repressors, responding to divalent and multivalent heavy metal ions and controlling the expression of genes involved in metal uptake, efflux, sequestration, or detoxification [40]. In the presence of heavy metal ions, their DNA binding activity are inhibited, allowing derepression of target genes [40].…”

Structural analysis of the transcriptional regulator homolog protein from Pyrococcus horikoshii OT3

“…Although the functions in these organisms are still unknown, a Pfam 2 database search revealed that PH1061 has a helix-turnhelix (HTH) motif ("HTH_5 motif" in the Pfam database), which may suggest that it is a DNA-binding protein like members of the ArsR family. 3 ArsR is a transcriptional repressor of the arsenic resistance operon in Escherichia coli, and other members of this family involve zinc-sensing transcriptional repressors, such as CzrA 4 from Staphylococcus aureus and SmtB 5 from Synechococcus pcc7942. The primary sequence similarities to PH1061 are 20%, 21%, and 6.6% with ArsR, CzrA, and SmtB, respectively.…”