title: Predicting metals sensed by ArsR-SmtB repressors: allosteric interference by a non-effector metal.
abstract: Many bacterial genomes encode multiple metal-sensing ArsR-SmtB transcriptional repressors. There is interest in understanding and predicting their metal specificities. Here we analyse two arsR-smtB genes, ydeT and yozA (now aseR and czrA) from Bacillus subtilis. Purified AseR and CzrA formed complexes in gel-retardation and fluorescence-anisotropy assays with fragments of promoters that were derepressed in DeltaaseR and DeltaczrA cells. Candidate (i) partly thiolate, alpha3-helix (for AseR) and (ii) tetrahedral, non-thiolate, alpha5-helix (for CzrA) metal binding sites were predicted then tested in vitro and/or in vivo. The precedents are for such sites to sense arsenite/antimonite (alpha3) and zinc (alpha5). This correlated with the respective metal inducers of AseR and CzrA repressed promoters in B. subtilis and matched the metals that impaired formation of protein-DNA complexes in vitro. The putative sensory sites of 1024 ArsR-SmtB homologues are reported. Although AseR did not sense zinc in vivo, it bound zinc in vitro exploiting alpha3 thiols, but AseR DNA binding was not impaired by zinc. If selectivity relies on discriminatory triggering of allostery not just selective metal binding, then tight non-effector metal complexes could theoretically inhibit metal sensing. AseR remained arsenite-sensitive in equimolar zinc, while CzrA remained zinc-sensitive in equimolar arsenite in vitro. However, cupric ions did not impair CzrA-DNA complex formation but did inhibit zinc-mediated allostery in vitro and prevent zinc binding. Access to copper must be controlled in vivo to avoid formation of cupric CzrA.
authors.affiliation: Cell and Molecular Biosciences, Medical School, University of Newcastle, Newcastle NE2 4HH, UK.
authors.forename: Duncan R
authors.initials: DR
authors.lastname: Harvie
authors.forename: Claudia
authors.initials: C
authors.lastname: Andreini
authors.forename: Gabriele
authors.initials: G
authors.lastname: Cavallaro
authors.forename: Wenmao
authors.initials: W
authors.lastname: Meng
authors.forename: Bernard A
authors.initials: BA
authors.lastname: Connolly
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Yoshida
authors.forename: Yasutaro
authors.initials: Y
authors.lastname: Fujita
authors.forename: Colin R
authors.initials: CR
authors.lastname: Harwood
authors.forename: David S
authors.initials: DS
authors.lastname: Radford
authors.forename: Stephen
authors.initials: S
authors.lastname: Tottey
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Cavet
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Robinson
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D018053:Arsenites / Q000378:metabolism / Q000494:pharmacology; D001412:Bacillus subtilis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D003300:Copper / Q000378:metabolism / Q000494:pharmacology; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008670:Metals / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology
chemical_list: D018053:Arsenites; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D008670:Metals; D012097:Repressor Proteins; D013438:Sulfhydryl Compounds; D015534:Trans-Activators; D003300:Copper; D015032:Zinc; C015001:arsenite