title: Generating a Metal-responsive Transcriptional Regulator to Test What Confers Metal Sensing in Cells.
abstract: FrmR from Salmonella enterica serovar typhimurium (a CsoR/RcnR-like transcriptional de-repressor) is shown to repress the frmRA operator-promoter, and repression is alleviated by formaldehyde but not manganese, iron, cobalt, nickel, copper, or Zn(II) within cells. In contrast, repression by a mutant FrmRE64H (which gains an RcnR metal ligand) is alleviated by cobalt and Zn(II). Unexpectedly, FrmR was found to already bind Co(II), Zn(II), and Cu(I), and moreover metals, as well as formaldehyde, trigger an allosteric response that weakens DNA affinity. However, the sensory metal sites of the cells' endogenous metal sensors (RcnR, ZntR, Zur, and CueR) are all tighter than FrmR for their cognate metals. Furthermore, the endogenous metal sensors are shown to out-compete FrmR. The metal-sensing FrmRE64H mutant has tighter metal affinities than FrmR by approximately 1 order of magnitude. Gain of cobalt sensing by FrmRE64H remains enigmatic because the cobalt affinity of FrmRE64H is substantially weaker than that of the endogenous cobalt sensor. Cobalt sensing requires glutathione, which may assist cobalt access, conferring a kinetic advantage. For Zn(II), the metal affinity of FrmRE64H approaches the metal affinities of cognate Zn(II) sensors. Counter-intuitively, the allosteric coupling free energy for Zn(II) is smaller in metal-sensing FrmRE64H compared with nonsensing FrmR. By determining the copies of FrmR and FrmRE64H tetramers per cell, then estimating promoter occupancy as a function of intracellular Zn(II) concentration, we show how a modest tightening of Zn(II) affinity, plus weakened DNA affinity of the apoprotein, conspires to make the relative properties of FrmRE64H (compared with ZntR and Zur) sufficient to sense Zn(II) inside cells.
authors.affiliation: From the School of Biological and Biomedical Sciences and Department of Chemistry, Durham University, Durham DH1 3LE, United Kingdom.
authors.forename: Deenah
authors.initials: D
authors.lastname: Osman
authors.affiliation: From the School of Biological and Biomedical Sciences and Department of Chemistry, Durham University, Durham DH1 3LE, United Kingdom.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Piergentili
authors.affiliation: Procter and Gamble, Mason Business Centre, Cincinnati, Ohio 45040, and.
authors.forename: Junjun
authors.initials: J
authors.lastname: Chen
authors.affiliation: From the School of Biological and Biomedical Sciences and Department of Chemistry, Durham University, Durham DH1 3LE, United Kingdom.
authors.forename: Buddhapriya
authors.initials: B
authors.lastname: Chakrabarti
authors.affiliation: From the School of Biological and Biomedical Sciences and Department of Chemistry, Durham University, Durham DH1 3LE, United Kingdom.
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Foster
authors.affiliation: Life Sciences Open Innovation, London Innovation Centre, Procter and Gamble Technical Centres, Ltd., Egham TW20 9NW, United Kingdom.
authors.forename: Elena
authors.initials: E
authors.lastname: Lurie-Luke
authors.affiliation: Procter and Gamble, Mason Business Centre, Cincinnati, Ohio 45040, and.
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Huggins
authors.affiliation: From the School of Biological and Biomedical Sciences and Department of Chemistry, Durham University, Durham DH1 3LE, United Kingdom, nigel.robinson@durham.ac.uk.
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Robinson
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002413:Cations, Divalent; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005557:Formaldehyde / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009532:Nickel / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D014158:Transcription, Genetic; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D011994:Recombinant Proteins; D012097:Repressor Proteins; D005557:Formaldehyde; D003035:Cobalt; D008345:Manganese; D003300:Copper; D009532:Nickel; D015032:Zinc
keywords: FrmR; RcnR-CsoR; Salmonella enterica; cobalt; copper; metal; metal homeostasis; metal-sensor; metalloprotein; zinc