D. Osman
Generating a metal-responsive transcriptional regulator to test what confers metal-sensing in cells
Osman, D.; Piergentili, C.; Chen, J.; Chakrabarti, B.; Foster, A.; Lurie-Luke, E.; Huggins, T.; Robinson, N.
Authors
C. Piergentili
J. Chen
B. Chakrabarti
A. Foster
E. Lurie-Luke
T. Huggins
Professor Nigel Robinson nigel.robinson@durham.ac.uk
Professor
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.
Citation
Osman, D., Piergentili, C., Chen, J., Chakrabarti, B., Foster, A., Lurie-Luke, E., …Robinson, N. (2015). Generating a metal-responsive transcriptional regulator to test what confers metal-sensing in cells. Journal of Biological Chemistry, 290(32), 19806-19822. https://doi.org/10.1074/jbc.m115.663427
Journal Article Type | Article |
---|---|
Acceptance Date | Jun 24, 2015 |
Online Publication Date | Jun 24, 2015 |
Publication Date | Aug 7, 2015 |
Deposit Date | Jun 29, 2015 |
Publicly Available Date | Aug 20, 2015 |
Journal | Journal of Biological Chemistry |
Print ISSN | 0021-9258 |
Electronic ISSN | 1083-351X |
Publisher | American Society for Biochemistry and Molecular Biology |
Peer Reviewed | Peer Reviewed |
Volume | 290 |
Issue | 32 |
Pages | 19806-19822 |
DOI | https://doi.org/10.1074/jbc.m115.663427 |
Keywords | Copper, Metal, Metal homeostasis, Metalloprotein, Salmonella enterica, Zinc, FrmR, RcnR-CsoR, Cobalt, Metal-sensor. |
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Copyright Statement
Author's Choice—Final version free via Creative Commons CC-BY license.
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