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Glutamate Ligation in the Ni(II)- and Co(II)-Responsive Escherichia coli Transcriptional Regulator, RcnR

Carr, CE; Musiani, F; Huang, H-T; Chivers, PT; Ciurli, S; Maroney, MJ

Glutamate Ligation in the Ni(II)- and Co(II)-Responsive Escherichia coli Transcriptional Regulator, RcnR Thumbnail


Authors

CE Carr

F Musiani

H-T Huang

S Ciurli

MJ Maroney



Abstract

Escherichia coli RcnR (resistance to cobalt and nickel regulator, EcRcnR) is a metal-responsive repressor of the genes encoding the Ni(II) and Co(II) exporter proteins RcnAB by binding to PRcnAB. The DNA binding affinity is weakened when the cognate ions Ni(II) and Co(II) bind to EcRcnR in a six-coordinate site that features a (N/O)5S ligand donor-atom set in distinct sites: while both metal ions are bound by the N terminus, Cys35, and His64, Co(II) is additionally bound by His3. On the other hand, the noncognate Zn(II) and Cu(I) ions feature a lower coordination number, have a solvent-accessible binding site, and coordinate protein ligands that do not include the N-terminal amine. A molecular model of apo-EcRcnR suggested potential roles for Glu34 and Glu63 in binding Ni(II) and Co(II) to EcRcnR. The roles of Glu34 and Glu63 in metal binding, metal selectivity, and function were therefore investigated using a structure/function approach. X-ray absorption spectroscopy was used to assess the structural changes in the Ni(II), Co(II), and Zn(II) binding sites of Glu → Ala and Glu → Cys variants at both positions. The effect of these structural alterations on the regulation of PrcnA by EcRcnR in response to metal binding was explored using LacZ reporter assays. These combined studies indicate that while Glu63 is a ligand for both metal ions, Glu34 is a ligand for Co(II) but possibly not for Ni(II). The Glu34 variants affect the structure of the cognate metal sites, but they have no effect on the transcriptional response. In contrast, the Glu63 variants affect both the structure and transcriptional response, although they do not completely abolish the function of EcRcnR. The structure of the Zn(II) site is not significantly perturbed by any of the glutamic acid variations. The spectroscopic and functional data obtained on the mutants were used to calculate models of the metal-site structures of EcRcnR bound to Ni(II), Co(II), and Zn(II). The results are interpreted in terms of a switch mechanism, in which a subset of the metal-binding ligands is responsible for the allosteric response required for DNA release.

Journal Article Type Article
Acceptance Date May 3, 2017
Online Publication Date May 18, 2017
Publication Date May 18, 2017
Deposit Date Jul 17, 2017
Publicly Available Date May 18, 2018
Journal Inorganic Chemistry
Print ISSN 0020-1669
Electronic ISSN 1520-510X
Publisher American Chemical Society
Peer Reviewed Peer Reviewed
Volume 56
Issue 11
Pages 6459-6476
DOI https://doi.org/10.1021/acs.inorgchem.7b00527
Public URL https://durham-repository.worktribe.com/output/1353133
Publisher URL https://10.1021/acs.inorgchem.7b00527

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Copyright Statement
This document is the Accepted Manuscript version of a Published Work that appeared in final form in Inorganic chemistry copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://10.1021/acs.inorgchem.7b00527






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