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Co(II) and Ni(II) binding of the Escherichia coli transcriptional repressor RcnR orders its N-terminus, alters helix dynamics, and reduces DNA affinity

Huang, H-T.; Bobst, C.E.; Iwig, J.S.; Chivers, P.T.; Kaltashov, I.; Maroney, M.J.

Co(II) and Ni(II) binding of the Escherichia coli transcriptional repressor RcnR orders its N-terminus, alters helix dynamics, and reduces DNA affinity Thumbnail


Authors

H-T. Huang

C.E. Bobst

J.S. Iwig

I. Kaltashov

M.J. Maroney



Abstract

RcnR, a transcriptional regulator in Escherichia coli, derepresses the expression of the export proteins RcnAB upon binding Ni(II) or Co(II). Lack of structural information has precluded elucidation of the allosteric basis for the decreased DNA affinity in RcnR’s metal-bound states. Here, using hydrogen–deuterium exchange coupled with MS (HDX-MS), we probed the RcnR structure in the presence of DNA, the cognate metal ions Ni(II) and Co(II), or the noncognate metal ion Zn(II). We found that cognate metal binding altered the flexibility from the N-terminus through helix 1 and modulated the RcnR–DNA interaction. Apo-RcnR and RcnR–DNA complexes and the Zn(II)-RcnR complex exhibited similar 2H uptake kinetics, with fast-exchanging segments located in the N terminus, in helix 1 (residues 14–24), and at the C terminus. The largest difference in 2H incorporation between apo- and Ni(II)- and Co(II)-bound RcnR was observed in helix 1, which contains the N-terminus and His3, and has been associated with cognate metal binding. 2H uptake in helix 1 was suppressed in the Ni(II)- and Co(II)-bound RcnR complexes, in particular in the peptide corresponding to residues 14–24, containing Arg-14 and Lys-17. Substitution of these two residues drastically affected DNA-binding affinity, resulting in rcnA expression in the absence of metal. Our results suggest that cognate metal binding to RcnR orders its N-terminus, decreases helix 1 flexibility, and induces conformational changes that restrict DNA interactions with the positively charged residues Arg14 and Lys17. These metal-induced alterations decrease RcnR–DNA binding affinity, leading to rcnAB expression.

Citation

Huang, H., Bobst, C., Iwig, J., Chivers, P., Kaltashov, I., & Maroney, M. (2018). Co(II) and Ni(II) binding of the Escherichia coli transcriptional repressor RcnR orders its N-terminus, alters helix dynamics, and reduces DNA affinity. Journal of Biological Chemistry, 293(1), 324-332. https://doi.org/10.1074/jbc.ra117.000398

Journal Article Type Article
Acceptance Date Nov 17, 2017
Online Publication Date Nov 17, 2017
Publication Date Jan 5, 2018
Deposit Date Nov 22, 2017
Publicly Available Date Nov 22, 2017
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 293
Issue 1
Pages 324-332
DOI https://doi.org/10.1074/jbc.ra117.000398

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Copyright Statement
This research was originally published in Journal of Biological Chemistry. Huang, H-T., Bobst, C. E., Iwig, J. S., Chivers, P. T., Kaltashov, I. & Maroney, M. J. (2017). Co(II) and Ni(II) binding of the Escherichia coli transcriptional repressor RcnR orders its N-terminus, alters helix dynamics, and reduces DNA affinity. Journal of Biological Chemistry. © 2017, the American Society for Biochemistry and Molecular Biology





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