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Durham University

Department of Biosciences

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Publication details for Dr Peter Chivers

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

Author(s) from Durham

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.