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

Abstract

RcnR, a transcriptional regulator in , 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 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 similarH 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 H incorporation between apo- and Ni(II)- and Co(II)-bound RcnR was observed in helix 1, which contains the N terminus and His-3, and has been associated with cognate metal binding.H 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 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 Arg-14 and Lys-17. These metal-induced alterations decrease RcnR-DNA binding affinity, leading to expression.

Molecular Modelling of the Ni(II)-Responsive Synechocystis PCC 6803 Transcriptional Regulator InrS in the Metal Bound Form

“…Thus, a quaternary structure rearrangement is necessary for metal binding upon shifting from the apo-to the holo-form of the protein. This result agrees with what has been proposed for other members of the RcnR/CsoR family [8,9,12,20,22,35]. The cross-link between two monomer chains occurring at the dimer interface appears to stabilize a specific protein conformation, interfering with the capability of SyInrS to bind the DNA.…”

Nickel Metalloregulators and Chaperones

“…Mass spectrometry, NMR, metal binding studies, and modeling studies suggest that cognate metal binding remodels the shape of the tetramer in a way that does not allow for the relevant positively charged residues to interact with the DNA [36,74,75]. Hydrogen-deuterium exchange studies coupled with mass spectrometry (HDX-MS) was used to probe the RcnR structure in the presence of DNA, Ni(II), Co(II), or Zn(II) [76]. These experiments determined that Ni(II) and Co(II) binding to RcnR orders the N-terminus, decreases the flexibility of helix 1, and induces conformational changes in the protein that restricts DNA interactions with Arg14 and Lys17 [76].…”

“…Hydrogen-deuterium exchange studies coupled with mass spectrometry (HDX-MS) was used to probe the RcnR structure in the presence of DNA, Ni(II), Co(II), or Zn(II) [76]. These experiments determined that Ni(II) and Co(II) binding to RcnR orders the N-terminus, decreases the flexibility of helix 1, and induces conformational changes in the protein that restricts DNA interactions with Arg14 and Lys17 [76].…”

Complexation of the nickel and cobalt transcriptional regulator RcnR with DNA

“…Biochemical studies of several members of this family, including CsoR, a copper regulator (Chang et al, 2014;Liu et al, 2007), RcnR, a nickel/cobalt regulator (Iwig et al, 2008;Higgins et al, 2012;Carr et al, 2017;Huang et al, 2018;Huang & Maroney, 2019), and FrmR, a formaldehyde regulator (Osman et al, 2016;Denby et al, 2016), have previously been conducted. Crystal structures of CsoR and FrmR are available in both apo and effector-bound forms (Chang et al, 2014;Dwarakanath et al, 2012;Liu et al, 2007;Porto et al, 2015;Sakamoto et al, 2010;Denby et al, 2016;Osman et al, 2016).…”

“…# 2020 International Union of Crystallography exchange identified Arg14 and Lys17 of the protein as key to DNA binding (Huang et al, 2018).…”