Ni(II) Sensing by RcnR Does Not Require an FrmR-Like Intersubunit Linkage

Abstract

E. coli RcnR (resistance to cobalt and nickel regulator) is a homotetrameric DNA binding protein that regulates the expression of a Ni(II) and Co(II) exporter (RcnAB) by derepressing expression of rcnA and rcnB in response to binding Co(II) or Ni(II). Prior studies have shown that the cognate metal ions, Ni(II) and Co(II), bind in six-coordinate sites at subunit interfaces and are distinguished from noncognate metals (Cu(I), Cu(II), and Zn(II)) by coordination number and ligand selection. In analogy with FrmR, a formaldehyde-responsive transcriptional regulator in the RcnR/CsoR family, the interfacial site allows the metal ions to "cross-link" the N-terminal domain of one subunit with the invariant Cys35 residue in another, which has been deemed to be key to mediating the allosteric response of the tetrameric protein to metal binding. Through the use of mutagenesis to disconnect one subunit from the metal-mediated cross-link, X-ray absorption spectroscopy (XAS) as a structural probe, LacZ reporter assays, and metal binding studies using isothermal titration calorimetry (ITC), the work presented here shows that neither the interfacial binding site nor the coordination number of Ni(II) is important to the allosteric response to binding of this cognate metal ion. The opposite is found for the other cognate metal ion, Co(II), with respect to the interfacial binding site, suggesting that the molecular mechanisms for transcriptional regulation by the two ions are distinct. The metal binding studies reveal that tight metal binding is maintained in the variant. XAS is further used to demonstrate that His33 is not a ligand for Co(II), Ni(II), or Zn(II) in WT-RcnR. The results are discussed in the context of the overall understanding of the molecular mechanisms of metallosensors.

Allosteric control of metal-responsive transcriptional regulators in bacteria

“…Altogether, the results suggest that re-organization upon metal binding would impact key nonspecific electrostatic interactions with the DNA. Another recent study demonstrated that the molecular mechanisms for responding to Co(II) and Ni(II) are distinct, because coordination of Co(II)-but not Ni(II)-forms an inter-subunit linkage to drive allostery in a manner similar to that of formaldehyde-responsive FrmR from the same family (91,92). The response to Ni(II) is still being investigated but does not appear to depend on its coordination geometry (91).…”

Direct Detection of the Labile Nickel Pool in Escherichia coli: New Perspectives on Labile Metal Pools

“…Nickel homeostasis in E. coli involves two DNA-binding metalloregulatory proteins, NikR and RcnR; NikR controls Ni import, whereas RcnR controls Ni export; both bind aqueous Ni ions with nM affinities. , The K d for Ni binding to the InrS metalloregulator in cyanobacteria is in the pM range, again determined using aqueous nickel as a titrant . The proper functioning of these metalloregulators in Ni homeostasis is thought to require that the concentration of the LNiP be in the same nM or pM range, far less than one Ni atom per cell.…”

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).…”