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.