Ni(II) Sensing by RcnR Does Not Require an FrmR-Like Intersubunit Linkage Hsin-Ting Huang † and Michael J. Maroney* ,†,‡ † Department of Chemistry and ‡ Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003, United States * S Supporting Information 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. ■ INTRODUCTION Organisms have developed systems to maintain metal ion homeostasis in response to changing metal ion availability. 1,2 These systems rely on proteins, collectively referred to here as metal trafficking proteins, to acquire metals, maintain their availability within optimum levels, and provide mechanisms for metal ion incorporation in metalloenzymes. 2 Biological responses to specific metals (cognate metals) are a necessary feature of such systems, and a number of mechanisms for metal discrimination by trafficking proteins have been elucidated, 3-6 including those based on metal coordination number/ geometry. 4,7-9 In the case of bacterial transition metal ion homeostasis, strategies employing specific metal uptake and export proteins that are regulated by metal-responsive transcriptional regulators are commonly employed. 10-15 These metallosensors are multisubunit proteins that typically bind metals at a subunit interface. 4,14,16,17 The nickel trafficking pathway in E. coli supplies Ni(II) for the maturation of hydrogenases under anaerobic condi- tions 18,19 and includes the nickel-specific importer, Ni- kABCDE; 20-24 metallochaperones; accessory proteins for delivering Ni(II) to the target enzymes, 2 HypA, 25, 26 HypB, 27,28 and SlyD; 29,30 and exporter proteins, RcnAB (resistance to cobalt and nickel). 31-33 Two transcriptional regulators, NikR (nickel-responsive regulator) 34 and RcnR, 35,36 regulate the expression of NikABCDE and RcnAB, respec- tively, in response to binding Ni(II) ions. Typically, the DNA affinity of the proteins is altered in response to binding the regulated metal ion (cognate metal ion), and this is the case for NikR, whose affinity for DNA is enhanced by binding Ni(II). 37,38 In the case of RcnR, the protein responds to both Ni(II) and Co(II) ions by decreasing the affinity for DNA. 13,35,39 To date, there is no known cobalt trafficking system in E. coli, likely due to its inability to synthesize B12/ cobalamins 40,41 and the lack of any confirmed cobalt- containing enzymes, 42,43 and thus it is generally assumed that RcnR plays a dual role in maintaining Ni(II) homeostasis and in detoxification of Co(II) ions in E. coli. E. coli RcnR is a 40 kDa α-helical tetrameric transcriptional repressor that is a founding member of the RcnR/CsoR family Special Issue: Metals in Biology: From Metallomics to Trafficking Received: April 15, 2019 Forum Article pubs.acs.org/IC Cite This: Inorg. Chem. XXXX, XXX, XXX-XXX © XXXX American Chemical Society A DOI: 10.1021/acs.inorgchem.9b01096 Inorg. Chem. XXXX, XXX, XXX-XXX Downloaded by UNIV OF SOUTHERN INDIANA at 22:20:56:614 on June 17, 2019 from https://pubs.acs.org/doi/10.1021/acs.inorgchem.9b01096.