Specificity of the Metalloregulator CueR for Monovalent Metal Ions: Possible Functional Role of a Coordinated Thiol?

Abstract

Metal ion responsive transcriptional regulators within the MerR family effectively discriminate between mono-and divalent metal ions. In this work we address the origin of the specificity of the CueR protein for monovalent metal ions. Several spectroscopic techniques were employed to study Ag I , Zn II , and Hg II binding to model systems encompassing the metal ion binding loop of CueR from E. coli and V. cholerae. In the presence of Ag I a conserved cysteine displays a pKa for deprotonation of the thiol close to physiological pH. This is unique to the monovalent metal ion. Quantum chemically optimized structures of the CueR metal site with Cys112 protonated demonstrate that the Ser77 backbone carbonyl oxygen is "pulled" towards the metal site. A common allosteric mechanism of the metalloregulatory members of the MerR family is proposed. For CueR the mechanism relies on a protonation switch of Cys112.

C‐terminal Cysteines of CueR Act as Auxiliary Metal Site Ligands upon HgII Binding—A Mechanism To Prevent Transcriptional Activation by Divalent Metal Ions?

“…Our previous studies on model peptides of the metal binding loop of CueR also showed that these fragments bind Cu I with a high affinity . However, according to model peptide studies and QM/MM calculations, Hg II ions may be coordinated even more efficiently. Moreover, Hg II is also able to bind to a CC sequence, and therefore coordination of Hg II ion by the CCHH motif is also highly probable.…”

“…199m Hg‐perturbed angular correlation (PAC) spectroscopy was used to elucidate the metal site structures and dynamics at the nanosecond timescale, see Figure and Supporting Information Figure S6. At pH 6.0 and Hg II :CueR of 0.2 and 1.0, the signals agree well with a HgS 2 coordination geometry, that is, coordination of Hg II by two cysteinates .…”

Short oligopeptides with three cysteine residues as models of sulphur-rich Cu(i)- and Hg(ii)-binding sites in proteins

“…The metal-binding features of the metalloregulator CueR and the metallochaperone Atx1, both encompassing 2 cysteines in their metal-binding domain, with different sequences between the Cys residues, were investigated via oligopeptide models of the relevant metal-binding loops. [27][28][29][30][31][32][33] A general finding was that the linear unstructured ligands, while displaying high affinity and interesting metal ion selectivity for Hg(II) or Cu(I) chelation, could not fully reproduce the efficiency of the modelled proteins. 28,31,33 Cyclisation of the linear Atx1 model, resulting in a more rigid skeleton with preoriented Cys-sidechains, was an important step forward leading to notably increased metal binding affinities.…”

“…[27][28][29][30][31][32][33] A general finding was that the linear unstructured ligands, while displaying high affinity and interesting metal ion selectivity for Hg(II) or Cu(I) chelation, could not fully reproduce the efficiency of the modelled proteins. 28,31,33 Cyclisation of the linear Atx1 model, resulting in a more rigid skeleton with preoriented Cys-sidechains, was an important step forward leading to notably increased metal binding affinities. 27,28 The more challenging imitation of the HgS 3 type metal coordination sites was successfully achieved by different approaches.…”

Advanced purification strategy for CueR, a cysteine containing copper(I) and DNA binding protein

“…Recently, the crystal structures of the metal ion free modified protein and the Ag I -bound form, both cocrystallized with DNA, have been published providing more insight into the influence of metal ion binding on the structure of the DNA [12]. Based on the combination of experimental studies with CueR model peptides and quantum chemical calculations we have proposed the participation of a protonated Cys thiol in the metal ion binding domain of CueR and the operation of a protonation switch in the mechanism of the protein [13]. However, the potential role of the protonation/deprotonation of Cys112 in the function of CueR has not been proved, yet.…”