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

Intracellular CuI is controlled by the transcriptional regulator CueR, which effectively discriminates between monovalent and divalent metal ions. It is intriguing that HgII does not activate transcription, as bis-thiolate metal sites exhibit high affinity for HgII . Here the binding of HgII to CueR and a truncated variant, ΔC7-CueR, without the last 7 amino acids at the C-terminus including a conserved CCHH motif is explored. ESI-MS demonstrates that up to two HgII bind to CueR, while ΔC7-CueR accommodates only one HgII . 199m Hg PAC and UV absorption spectroscopy indicate HgS2 structure at both the functional and the CCHH metal site. However, at sub-equimolar concentrations of HgII at pH 8.0, the metal binding site displays an equilibrium between HgS2 and HgS3 , involving cysteines from both sites. We hypothesize that the C-terminal CCHH motif provides auxiliary ligands that coordinate to HgII and thereby prevents activation of transcription.