An atypical linear Cu(I)-S₂ center constitutes the high-affinity metal-sensing site in the CueR metalloregulatory protein

CueR is a copper-responsive genetic switch that regulates transcription of genes encoding the primary copper-exporting system in E. coli, CopA and CueO. Although a member of the MerR family of regulatory proteins, CueR has four cysteines in an array that is distinct from those in Hg(II)-sensing MerR or in Zn-sensing ZntR. A recent crystal structure showed one copper atom in CueR bound to cysteines Cys112 and Cys120 in a linear CuS₂ structure, but left open the questions of whether the other half of the CueR dimer has the same structure, and of whether these structures depend on the two additional C-terminal cysteines. Metal binding, transcription runoff, and cysteine modification studies show that only Cys112 and Cys 120 are necessary and sufficient to make the transcriptionally active Cu-sensing site. X-ray absorption spectroscopy shows that this site binds Cu(I) in a strictly linear CuS₂ site in solution, a structure that is rarely observed in copper proteins. This structure does not depend either on additional metal loading or upon the presence of additional C-terminal cysteine ligands and is well suited for an ultrasensitve receptor site that discriminates against the binding of other metal ions.