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Resistance to copper (Cu) toxicity in the respiratory pathogen Streptococcus pneumoniae is regulated by the Cu-specific metallosensor CopY. CopY is structurally related to the antibiotic-resistance regulatory proteins MecI and BlaI from Staphylococcus aureus , but is otherwise poorly characterized. Here we employ a multi-pronged experimental strategy to define the Spn CopY coordination chemistry and the unique mechanism of allosteric activation by Zn(ii) and allosteric inhibition by Cu(i) of cop promoter DNA binding. We show that Zn(ii) is coordinated by a subunit-bridging 3S 1H 2 O complex formed by the same residues that coordinate Cu(i), as determined by X-ray absorption spectroscopy and ratiometric pulsed alkylation-mass spectrometry (rPA-MS). Apo- and Zn-bound CopY are homodimers by small angle X-ray scattering (SAXS); however, Zn stabilizes the dimer, narrows the conformational ensemble of the apo-state as revealed by ion mobility-mass spectroscopy (IM-MS), and activates DNA binding in vitro and in cells. In contrast, Cu(i) employs the same Cys pair to form a subunit-bridging, kinetically stable, multi-metallic Cu·S cluster ( K  Cu ≈ 10 16 M -1 ) that induces oligomerization beyond the dimer as revealed by SAXS, rPA-MS and NMR spectroscopy, leading to inhibition of DNA binding. These studies suggest that CopY employs conformational selection to drive Zn-activation of DNA binding, and a novel Cu(i)-mediated assembly mechanism that dissociates CopY from the DNA via ligand exchange-catalyzed metal substitution, leading to expression of Cu resistance genes. Mechanistic parallels to antibiotic resistance repressors MecI and BlaI are discussed.

Metal-dependent allosteric activation and inhibition on the same molecular scaffold: the copper sensor CopY from Streptococcus pneumoniae