Metal-dependent allosteric activation and inhibition on the same molecular scaffold: the copper sensor CopY from Streptococcus pneumoniae† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc04396a

• Published in: Chemical science (Cambridge) 2017-11, Vol.9 (1), p.105-118
• Main Authors: Glauninger, Hendrik, Zhang, Yifan, Higgins, Khadine A., Jacobs, Alexander D., Martin, Julia E., Fu, Yue, Coyne, 3rd, H. Jerome, Bruce, Kevin E., Maroney, Michael J., Clemmer, David E., Capdevila, Daiana A., Giedroc, David P.
• Format: Article
• Language: English
• Subjects: Chemistry
• ISSN: 2041-6520; 2041-6539
• DOI: 10.1039/c7sc04396a

The dynamics and marginal stability of CopY enable allosteric activation of DNA binding by Zn( ii ) and inhibition by Cu( i ). 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.