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

Resistance to copper (Cu) toxicity in the respiratory pathogen is regulated by the Cu-specific metallosensor CopY. CopY is structurally related to the antibiotic-resistance regulatory proteins MecI and BlaI from , but is otherwise poorly characterized. Here we employ a multi-pronged experimental str...

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Published in:Chemical science (Cambridge) 2018-01, Vol.9 (1), p.105-118
Main Authors: Glauninger, Hendrik, Zhang, Yifan, Higgins, Khadine A, Jacobs, Alexander D, Martin, Julia E, Fu, Yue, Coyne Rd, H Jerome, Bruce, Kevin E, Maroney, Michael J, Clemmer, David E, Capdevila, Daiana A, Giedroc, David P
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cited_by cdi_FETCH-LOGICAL-c474t-afc2892370aec54075c3b831d65d1abfeca303761ec9ec023dfeb759b96dcc2f3
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container_title Chemical science (Cambridge)
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creator Glauninger, Hendrik
Zhang, Yifan
Higgins, Khadine A
Jacobs, Alexander D
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Bruce, Kevin E
Maroney, Michael J
Clemmer, David E
Capdevila, Daiana A
Giedroc, David P
description Resistance to copper (Cu) toxicity in the respiratory pathogen is regulated by the Cu-specific metallosensor CopY. CopY is structurally related to the antibiotic-resistance regulatory proteins MecI and BlaI from , but is otherwise poorly characterized. Here we employ a multi-pronged experimental strategy to define the CopY coordination chemistry and the unique mechanism of allosteric activation by Zn(ii) and allosteric inhibition by Cu(i) of promoter DNA binding. We show that Zn(ii) is coordinated by a subunit-bridging 3S 1H 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 and in cells. In contrast, Cu(i) employs the same Cys pair to form a subunit-bridging, kinetically stable, multi-metallic Cu·S cluster ( ≈ 10 M ) 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 ligand exchange-catalyzed metal substitution, leading to expression of Cu resistance genes. Mechanistic parallels to antibiotic resistance repressors MecI and BlaI are discussed.
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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 and in cells. In contrast, Cu(i) employs the same Cys pair to form a subunit-bridging, kinetically stable, multi-metallic Cu·S cluster ( ≈ 10 M ) 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 ligand exchange-catalyzed metal substitution, leading to expression of Cu resistance genes. 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We show that Zn(ii) is coordinated by a subunit-bridging 3S 1H 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 and in cells. In contrast, Cu(i) employs the same Cys pair to form a subunit-bridging, kinetically stable, multi-metallic Cu·S cluster ( ≈ 10 M ) that induces oligomerization beyond the dimer as revealed by SAXS, rPA-MS and NMR spectroscopy, leading to inhibition of DNA binding. 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subjects Activation
Alkylation
Antibiotics
Binding
Chemistry
Copper
Deoxyribonucleic acid
DNA
Gene expression
Ionic mobility
Mass spectrometry
NMR spectroscopy
Oligomerization
Proteins
Small angle X ray scattering
Streptococcus infections
Toxicity
title Metal-dependent allosteric activation and inhibition on the same molecular scaffold: the copper sensor CopY from Streptococcus pneumoniae
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