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bioRxiv · 10.1101/2021.07.25.453692

Functional asymmetry and chemical reactivity of CsoR family persulfide sensors

Fakhoury, J.·Zhang, Y.·Edmonds, K. A.·Bringas, M.·Luebke, J.·Gonzalez-Gutierrez, G.·Capdevila, D. A.·Giedroc, D. P.
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Abstract

CstR is a persulfide-sensing member of the functionally diverse copper-sensitive operon repressor (CsoR) superfamily that regulates the bacterial response to hydrogen sulfide (H2S) and more oxidized reactive sulfur species (RSS) in Gram-positive pathogens. A cysteine thiol pair on CstR reacts with RSS to form a mixture of interprotomer di-, tri- and tetrasulfide crosslinks, which drives transcriptional derepression of CstR-regulated genes. In some bacteria, notably methicillin-resistant Staphylococcus aureus (MRSA), CstR and CsoR, a Cu(I)-sensor, exhibit no regulatory crosstalk in cells, despite maintaining an identical pair of cysteines. We report a sequence similarity network (SSN) analysis of the entire CsoR superfamily, together with the first crystallographic structure of a CstR protein and mass spectrometry-based kinetic profiling experiments to obtain new insights into the molecular basis of RSS specificity in CstRs. The more N-terminal cysteine is the attacking Cys in CstR and is far more nucleophilic than in a CsoR. This cysteine, C30 in SpCstR, is separated from the resolving thiol, C59, by an Asn55 wedge. Chemical reactivity experiments reveal a striking asymmetry of reactivity, preserved in all CstRs and with all oxidants tested; however, the distribution of crosslinked products varies markedly among CstRs. Substitution of N55 with Ala in SpCstR significantly impacts the distribution of species, despite adopting the same structure as the parent repressor. We show that CstRs react with hydrogen peroxide, a finding that contrasts sharply with other structurally distinct persulfide sensors from Gram-negative bacteria. This suggests that other factors may enhance the specificity and repressor activity of CstRs in cells.

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Fakhoury, J., Zhang, Y., Edmonds, K. A., Bringas, M., Luebke, J., Gonzalez-Gutierrez, G., Capdevila, D. A., Giedroc, D. P.. 2021-07-25. Functional asymmetry and chemical reactivity of CsoR family persulfide sensors. https://doi.org/10.1101/2021.07.25.453692

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