Abstract
Staphylococcus aureus CstR (CsoR-like sulfur transferase repressor) is a member of the CsoR family of transition metal sensing metalloregulatory proteins. Unlike CsoR, CstR does not form a stable complex with transition metals but instead reacts with sulfite to form a mixture of di- and trisulfide species, CstR2(RS-SR') and CstR2(RS-S-SR')n)n=1 or 2, respectively. Here, we investigate if CstR performs similar chemistry with related chalcogen oxyanions selenite and tellurite. In this work we show by high resolution tandem mass spectrometry that CstR is readily modified by selenite (SeO3(2-)) or tellurite (TeO3(2-)) to form a mixture of intersubunit disulfides and selenotrisulfides or tellurotrisulfides, respectively, between Cys31 and Cys60'. Analogous studies with S. aureus CsoR reveals no reaction with selenite and minimal reaction with tellurite. All cross-linked forms of CstR exhibit reduced DNA binding affinity. We show that Cys31 initiates the reaction with sulfite through the formation of S-sulfocysteine (RS-SO3(2-)) and Cys60 is required to fully derivatize CstR to CstR2(RS-SR') and CstR2(RS-S-SR'). The modification of Cys31 also drives an allosteric switch that negatively regulates DNA binding while derivatization of Cys60 alone has no effect on DNA binding. These results highlight the differences between CstRs and CsoRs in chemical reactivity and metal ion selectivity and establish Cys31 as the functionally important cysteine residue in CstRs.