Selenite and tellurite form mixed seleno- and tellurotrisulfides with CstR from Staphylococcus aureus

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 (SeO32−) or tellurite (TeO32−) 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-SO32−) 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.

Insights into Protein Allostery in the CsoR/RcnR Family of Transcriptional Repressors

“…Similarly, CstR will form disulfides, selenotrisulfides, tellurotrisulfides when reacted with the calcogen oxyanions selenite and tellurite, respectively (Figure 3B). 29 These crosslinked products were not observed in CsoR. 6,29 Work done by Luebke et al showed that Cys31 initiates the reaction with sulfite through the formation of S -sulfocysteine (Figure 3B).…”

“…29 These crosslinked products were not observed in CsoR. 6,29 Work done by Luebke et al showed that Cys31 initiates the reaction with sulfite through the formation of S -sulfocysteine (Figure 3B). 29 They also determined that modify-cation of Cys31 (in a Cys60A background) with the cysteine-modifying reagent methylmethanethiosulfonate (MMTS) resulted in a decrease in DNA binding affinity while similar modifications to Cys60 had no effect on DNA binding affinity.…”

“…29 They also determined that modify-cation of Cys31 (in a Cys60A background) with the cysteine-modifying reagent methylmethanethiosulfonate (MMTS) resulted in a decrease in DNA binding affinity while similar modifications to Cys60 had no effect on DNA binding affinity. 29 Collectively, these studies show that CstR and CsoR function via different derepression mechanisms and that Cys31 is the functionally important cysteine residue in CstR.…”

Cu(I)-mediated Allosteric Switching in a Copper-sensing Operon Repressor (CsoR)

“…Normalized r values (ranging from 0 to 1) represent fractional saturation of the DNA and were calculated from the ratio (r obs Ϫ r DNA )/(r max Ϫ r DNA ), where r max is the maximum anisotropy obtained at saturating protein concentrations, and r DNA is the anisotropy of the free DNA. The resultant data were subjected to an unweighted nonlinear least squares fit to a two-tetramer binding model (defined by K 1 and K 2 ) as described earlier (16,25) using the program DynaFit (43) and assuming a linear relationship between r obs and fractional saturation of the DNA as verified previously (16). The macroscopic DNA binding constant, A 2 , is determined from A 2 ϭ K 1 ⅐K 2 (see Table 1 …”

“…A second M. tuberculosis CsoR paralog, RicR, was shown to be involved in the regulation of several genes only found in pathogenic mycobacteria in response to copper stress (2). Several non-copper-sensing CsoR-like proteins, including the nickel-sensing repressors RcnR in Escherichia coli (21,22) and InrS in Synechocystis (23) and the sulfur transferase regulator CstR in S. aureus (24,25), have also been identified, revealing that this family of proteins has evolved to sense a range of inducers.…”