The CsoR-like sulfurtransferase repressor, CstR, is a member of the CsoR (Copper-sensitive operon repressor) family of bacterial transcriptional regulators. Many members of this family respond to metals such as cobalt, nickel, or copper, whereas CstR responds to persulfide stress. Structures of proteins in this family lack canonical DNA binding motifs like the helix-turn-helix, but instead feature a disc-like arrangement of 3-helix bundles packed into a dimer of dimers. Although several structures of CsoR-family proteins have been solved, we still do not have a matched pair of structures of the active and inactive states of the same protein to help us to understand how DNA is released. CstR has been characterized in Staphylococcus aureus and in Enterococcus faecalis, but neither protein has been suitable for structural biology due to low solubility. Here we show that CstR from Streptococcus pneumoniae is unusually stable and that we obtained the first crystal structures of Spn CstR C9A and C9A N55A at multiple pH’s. SAXS, and CD confirm that the assembly state of Spn CstR C9A is a tetramer with mostly α-helical characteristics. Fluorescence anisotropy measurements suggest that Spn CstR C9A binds to DNA with a ratio of 2:1 protein:DNA. Upon oxidation with glutathione disulfide, Spn CstR C9A forms interprotomer disulfide linkages that are structurally and functionally reversible based on NMR and fluorescence anisotropy experiments. Reactivity of Spn CstR C9A and C9A/N55A with a persulfide donor yield diverging pathways, yet ultimately favoring the formation of tetrasulfide linkages. These new insights provide a clearer picture of how persulfide sensors in the CsoR family behave.

Structural and mechanistic characterization of persulfide sensing transcriptional regulators

ACS Spring Meeting 2021

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