title: Conformational analysis and chemical reactivity of the multidomain sulfurtransferase, Staphylococcus aureus CstA.
abstract: The cst operon of the major human pathogen Staphylococcus aureus (S. aureus) is under the transcriptional control of CsoR-like sulfurtransferase repressor (CstR). Expression of this operon is induced by hydrogen sulfide, and two components of the cst operon, cstA and cstB, protect S. aureus from sulfide toxicity. CstA is a three-domain protein, and each domain harbors a single cysteine that is proposed to function in vectorial persulfide shuttling. We show here that single cysteine substitution mutants of CstA fail to protect S. aureus against sulfide toxicity in vivo. The N-terminal domain of CstA exhibits thiosulfate sulfurtransferase (TST; rhodanese) activity, and a Cys66 (34)S-persulfide is formed as a catalytic intermediate in both the presence and absence of the adjacent TusA-like domain using (34)S-SO3(2-) as a substrate. Cysteine persulfides can be trapped on both C66 in CstA(Rhod) and on C66 and C128 in CstA(Rhod-TusA) when incubated with thiosulfate, sodium tetrasulfide (Na2S4), and in situ persulfurated SufS. C66A substitution in CstA(Rhod-TusA) abolishes C128 S-sulfhydration, consistent with directional persulfide shuttling in CstA. Fully reduced CstA(Rhod-TusA) is predominately monomeric, and high resolution tandem mass spectrometry reveals that Cys66 and Cys128 can form a C66-C128 disulfide bond using a number of oxidants, which leads to a significant change in conformation. A competing intermolecular C128-C128' disulfide bond is also formed. Small-angle X-ray scattering measurements and gel filtration chromatography of reduced CstA(Rhod-TusA) reveal an elongated molecule (Rg ≈ 30 Å, 21.6 kDa) where the two domains pack "side-by-side" that likely places Cys66 and Cys128 far apart. These studies are consistent with the low yield of C66-C128 cross-link as a mimic of a persulfide transfer intermediate in CstA, and small, but measurable persulfide transfer from Cys66 to Cys128 within the CstA(Rhod-TusA) with inorganic sulfur donors.
authors: †Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102, United States.
authors: Khadine A
authors: KA
authors: Higgins
authors: †Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102, United States.|‡Graduate Program in Biochemistry, Indiana University, Bloomington, Indiana 47405, United States.
authors: Hui
authors: H
authors: Peng
authors: †Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102, United States.
authors: Justin L
authors: JL
authors: Luebke
authors: †Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102, United States.
authors: Feng-Ming James
authors: FM
authors: Chang
authors: †Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102, United States.
authors: David P
authors: DP
authors: Giedroc
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D013437:Carbon-Sulfur Lyases / Q000378:metabolism; D003545:Cysteine / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D006862:Hydrogen Sulfide / Q000494:pharmacology; D009154:Mutation; D017434:Protein Structure, Tertiary; D017670:Sodium Compounds / Q000494:pharmacology; D013211:Staphylococcus aureus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D013440:Sulfides / Q000494:pharmacology; D013466:Sulfurtransferases / Q000235:genetics / Q000378:metabolism*; D013884:Thiosulfate Sulfurtransferase / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D017670:Sodium Compounds; D013440:Sulfides; C519323:sodium tetrasulfide; C116840:cysteine persulfide; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase; D013437:Carbon-Sulfur Lyases; C080546:cysteine desulfurase; C025451:sodium bisulfide; D003545:Cysteine; D006862:Hydrogen Sulfide