Conformational Analysis and Chemical Reactivity of the Multidomain Sulfurtransferase, Staphylococcus aureus CstA
Conformational Analysis and Chemical Reactivity of the Multidomain Sulfurtransferase, Staphylococcus aureus CstA
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DOI：
10.1021/acs.biochem.5b00056
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发表时间：
2015-04-14
期刊：
影响因子：
2.9
通讯作者：
Giedroc, David P.
中科院分区：
文献类型：
--
作者：
Higgins, Khadine A.;Peng, Hui;Giedroc, David P.
主要人类病原体金黄色葡萄球菌 (S. aureus) 的 cst 操纵子受到 CsoR 样硫转移酶阻遏物 (CstR) 的转录控制。该操纵子的表达由硫化氢诱导，并且 cst 操纵子的两种成分 cstA 和 cstB 可保护金黄色葡萄球菌免受硫化物毒性。 CstA 是一种三结构域蛋白，每个结构域都包含一个半胱氨酸，该半胱氨酸被认为在矢量过硫化物穿梭中发挥作用。我们在此表明，CstA 的单个半胱氨酸取代突变体无法保护金黄色葡萄球菌免受体内硫化物毒性。 CstA 的 N 末端结构域表现出硫代硫酸盐硫转移酶（TST；硫氰酸酶）活性，并且使用 S-34-SO32- 作为底物，在存在和不存在相邻 TusA 样结构域的情况下形成 Cys66 S-34-过硫化物作为催化中间体。当与硫代硫酸盐、四硫化钠 (Na2S4) 和原位过硫酸 SufS 一起孵育时，半胱氨酸过硫化物可被捕获在 CstA(Rhod) 中的 C66 上以及 CstA(Rhod-TusA) 中的 C66 和 C128 上。 CstA(Rhod-TusA) 中的 C66A 取代消除了 C128 S-硫化作用，与 CstA 中的定向过硫化物穿梭一致。完全还原的CstA(Rhod-TusA)主要是单体，高分辨率串联质谱分析表明Cys66和Cys128可以使用多种氧化剂形成C66-C128二硫键，从而导致构象发生显着变化。还形成竞争性分子间C128-C128'二硫键。还原 CstA (Rhod-TusA) 的小角 X 射线散射测量和凝胶过滤色谱揭示了一个细长的分子（R-g 大约为 30 埃，21.6 kDa），其中两个结构域并排堆积，可能使 Cys66 和 Cys128 相距很远。这些研究与 CstA 中过硫化物转移中间体的模拟物 C66-C128 交联的低收率一致，以及在无机硫供体的 CstA(Rhod-TusA) 内从 Cys66 到 Cys128 的小但可测量的过硫化物转移。
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 S-34-persulfide is formed as a catalytic intermediate in both the presence and absence of the adjacent TusA-like domain using S-34-SO32- 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 (R-g approximate to 30 angstrom, 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.