Arsenic binding and transfer by the ArsD As(III) metallochaperone.
Arsenic binding and transfer by the ArsD As(III) metallochaperone.
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DOI：
10.1021/bi100026a
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发表时间：
2010-05-04
期刊：
影响因子：
2.9
通讯作者：
Rosen BP
中科院分区：
文献类型：
--
作者：
Yang J;Rawat S;Stemmler TL;Rosen BP
ArsD是一种金属伴侣蛋白，可将三价类金属[As（III）或Sb（III）]递送至ArsA ATP酶，ArsAB泵的催化亚基由大肠杆菌质粒R773的arsRDABC操纵子编码。与ArsD的相互作用增加了ArsA对As（III）的亲和力，从而对环境中的砷浓度产生抗性。先前的遗传分析表明，ArsD残基Cys 12，Cys 13和Cys 18参与As（III）转移到ArsA。在这里，X射线吸收光谱被用来显示，作为（III）是与三个硫原子，与三个半胱氨酸残基形成的As（III）结合位点相一致的协调。两个单一的色氨酸衍生物的ArsD表现出内在的蛋白质荧光淬灭后，结合的As（III）或Sb（III），这允许估计的结合率和亲和力的类金属。Cys 12、Cys 13或Cys 18的取代使As（III）的亲和力降低10倍以上。还原型谷胱甘肽大大增加了结合率的As（III）的ArsD，但不影响结合的As（III）的ArsA。这表明，在体内胞质As（III）可能最初绑定到GSH和转移到ArsD，然后ArsAB，泵的类金属细胞。As（III）螯合剂二巯基琥珀酸没有阻断从ArsD到ArsA的转移，这与类金属从一种蛋白质到另一种蛋白质的通道相一致，而不是类金属的释放和重新结合。最后，在MgATP存在下，As（III）从ArsD向ArsA的转移发生在23 °C，而不是在4 °C。MgADP和MgATP-γ-S均不能替代MgATP。这些结果表明，转移发生在一个构象的ArsA，短暂的催化循环过程中形成。
ArsD is a metallochaperone that delivers trivalent metalloids [As(III) or Sb(III)] to the ArsA ATPase, the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. Interaction with ArsD increases the affinity of ArsA for As(III), conferring resistance to environmental concentrations of arsenic. Previous genetic analysis suggested that ArsD residues Cys12, Cys13, and Cys18 are involved in the transfer of As(III) to ArsA. Here X-ray absorption spectroscopy was used to show that As(III) is coordinated with three sulfur atoms, consistent with the three cysteine residues forming the As(III) binding site. Two single-tryptophan derivatives of ArsD exhibited quenching of intrinsic protein fluorescence upon binding of As(III) or Sb(III), which allowed estimation of the rates of binding and affinities for metalloids. Substitution of Cys12, Cys13, or Cys18 decreased the affinity for As(III) more than 10-fold. Reduced glutathione greatly increased the rate of binding of As(III) to ArsD but did not affect binding of As(III) to ArsA. This suggests that in vivo cytosolic As(III) might be initially bound to GSH and transferred to ArsD and then to ArsAB, which pumps the metalloid out of the cell. The As(III) chelator dimercaptosuccinic acid did not block the transfer from ArsD to ArsA, consistent with channeling of the metalloid from one protein to the other, as opposed to release and rebinding of the metalloid. Finally, transfer of As(III) from ArsD to ArsA occurred in the presence of MgATP at 23 °C but not at 4 °C. Neither MgADP nor MgATP-γ-S could replace MgATP. These results suggest that transfer occurs with a conformation of ArsA that transiently forms during the catalytic cycle.
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DOI：
10.1016/0304-4165(75)90205-6
发表时间：
1975-01-01
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
APONTOWEIL, P;BERENDS, W
通讯作者：
BERENDS, W
影响因子：
4.2
作者：
Abernathy, CO;Thomas, DJ;Calderon, RL
通讯作者：
Calderon, RL
影响因子：
5.1
作者：
DELNOMDEDIEU, M;BASTI, MM;THOMAS, DJ
通讯作者：
THOMAS, DJ
影响因子：
3
作者：
Field, LS;Luk, E;Culotta, VC
通讯作者：
Culotta, VC
影响因子：
3.6
作者：
Li, JX;Rosen, BP
通讯作者：
Rosen, BP