ArsD residues Cys12, Cys13, and Cys18 form an As(III)-binding site required for arsenic metallochaperone activity
ArsD residues Cys12, Cys13, and Cys18 form an As(III)-binding site required for arsenic metallochaperone activity
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DOI：
10.1074/jbc.m700886200
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发表时间：
2007-06-08
影响因子：
4.8
通讯作者：
Rosen, Barry P.
中科院分区：
文献类型：
--
作者：
Lin, Yung-Feng;Yang, Jianbo;Rosen, Barry P.
ARSA ATPase是由大肠杆菌质粒R773的arsRDABC操纵子编码的ArsAB泵的催化亚单位。ARSD是一种金属配位体，能将As(III)传递给ARSA，增加其对As(III)的亲和力，从而对环境中的砷浓度产生抵抗力。R773 ARSD是一个同源二聚体，在每个亚基上有三个邻近的半胱氨酸对，即Cys(12)-Cys(13)，Cys(112)-Cys(113)和Cys(119)-Cys(120)。每个相邻对都绑定为AS(III)或SB(III)。ARSD同源物的初级序列比对表明，只有第一个邻近的半胱氨酸对Cys(12)-Cys(13)和另外一个半胱氨酸Cys(18)是保守的。考察了半胱氨酸到丙氨酸的取代和截断的影响。通过酵母双杂交分析，几乎所有ARSD突变体都能与野生型ARSD相互作用，表明突变不干扰二聚化。用丙氨酸取代半胱氨酸(112)、半胱氨酸(113)、半胱氨酸(119)或半胱氨酸(120)的ARSD突变体保留了与ARSA相互作用并激活其ATPase活性的能力。表达这些突变体的细胞保留了ARSD增强的AS(III)外排和抗性。相反，带有保守的半胱氨酸(12)、半胱氨酸(13)或半胱氨酸(18)替换的突变体，无论是单独或成对替换，都不能激活ARSA或增强ArsAB泵的活性。我们认为，ARSD残基Cys(12)、Cys(13)和Cys(18)，而不是Cys(112)、Cys(113)、Cys(119)或Cys(120)，是As(III)输送到ArsAB泵和激活ArsAB泵所必需的。
The ArsA ATPase is the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. ArsD is a metallochaperone that delivers As(III) to ArsA, increasing its affinity for As(III), thus conferring resistance to environmental concentrations of arsenic. R773 ArsD is a homodimer with three vicinal cysteine pairs, Cys(12)-Cys(13), Cys(112)-Cys(113), and Cys(119)-Cys(120), in each subunit. Each vicinal pair binds As(III) or Sb(III). Alignment of the primary sequence of homologues of ArsD indicates that only the first vicinal cysteine pair, Cys(12)-Cys(13), and an additional cysteine, Cys(18), are conserved. The effect of cysteine-to-alanine substitutions and truncations were examined. By yeast two-hybrid analysis, nearly all of the ArsD mutants were able to interact with wild type ArsD, indicating that the mutations do not interfere with dimerization. ArsD mutants with alanines substituting for Cys(112), Cys(113), Cys(119), or Cys(120) individually or in pairs or truncations lacking the vicinal pairs retained ability to interact with ArsA and to activate its ATPase activity. Cells expressing these mutants retained ArsD-enhanced As(III) efflux and resistance. In contrast, mutants with substitutions of conserved Cys(12), Cys(13), or Cys(18), individually or in pairs, were unable to activate ArsA or to enhance the activity of the ArsAB pump. We propose that ArsD residues Cys(12), Cys(13), and Cys(18), but not Cys(112), Cys(113), Cys(119), or Cys(120), are required for delivery of As(III) to and activation of the ArsAB pump.