Genetic mapping of the interface between the ArsD metallochaperone and the ArsA ATPase.
Genetic mapping of the interface between the ArsD metallochaperone and the ArsA ATPase.
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ArsD 金属伴侣和 ArsA ATP 酶之间界面的遗传图谱。
DOI：
10.1111/j.1365-2958.2010.07494.x
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发表时间：
2011-02
影响因子：
3.6
通讯作者：
Rosen BP
中科院分区：
文献类型：
--
作者：
Yang J;Salam AA;Rosen BP
ArsD金属伴侣将三价类金属As（III）或Sb（III）传递给ArsA atp酶，ArsAB As（III）外排泵的催化亚基。As（III）的转移增加了ArsA对As（III）的亲和力，使其能够抵抗环境砷浓度。As（III）从伴侣转移到atp酶，这意味着ArsD和ArsA在其金属结合位点形成了一个界面。一种遗传学方法被用来检验这一假设。通过抑制转激活酵母双杂交或反向酵母双杂交试验，选择了13个与ArsA相互作用较弱或较强的ArsD突变体。此外，Lys-37和Lys-62通过定点诱变和化学修饰被鉴定为参与ArsD功能。在任何位置上用精氨酸取代都是可以容忍的，这表明有正电荷的参与。通过酵母双杂交分析，K37A和K62A突变体失去了与ArsA的相互作用。所有15个突变都被映射到ArsD结构表面，它们的位置与通过计算机对接生成的结构模型一致。其中4个位于类金属结合位点Cys-12、Cys-13和Cys18附近，7个位于螺旋1表面。这些结果表明，该界面包括螺旋1的一个表面和类金属结合位点。
The ArsD metallochaperone delivers trivalent metalloids, As(III) or Sb(III), to the ArsA ATPase, the catalytic subunit of the ArsAB As(III) efflux pump. Transfer of As(III) increases the affinity of ArsA for As(III), allowing resistance to environmental arsenic concentrations. As(III) transfer is channeled from chaperone to ATPase, implying that ArsD and ArsA form an interface at their metal binding sites. A genetic approach was used to test this hypothesis. Thirteen ArsD mutants exhibiting either weaker or stronger interaction with ArsA were selected by either repressed transactivator yeast two-hybrid or reverse yeast two-hybrid assays. Additionally, Lys-37 and Lys-62 were identified as being involved in ArsD function by site-directed mutagenesis and chemical modification. Substitution at either position with arginine was tolerated, suggesting participation of a positive charge. By yeast two-hybrid analysis K37A and K62A mutants lost interaction with ArsA. All fifteen mutations were mapped on the surface of the ArsD structure, and their locations are consistent with a structural model generated by in silico docking. Four are close to metalloid binding site residues Cys-12, Cys-13 and Cys18, and seven are on the surface of helix 1. These results suggest that the interface involves one surface of helix 1 and the metalloid binding site.
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影响因子：
2.9
作者：
Yang J;Rawat S;Stemmler TL;Rosen BP
通讯作者：
Rosen BP
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
影响因子：
2.9
作者：
Ye, Jun;Ajees, A. Abdul;Rosen, Barry P.
通讯作者：
Rosen, Barry P.
影响因子：
2.6
作者：
Hua, SB;Qiu, MS;Luo, Y
通讯作者：
Luo, Y