Direct evidence that a conserved arginine in RuvB AAA+ ATPase acts as an allosteric effector for the ATPase activity of the adjacent subunit in a hexamer
Direct evidence that a conserved arginine in RuvB AAA+ ATPase acts as an allosteric effector for the ATPase activity of the adjacent subunit in a hexamer
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DOI：
10.1073/pnas.0403584101
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发表时间：
2004-06-29
影响因子：
11.1
通讯作者：
Shinagawa, H
中科院分区：
文献类型：
--
作者：
Hishida, T;Han, YW;Shinagawa, H
大肠杆菌 RuvA 和 RuvB 蛋白复合物在重组修复和同源重组期间以及在复制叉停滞时促进霍利迪连接体的分支迁移。 RuvB蛋白属于AAA(+)（与各种细胞活动相关的ATP酶）ATP酶家族，并以ATP依赖性方式形成六聚环。对寡聚 AAA+ 类 ATP 酶的研究表明，保守的精氨酸残基位于邻近亚基的 ATP 酶位点附近，在 ATP 水解过程中发挥重要作用。这项研究提供了直接证据，表明 RuvB 的 Arg-174 变构刺激 RuvB 六聚体中相邻亚基的 ATP 酶。 RuvBR174A 对体内 DNA 修复表现出显性失活表型，并抑制野生型 RuvB 催化的分支迁移。 RuvBK68A（Walker A 突变）也观察到显性失活表型。 RuvB K68A-R174A 双突变体表现出比单突变体 RuvB K68A 或 R174A 更严重的显性负效应。此外，尽管RuvB K68A和R174A的ATP酶活性完全缺陷，但当这两种突变蛋白以1:1的比例混合时，ATP酶活性恢复。这些结果表明，两种突变体均具有明显的功能缺陷，并且 ATP 酶活性的恢复是通过异六聚体中突变体亚基之间的互补相互作用实现的。本研究表明，R174 在 RuvB 水解 ATP 过程中发挥分子间催化作用。这种作用可能是寡聚 AAA/AAA(+) ATP 酶的一般特征。
The Escherichia coli RuvA and RuvB protein complex promotes branch migration of Holliday junctions during recombinational repair and homologous recombination and at stalled replication forks. The RuvB protein belongs to the AAA(+) (ATPase associated with various cellular activities) ATPase family and forms a hexameric ring in an ATP-dependent manner. Studies on the oligomeric AAA+ class ATPases suggest that a conserved arginine residue is located in close proximity to the ATPase site of the adjacent subunit and plays an essential role during ATP hydrolysis. This study presents direct evidence that Arg-174 of RuvB allosterically stimulates the ATPase of the adjacent subunit in a RuvB hexamer. RuvBR174A shows a dominant negative phenotype for DNA repair in vivo and inhibits the branch migration catalyzed by wild-type RuvB. A dominant negative phenotype was also observed with RuvBK68A (Walker A mutation). RuvB K68A-R174A double mutant demonstrates a more severe dominant negative effect than the single mutants RuvB K68A or R174A. Moreover, although RuvB K68A and R174A are totally defective in ATPase activity, ATPase activity is restored when these two mutant proteins are mixed at a 1:1 ratio. These results suggest that each of the two mutants has distinct functional defects and that restoration of the ATPase activity is brought by complementary interaction between the mutant subunits in the heterohexamers. This study demonstrates that R174 plays an intermolecular catalytic role during ATP hydrolysis by RuvB. This role may be a general feature of the oligomeric AAA/AAA(+) ATPases.