Co-chaperone regulation of conformational switching in the Hsp90 ATPase cycle
Co-chaperone regulation of conformational switching in the Hsp90 ATPase cycle
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DOI：
10.1074/jbc.m410562200
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发表时间：
2004-12-10
影响因子：
4.8
通讯作者：
Prodromou, C
中科院分区：
文献类型：
--
作者：
Siligardi, G;Hu, B;Prodromou, C
Hsp 90分子伴侣的ATP水解需要一组连接的构象开关，由ATP结合到Hsp 90二聚体的N-末端结构域触发。其核心是结构的一部分，其像“盖子”一样在结合的ATP上关闭，促进N-末端二聚化和活性位点的组装。影响这些构象转换的Hsp 90突变体对ATP酶活性有很强的影响。ATP酶活性受Hsp 90辅伴侣的特异性调节，其直接影响构象转换。在这里，我们已经分析了热休克蛋白90突变的结合（使用等温滴定量热法和差圆二色性）和ATP酶的调节辅伴侣Aha 1，Sti 1（啤酒花），和Sba 1（p23）的效果。Sti 1结合Hsp 90并抑制其ATP酶活性的能力不受筛选的任何突变体的影响。Sba 1绑定在AMPPNP的存在下，野生型和ATP酶高活性的突变体具有相似的亲和力，但只有非常弱的低活性的突变体，尽管他们的野生型ATP亲和力。出乎意料的是，在所有情况下，Sba 1以1：2的摩尔化学计量与Hsp 90结合。Aha 1结合突变体是类似的野生型，但其ATP酶的激活倍数突变体之间的差异很大。与共分子伴侣混合物的复合物形成的分析表明，Aha 1和p50（cdc 37）能够同时结合Hsp 90，但没有直接的相互作用。Sba 1和p50 cdc 37独立地结合Hsp 90-AMPPNP，但不一起结合。这些数据表明，Sba 1和Aha 1调节热休克蛋白90通过影响构象状态的“ATP盖”和随之而来的N-末端二聚化，而Sti 1不。
ATP hydrolysis by the Hsp90 molecular chaperone requires a connected set of conformational switches triggered by ATP binding to the N-terminal domain in the Hsp90 dimer. Central to this is a segment of the structure, which closes like a "lid" over bound ATP, promoting N-terminal dimerization and assembly of a competent active site. Hsp90 mutants that influence these conformational switches have strong effects on ATPase activity. ATPase activity is specifically regulated by Hsp90 co-chaperones, which directly influence the conformational switches. Here we have analyzed the effect of Hsp90 mutations on binding ( using isothermal titration calorimetry and difference circular dichroism) and ATPase regulation by the co-chaperones Aha1, Sti1 (Hop), and Sba1 (p23). The ability of Sti1 to bind Hsp90 and arrest its ATPase activity was not affected by any of the mutants screened. Sba1 bound in the presence of AMPPNP to wild-type and ATPase hyperactive mutants with similar affinity but only very weakly to hypoactive mutants despite their wild-type ATP affinity. Unexpectedly, in all cases Sba1 bound to Hsp90 with a 1: 2 molar stoichiometry. Aha1 binding to mutants was similar to wild-type, but the - fold activation of their ATPase varied substantially between mutants. Analysis of complex formation with co-chaperone mixtures showed Aha1 and p50(cdc37) able to bind Hsp90 simultaneously but without direct interaction. Sba1 and p50cdc37 bound independently to Hsp90-AMPPNP but not together. These data indicated that Sba1 and Aha1 regulate Hsp90 by influencing the conformational state of the "ATP lid" and consequent N-terminal dimerization, whereas Sti1 does not.