Department of Cell Biology, 514 Medical Sciences Building, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Department of Biochemistry, 416 Medical Sciences Building, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Sci Rep. 2016 Sep 12;6:33179. doi: 10.1038/srep33179.
Hsp90 is a dimeric molecular chaperone responsible for the folding, maturation, and activation of hundreds of substrate proteins called 'clients'. Numerous co-chaperone proteins regulate progression through the ATP-dependent client activation cycle. The most potent stimulator of the Hsp90 ATPase activity is the co-chaperone Aha1p. Only one molecule of Aha1p is required to fully stimulate the Hsp90 dimer despite the existence of two, presumably identical, binding sites for this regulator. Using ATPase assays with Hsp90 heterodimers, we find that Aha1p stimulates ATPase activity by a three-step mechanism via the catalytic loop in the middle domain of Hsp90. Binding of the Aha1p N domain to the Hsp90 middle domain exerts a small stimulatory effect but also drives a separate conformational rearrangement in the Hsp90 N domains. This second event drives a rearrangement in the N domain of the opposite subunit and is required for the stimulatory action of the Aha1p C domain. Furthermore, the second event can be blocked by a mutation in one subunit of the Hsp90 dimer but not the other. This work provides a foundation for understanding how post-translational modifications regulate co-chaperone engagement with the Hsp90 dimer.
Hsp90 是一种二聚体分子伴侣，负责数百种称为“客户”的底物蛋白的折叠、成熟和激活。许多共伴侣蛋白调节通过 ATP 依赖性客户激活循环的进展。Hsp90 ATP 酶活性的最强刺激剂是共伴侣 Aha1p。尽管存在两个可能相同的结合位点，但只需要一个 Aha1p 分子即可完全刺激 Hsp90 二聚体。使用 Hsp90 异二聚体的 ATPase 测定，我们发现 Aha1p 通过 Hsp90 中间域中的催化环通过三步机制刺激 ATP 酶活性。Aha1p N 结构域与 Hsp90 中间域的结合仅产生较小的刺激作用，但也驱动 Hsp90 N 结构域中的单独构象重排。第二个事件驱动相反亚基的 N 结构域的重排，并且是 Aha1p C 结构域的刺激作用所必需的。此外，第二个事件可以被 Hsp90 二聚体的一个亚基中的突变阻断，但不能被另一个亚基中的突变阻断。这项工作为理解翻译后修饰如何调节共伴侣与 Hsp90 二聚体的结合提供了基础。
