Weikl T, Muschler P, Richter K, Veit T, Reinstein J, Buchner J
Institut für Organische Chemie & Biochemie, Garching, 85747, Germany.
J Mol Biol. 2000 Nov 3;303(4):583-92. doi: 10.1006/jmbi.2000.4157.
Hsp90 is an abundant molecular chaperone that functions in an ATP-dependent manner in vivo. The ATP-binding site is located in the N-terminal domain of Hsp90. Here, we dissect the ATPase cycle of Hsp90 kinetically. We find that Hsp90 binds ATP with a two-step mechanism. The rate-limiting step of the ATPase cycle is the hydrolysis of ATP. Importantly, ATP becomes trapped and committed to hydrolyze during the cycle. In the isolated ATP-binding domain of Hsp90, however, the bound ATP was not committed and the turnover numbers were markedly reduced. Analysis of a series of truncation mutants of Hsp90 showed that C-terminal regions far apart in sequence from the ATP-binding domain are essential for trapping the bound ATP and for maximum hydrolysis rates. Our results suggest that ATP binding and hydrolysis drive conformational changes that involve the entire molecule and lead to repositioning of the N and C-terminal domains of Hsp90.
热休克蛋白90（Hsp90）是一种丰富的分子伴侣，在体内以ATP依赖的方式发挥作用。ATP结合位点位于Hsp90的N端结构域。在此，我们从动力学角度剖析了Hsp90的ATP酶循环。我们发现Hsp90通过两步机制结合ATP。ATP酶循环的限速步骤是ATP的水解。重要的是，ATP在循环过程中会被困住并注定要水解。然而，在分离的Hsp90的ATP结合结构域中，结合的ATP并不注定要水解，且周转数显著降低。对一系列Hsp90截短突变体的分析表明，与ATP结合结构域在序列上相距甚远的C端区域对于捕获结合的ATP和实现最大水解速率至关重要。我们的结果表明，ATP的结合和水解驱动了涉及整个分子的构象变化，并导致Hsp90的N端和C端结构域重新定位。
