Kinetics of the conformational cycle of Hsp70 reveals the importance of the dynamic and heterogeneous nature of Hsp70 for its function
Kinetics of the conformational cycle of Hsp70 reveals the importance of the dynamic and heterogeneous nature of Hsp70 for its function
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Hsp70 构象循环动力学揭示了 Hsp70 的动态和异质性对其功能的重要性
DOI：
10.1073/pnas.1914376117
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Perrett Sarah
中科院分区：
文献类型：
--
作者：
Wu Si;Hong Liu;Wang Yuqing;Yu Jieqiong;Yang Jie;Yang Jie;Zhang Hong;Perrett Sarah
热休克蛋白70（Heat shock protein 70，Hsp 70）在维持蛋白质稳态中起重要作用。它与辅伴侣Hsp 40合作，刺激Hsp 70 ATP酶活性并将蛋白质底物提供给Hsp 70以协助重折叠。Hsp 40调控Hsp 70分子内和分子间变化的机制仍不清楚。在这里，散装和单分子FRET，我们报告的构象动力学的Hsp 70和Hsp 40的调节，以及动力学的多步Hsp 70-Hsp 40功能循环。我们发现，热休克蛋白40调制的构象的ATP结合的热休克蛋白70的结构域未对接的ATP酶刺激的状态，并促进异源四聚体热休克蛋白70-热休克蛋白40复合物的形成。我们的研究结果提供了深入了解这一核心分子伴侣机制的功能机制。热休克蛋白70是一种保守的分子伴侣，在调节蛋白质折叠、转位和降解中起着不可或缺的作用。热休克蛋白70的构象动力学和辅伴侣对其的调节是其功能的关键。利用体相和单分子荧光共振能量转移（smFRET）技术，研究了人应激诱导型Hsp 70 A1的结构域间构象分布以及核苷酸和Hsp 40辅助分子伴侣Hdj 1诱导的构象变化动力学。我们发现，核苷酸和底物结合域之间和内部的构象显示出异质性。在ATP结合状态下的构象分布可以被Hdj 1诱导形成“ADP样”未对接构象，这是ATP酶刺激状态。动力学测量表明，Hdj 1结合单体热休克蛋白70作为第一步，然后诱导的两个域的解对接和关闭的基板结合裂缝。二聚体Hdj 1然后促进Hsp 70的二聚化和异源四聚体Hsp 70-Hsp 40复合物的形成。我们的研究结果提供了一个动力学视图的热休克蛋白70的构象周期，并揭示了其功能的热休克蛋白70的动态性质的重要性。
Significance Heat shock protein 70 kDa (Hsp70) plays a central role in maintaining protein homeostasis. It cooperates with cochaperone Hsp40, which stimulates Hsp70 ATPase activity and presents protein substrates to Hsp70 to assist refolding. The mechanism by which Hsp40 regulates the intramolecular and intermolecular changes of Hsp70 is still largely unknown. Here, by bulk and single-molecule FRET, we report the conformational dynamics of Hsp70 and its regulation by Hsp40 as well as the kinetics of the multistep Hsp70–Hsp40 functional cycle. We show that Hsp40 modulates the conformations of ATP-bound Hsp70 to a domain-undocked ATPase-stimulated state, and facilitates the formation of a heterotetrameric Hsp70–Hsp40 complex. Our findings provide insights into the functional mechanism of this core chaperone machinery. Hsp70 is a conserved molecular chaperone that plays an indispensable role in regulating protein folding, translocation, and degradation. The conformational dynamics of Hsp70 and its regulation by cochaperones are vital to its function. Using bulk and single-molecule fluorescence resonance energy transfer (smFRET) techniques, we studied the interdomain conformational distribution of human stress-inducible Hsp70A1 and the kinetics of conformational changes induced by nucleotide and the Hsp40 cochaperone Hdj1. We found that the conformations between and within the nucleotide- and substrate-binding domains show heterogeneity. The conformational distribution in the ATP-bound state can be induced by Hdj1 to form an “ADP-like” undocked conformation, which is an ATPase-stimulated state. Kinetic measurements indicate that Hdj1 binds to monomeric Hsp70 as the first step, then induces undocking of the two domains and closing of the substrate-binding cleft. Dimeric Hdj1 then facilitates dimerization of Hsp70 and formation of a heterotetrameric Hsp70–Hsp40 complex. Our results provide a kinetic view of the conformational cycle of Hsp70 and reveal the importance of the dynamic nature of Hsp70 for its function.