Structural and Functional Studies of Hsp70 Molecular Chaperones

项目摘要 作为普遍存在且高度保守的分子伴侣，Hsp70在细胞周期中发挥着多种重要作用。 通过协助蛋白质折叠，组装，维持细胞蛋白质平衡(蛋白质平衡) 降解、解聚和跨膜运输。保持的根本重要性 蛋白质组学不可避免地将Hsp70与许多威胁生命的人类疾病联系在一起，最明显的是癌症和 神经退行性疾病。因此，阐明Hsp70的生化和结构特性将不会 这不仅加深了我们对HSP70辅助折叠的基本分子机制的理解，而且也为HSP70辅助折叠的分子机制研究提供了新的思路 合理设计癌症和神经退行性变新疗法的关键和坚实基础 精神错乱。所有的Hsp70都含有两个功能结构域，一个核苷酸结合域(NBD)和一个底物- 结合结构域(SBD)，对应于两个关键的内在生化活性：ATPase和多肽 底物结合。尽管NBD和SBD都可以独立地结合它们的底物，但伴侣分子 Hsp70的活性严格要求这两个结构域在ATP结合时紧密耦合。海流 Hsp70分子伴侣环的范式主要是基于这一基本的变构偶联而提出的 主要是关于不同的核苷酸结合状态如何控制多肽底物结合。尽管 由于缺乏大量的努力，Hsp70辅助蛋白质折叠的最基本的机制仍然不清楚 对两个关键问题的深入理解：1)多肽底物的结合和释放 建议发生在ATP结合状态(Hsp70-ATP)。Hsp70-ATP如何决定何时结合以及何时结合 释放底物以促进高效的伴侣循环？2)到目前为止，还没有可用的结构 与多肽底物结合的Hsp70-ATP。Hsp70-ATP如何与多肽底物结合 促进蛋白质折叠？因此，这项建议的总体目标是分析这两个关键 问题，以剖析Hsp70分子伴侣功能的基本分子机制。最近，我们有 成功解决了三个Hsp70-ATP结构，意外揭示了两个完全不同的新奇结构 多肽结合口袋的构象，表明ATP-1的多肽结合口袋- 束缚态是高度动态的。更重要的是，我们的解决方案研究受到这些结构的启发 通过三项改变范式的发现彻底改变了成熟的伴侣周期：1)一个活跃的 结合底物在ATP结合时的释放，2)HSP40辅助伴侣是启动有效底物的关键 与热休克蛋白70-三磷酸腺苷结合从而启动生产性伴侣循环；3)热休克蛋白70‘S的活性展开 多肽结合袋。基于这些原始发现，我们提出了以下两个具体目标： 1)表征Hsp70多肽结合口袋在活性伴侣环中的动力学，以及2) 确定多肽底物与Hsp70-ATP结合的分子机制。为了实现我们的目标，我们 使用结合生物化学、X射线结晶学、FRET、单分子的多学科方法 生物物理学、EPR、计算化学、酵母菌和大肠杆菌遗传学。我们期待这一成功 这项建议的完成将有助于我们实现我们的长期目标，即建立一个彻底的机制 了解Hsp70分子伴侣活性维持蛋白稳定的最基本机制。

Project Summary As ubiquitous and highly conserved molecular chaperones, Hsp70s play multiple essential roles in maintaining cellular protein homeostasis (proteostasis) through assisting in protein folding, assembly, degradation, disaggregation, and transportation across membrane. The fundamental importance of maintaining proteostasis inevitably links Hsp70s with many life-threatening human diseases, most notably cancers and neurodegenerative disorders. Thus, elucidating the biochemical and structural properties of Hsp70s will not only advance our understanding on the basic molecular mechanism of Hsp70-assited folding, but also provide a crucial and solid foundation for rational design of novel therapeutics for cancers and neurodegenerative disorders. All Hsp70s contain two functional domains, a nucleotide binding domain (NBD) and a substrate- binding domain (SBD), corresponding to two key intrinsic biochemical activities: ATPase and polypeptide substrate binding. Although NBD and SBD can each bind their substrates independently, the chaperone activity of Hsp70s strictly requires the tight coupling of these two domains upon ATP binding. The current paradigm for Hsp70 chaperone cycle was proposed primarily based on this essential allosteric coupling, which is mainly about how different nucleotide-bound states control polypeptide substrate binding. In spite of extensive efforts, the very basic mechanisms of Hsp70-assisted protein folding are still ill-defined due to a lack of in-depth understanding of two key questions: 1) Both binding and release of polypeptide substrates were proposed to occur in the ATP-bound state (Hsp70-ATP). How does Hsp70-ATP decide when to bind and when to release substrates to promote a productive chaperone cycle? and 2) Until now, no structure is available for an Hsp70-ATP with a polypeptide substrate bound. How does Hsp70-ATP bind polypeptide substrates to promote productive protein folding? Thus, the overall objective of this proposal is to analyze these two key questions in order to dissect the basic molecular mechanisms of Hsp70 chaperone function. Recently, we have successfully solved three Hsp70-ATP structures and unexpectedly revealed two novel and completely different conformations of the polypeptide-binding pocket, suggesting that the polypeptide-binding pocket of the ATP- bound state is highly dynamic. More importantly, our solution studies inspired by these structures have revolutionized the well-established chaperone cycle with three paradigm-shifting discoveries: 1) an active release of bound substrate upon ATP-binding, 2) Hsp40 co-chaperone is the key to initiate efficient substrate binding to Hsp70-ATP and thus start productive chaperone cycle, and 3) an active unfolding by Hsp70’s polypeptide-binding pocket. Based on these original discoveries, we propose the following two Specific Aims: 1) Characterize the dynamics of Hsp70s’ polypeptide-binding pocket in the active chaperone cycle, and 2) Determine the molecular mechanism of polypeptide substrate binding to Hsp70-ATP. To achieve our goal, we use a multidisciplinary approach combining biochemistry, X-ray crystallography, FRET, single-molecule biophysics, EPR, computational chemistry, and yeast and E.coli genetics. We expect that successful completion of this proposal will help us realize our long-term goal, which is to establish a thorough mechanism understanding of the very basic mechanism of Hsp70 chaperone activity in maintaining proteostasis.