Structural and Functional Studies of Hsp70 Molecular Chaperones

Hsp70 分子伴侣的结构和功能研究

• 批准号: 9913551
• 负责人: Qinglian Liu
• 金额: $ 31.83万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9913551
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Affinity; Binding; Biochemical; Biochemistry; Biophysics; Coupling; Escherichia coli; Fluorescence Resonance Energy Transfer; Foundations; Genetic; Goals; Hydrophobicity; Kinetics; Label; Life; Link; Malignant Neoplasms; Membrane; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Movement; Neurodegenerative Disorders; Nucleotides; Organism; Peptides; Play; Process; Property; Proteins; Research; Role; Solid; Structure; Substrate Domain; Transportation; X-Ray Crystallography; Yeasts; base; computational chemistry; design; human disease; innovation; insight; interdisciplinary approach; novel; novel therapeutics; polypeptide; protein folding; proteostasis; single molecule

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.

项目摘要 作为无处不在且高度保守的分子伴侣，HSP70在 通过协助蛋白质折叠，组装，维持细胞蛋白质稳态（蛋白质固醇）， 跨膜的降解，分解和运输。维护的基本重要性 蛋白质静态变性不可避免地将HSP70与许多威胁生命的人类疾病联系起来，最著名的是癌症和 神经退行性疾病。阐明HSP70的生化和结构特性不会 仅提高我们对HSP70分配的折叠的基本分子机制的理解，但也提供 为癌症和神经退行性的新型治疗合理设计的关键和坚实的基础 疾病。所有HSP70都包含两个功能域，一个核苷酸结合结构域（NBD）和底物 - 结合结构域（SBD），对应于两个关键的内在生化活动：ATPase和多肽 尽管NBD和SBD各自可以独立绑定其底物，但链条 HSP70的活性严格要求在ATP结合时这两个域的紧密耦合。电流 HSP70伴侣周期的范式主要是基于这种基本的变构耦合的，该耦合 主要是关于不同核苷酸的状态如何控制多肽底物结合的方式。尽管 由于缺乏 对两个关键问题的深入了解：1）多肽底物的结合和释放是 提议发生在ATP结合状态（HSP70-ATP）。 HSP70-ATP如何决定何时绑定以及何时绑定 释放底物以促进生产性伴侣周期？ 2）到目前为止，没有任何结构可用于 具有多肽底物结合的HSP70-ATP。 HSP70-ATP如何将多肽底物结合到 促进生产蛋白折叠？这是该提案的总体目的是分析这两个关键 问题是为了剖析HSP70伴侣函数的基本分子机制。最近，我们有 成功解决了三个HSP70-ATP结构，并意外地揭示了两种新颖，并且完全不同 多肽结合口袋的构象，表明ATP的多肽结合袋 界面是高度动态的。更重要的是，我们受这些结构启发的解决方案研究具有 通过三个范式转移发现彻底改变了公认的伴侣周期：1）活跃 在ATP结合时释放绑定的底物，2）HSP40副酮是启动有效底物的关键 与HSP70-ATP结合，从而启动生产性伴侣周期，3）HSP70的主动展开 多肽结合口袋。基于这些原始发现，我们提出以下两个具体目的： 1）表征活跃链循环中HSP70S多肽结合口袋的动力学，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 使用多学科方法，结合生物化学，X射线晶体学，fret，单分子 生物物理学，EPR，计算化学以及酵母和大肠杆菌遗传学。我们期望成功 该提案的完成将有助于我们实现长期目标，即建立彻底的机制 了解HSP70伴侣活性在维持蛋白质的基本机制。