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 Hsp 70作为一种普遍存在且高度保守的分子伴侣，在人类免疫系统中发挥着重要的作用。 通过帮助蛋白质折叠，装配， 降解、解聚和跨膜运输。维护的根本重要性 蛋白质稳态不可避免地将Hsp 70与许多危及生命的人类疾病联系起来，最明显的是癌症和 神经退行性疾病因此，阐明Hsp 70的生物化学和结构特性将不会 这不仅加深了我们对Hsp 70辅助折叠的基本分子机制的理解，而且也为我们提供了 为合理设计癌症和神经退行性疾病的新疗法奠定了重要而坚实的基础 紊乱所有的Hsp 70都含有两个功能结构域，一个核苷酸结合结构域（NBD）和一个底物结合结构域。 结合结构域（SBD），对应于两个关键的内在生物化学活性：ATP酶和多肽 底物结合虽然NBD和SBD可以各自独立地结合其底物，但是分子伴侣蛋白可以与它们的底物结合。 Hsp 70的活性严格要求这两个结构域在ATP结合时紧密偶联。当前 Hsp 70分子伴侣循环的模式主要是基于这种重要的变构偶联， 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 尽管经过大量的努力，Hsp 70辅助蛋白质折叠的基本机制仍然不明确，因为缺乏 深入理解两个关键问题：1）多肽底物的结合和释放都是 Hsp 70-ATP结合状态（Hsp 70-ATP）。Hsp 70-ATP如何决定何时结合以及何时结合 释放底物来促进生产性伴侣循环2）到目前为止，没有结构可用于 结合有多肽底物的Hsp 70-ATP。Hsp 70-ATP如何结合多肽底物， 促进蛋白质折叠因此，本提案的总体目标是分析这两个关键问题， 问题，以剖析热休克蛋白70分子伴侣功能的基本分子机制。最近我们 成功地解决了三个Hsp 70-ATP结构，出乎意料地揭示了两个新的，完全不同的 多肽结合口袋的构象，这表明ATP- 束缚态是高度动态的。更重要的是，我们受这些结构启发的解决方案研究， 彻底改变了完善的伴侣循环与三个范式转变的发现：1）一个积极的 2）Hsp 40共伴侣蛋白是启动有效底物的关键 与Hsp 70-ATP结合，从而启动生产性伴侣循环，以及3）Hsp 70的主动解折叠 多肽结合口袋。基于这些最初的发现，我们提出以下两个具体目标： 1)表征活性伴侣循环中Hsp 70 s多肽结合口袋的动力学，以及2） 确定多肽底物与Hsp 70-ATP结合的分子机制。为了实现我们的目标，我们 使用多学科的方法结合生物化学，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。我们期待成功 这一方案的完成将有助于我们实现建立一个彻底机制的长期目标 了解Hsp 70分子伴侣活性在维持蛋白质稳态中的基本机制。