Structural and Functional Studies of Hsp70 Molecular Chaperones

描述（由申请人提供）：hsp70是一种普遍存在的高度保守的分子伴侣，通过协助蛋白质折叠、组装、降解和跨膜运输，在维持细胞蛋白质稳态方面发挥多种重要作用。维持蛋白质稳态的根本重要性不可避免地将hsp70与许多破坏性的人类疾病联系起来，最明显的是癌症和神经退行性疾病，如帕金森病和阿尔茨海默病。因此，阐明hsp70的结构和生化特性不仅有助于我们了解hsp70辅助蛋白折叠的基本分子机制，而且为如何靶向hsp70进行癌症和神经退行性疾病的治疗干预提供重要见解。hsp70具有三种关键的生化活性，它们是伴侣活性的核心：atp酶、肽底物结合和atp诱导的变构偶联。尽管进行了大量的研究，但由于对两个关键的生化活动缺乏深入的了解，hsp70辅助蛋白折叠的基本机制仍然不明确：1)atp诱导的变构偶联是hsp70的伴侣活性的核心，但所有的研究都未能揭示其分子机制。2) Hsp70s上已建立的单肽结合位点使得其有效的伴侣活性难以解释。hsp70上是否存在额外的肽结合位点来解释高效率？因此，本提案的总体目标是分析这两个关键的生化活性，以剖析Hsp70伴侣蛋白功能的基本机制。最近，我们首次解决了一个完整的Hsp70在变构活性状态下的晶体结构，并发现了一个新的

DESCRIPTION (provided by applicant): Hsp70s are ubiquitous and highly conserved molecular chaperones that play multiple essential roles in maintaining cellular protein homeostasis through assisting in protein folding, assembly, degradation, and transportation across membrane. The fundamental importance of maintaining protein homeostasis inevitably links Hsp70s with many destructive human diseases, most notably cancers and neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Thus, elucidating the structural and biochemical properties of Hsp70s will not only advance our understanding of the basic molecular mechanism of Hsp70-assited protein folding, but also provide crucial insights regarding how to target Hsp70s for therapeutic interventions in treating cancers and neurodegenerative disorders. Hsp70s have three key biochemical activities that are at the heart of the chaperone activity: ATPase, peptide substrate binding, and ATP-induced allosteric coupling. In spite of extensive efforts, the very basic mechanism of Hsp70-assisted protein folding is still ill-defined due to the lack of in-depth understanding o two key biochemical activities: 1) ATP-induced allosteric coupling is central to Hsp70s' chaperone activity~ however, all previous studies had failed to reveal the molecular mechanism. 2) The well-established single peptide binding site on Hsp70s has made it difficult to explain the efficient chaperone activity. Could there be additional peptide binding sites on Hsp70s that account for the high efficiency? Thus, the overall objective of this proposal is to analyze these two key biochemical activities in order to dissect the basic mechanisms of Hsp70 chaperone function. Recently, we solved the first crystal structure of an intact Hsp70 in the allosteric active state, and discovered a novel peptide substrate binding site on Hsp70s. Based on these original discoveries, we propose the following two Specific Aims: 1) elucidate the molecular mechanism of the ATP-driven allosteric coupling in Hsp70s, and 2) characterize a novel peptide binding site on Hsp70s and investigate its role in protein folding. To achieve our goal, we use a multidisciplinary approach combining X-ray crystallography, biochemistry, NMR, 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 thoroug mechanism understanding of the very basic mechanism of Hsp70 chaperone activity in protein folding.