Structural and Functional Studies of Hsp70 Molecular Chaperones

描述（由申请人提供）：Hsp 70是普遍存在且高度保守的分子伴侣，其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中发挥多种重要作用。 维持蛋白质稳态的根本重要性不可避免地将Hsp 70与许多破坏性的人类疾病联系起来，最显著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Hsp 70的结构和生化特性不仅将促进我们对Hsp 70辅助蛋白折叠的基本分子机制的理解，而且还将为如何靶向Hsp 70用于治疗癌症和神经退行性疾病的治疗干预提供重要见解。Hsp 70具有三种关键的生物化学活性，它们是分子伴侣活性的核心：ATP酶、肽底物结合和ATP诱导的变构偶联。 尽管人们做了大量的工作，但由于对两个关键的生物化学活性缺乏深入的了解，Hsp 70辅助蛋白质折叠的基本机制仍然不清楚：1）ATP诱导的变构偶联是Hsp 70分子伴侣活性的核心，然而，以往的研究都未能揭示其分子机制。 2)热休克蛋白70 s上的单一肽结合位点使得很难解释有效的伴侣活性。 热休克蛋白70上是否有其他的肽结合位点，从而解释了这种高效率？ 因此，这一建议的总体目标是分析这两个关键的生化活动，以剖析的基本机制的Hsp 70分子伴侣功能。 最近，我们解决了第一个完整的热休克蛋白70在变构活性状态的晶体结构，并发现了一个新的 Hsp 70上的肽底物结合位点。基于这些发现，我们提出了以下两个具体目标：1）阐明Hsp 70 s中ATP驱动的变构偶联的分子机制; 2）表征Hsp 70 s上一个新的肽结合位点，并研究其在蛋白质折叠中的作用。 为了实现我们的目标，我们使用多学科方法，结合X射线晶体学，生物化学，NMR，EPR，计算化学，酵母和大肠杆菌遗传学。我们希望，成功完成这一建议将有助于我们实现我们的长期目标，这是建立一个非常基本的机制，热休克蛋白70分子伴侣活性在蛋白质折叠的理解。

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.