Analysis of the molecular mechanism of Hsp70 chaperones

Hsp70分子伴侣的分子机制分析

• 批准号: 183020176
• 负责人: Professor Dr. Bernd Bukau
• 金额: --
• 依托单位: Zentrum für Molekulare Biologie (ZMBH)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/183020176?language=en
• 关键词: Analysis; molecular; mechanism; Hsp70; chaperones

The 70 kDa heat shock proteins (Hsp70s) are involved in a wide variety of cellular protein folding processes. The remarkable versatility of Hsp70s is based on the transient interaction of their substrate binding domain (SBD) with a short degenerative motif within the substrate polypeptide. This interaction is regulated by an allosteric control mechanism between their nucleotide binding domain (NBD) and SBD. Thereby, Hsp70·ATP has a low affinity to substrates but high substrate association and dissociation rates and Hsp70·ADP has a high affinity for substrates with low substrate exchange rates. The intrinsic ATP hydrolysis rate is very low but is stimulated substantially by synergistic action of the protein substrate itself and a cochaperone of the family of J-domain proteins (JDP). Nucleotide exchange factors accelerate ADP release and, at physiological ATP concentrations, conversion to the low affinity state. They thus regulate the life-time of chaperone-substrate complexes.In the past funding period we were able to contribute substantially to a better understanding of the Hsp70 chaperone machine. Our investigations of the mechanics of substrate binding revealed that the SBD has a different conformation when protein substrates are bound as compared to the conformation with bound peptides, changing how we envision the interaction of Hsp70s with folded proteins, aggregates and amyloids. We were able to solve the crystal structure of Hsp70 in the ATP bound open conformation. A mutagenesis study base on the crystal structure revealed fundamental aspects of the allosteric regulatory mechanism and allowed us to propose a mechanical model of allostery in Hsp70s. We also discovered a synergistic action of JDPs of different classes in Hsp70-mediated disaggregation of protein aggregates and we characterized a Hsp70 machinery that efficiently dissolves alpha-synuclein aggregates.In the coming funding period we will address four issues of central importance to the understanding of the Hsp70 system: (i) based on the recent crystal structure we want to analyze the kinetics of conformational changes induced by ATP binding to nucleotide-free Hsp70 and by substrate binding to Hsp70·ATP and how they are modulated by substrates and cochaperones. These investigations should not only yield a dynamic model of Hsp70 action but also information on energy transfer to substrate proteins. (ii) To understand how JDP stimulate the ATPase activity of Hsp70s we will try to define the interaction site of the J-domain on the NBD of Hsp70s and J-domain induced conformational changes in the NBD. (iii) We will investigate the JDP-substrate interaction to clarify how JDPs of different classes bind to substrates, whether and how they change the conformation of substrates, and how they target Hsp70 to substrates. (iv) The cooperation of JDPs and nucleotide exchange factors will be studied to elucidate the synergistic action during the Hsp70 chaperone cycle.

70 kDa热休克蛋白(Hsp70)参与了多种细胞蛋白质折叠过程。Hsp70的显著多功能性是基于它们的底物结合结构域(SBD)与底物多肽中的短简并基序的瞬时相互作用。这种相互作用受其核苷酸结合域(NBD)和SBD之间的变构控制机制的调节。因此，Hsp70·ATP对底物的亲和力较低，但底物缔合和解离速率较高，而Hsp70·ADP对底物交换率较低的底物具有较高的亲和力。固有的ATP水解率非常低，但基本上是由蛋白质底物本身和J-结构域蛋白家族的辅伴侣蛋白(JDP)的协同作用刺激的。核苷酸交换因子加速ADP的释放，并在生理浓度下转换到低亲和力状态。因此，它们调节伴侣-底物复合体的寿命。在过去的资助期间，我们能够为更好地了解Hsp70伴侣机器做出重大贡献。我们对底物结合机理的研究表明，当蛋白质底物结合时，SBD的构象与结合的多肽的构象不同，改变了我们想象Hsp70与折叠蛋白质、聚集体和淀粉样蛋白相互作用的方式。我们能够在ATP结合的开放构象中解析出Hsp70的晶体结构。基于晶体结构的突变研究揭示了变构调节机制的基本方面，并使我们能够在Hsp70中提出变构的力学模型。我们还发现了不同类别的JDPs在Hsp70介导的蛋白质聚集体解聚中的协同作用，并描述了一种有效地溶解α-突触核蛋白聚集体的Hsp70机制。在即将到来的资助期间，我们将解决四个对理解Hsp70系统至关重要的问题：(I)基于最近的晶体结构，我们想要分析ATP与无核苷酸Hsp70结合和底物与Hsp70·ATP结合所引起的构象变化的动力学，以及它们是如何被底物和辅伴侣调节的。这些研究不仅应该产生Hsp70作用的动态模型，还应该提供关于能量转移到底物蛋白质的信息。(Ii)为了了解JDP是如何刺激Hsp70的ATPase活性的，我们将尝试定义Hsp70的NBD上J-结构域的相互作用位置和J-结构域在NBD中引起的构象变化。(Iii)我们将研究JDP与底物的相互作用，以澄清不同类别的JDP如何与底物结合、它们是否以及如何改变底物的构象，以及它们如何针对底物HSP70。(4)研究JDPs与核苷酸交换因子的协同作用，以阐明Hsp70分子伴侣循环中的协同作用。