Analysis of the molecular mechanism of Hsp70 chaperones

70 kDa热休克蛋白（Hsp 70）参与多种细胞蛋白质折叠过程。Hsp 70的显着多功能性是基于其底物结合结构域（SBD）与底物多肽内的短退行性基序的瞬时相互作用。这种相互作用是由它们的核苷酸结合结构域（NBD）和SBD之间的变构控制机制调节的。因此，Hsp 70·ATP对底物的亲和力低，但底物结合和解离速率高，Hsp 70·ADP对底物的亲和力高，底物交换速率低。固有的ATP水解速率是非常低的，但基本上由蛋白质底物本身和J结构域蛋白（JDP）家族的辅伴侣的协同作用刺激。核苷酸交换因子加速ADP释放，并且在生理ATP浓度下，转换为低亲和力状态。在过去的资助期内，我们能够为更好地理解Hsp 70分子伴侣机器做出重大贡献。我们对底物结合机制的研究表明，与结合肽的构象相比，当蛋白质底物结合时，SBD具有不同的构象，改变了我们对Hsp 70与折叠蛋白、聚集体和淀粉样蛋白相互作用的设想。我们能够在ATP结合的开放构象中解决Hsp 70的晶体结构。基于晶体结构的诱变研究揭示了热休克蛋白70变构调节机制的基本方面，并使我们能够提出一个热休克蛋白70变构的机械模型。我们还发现了不同类别的JDP在Hsp 70介导的蛋白质聚集体解聚中的协同作用，并且我们表征了Hsp 70有效溶解α-突触核蛋白聚集体的机制。在接下来的资助期内，我们将解决对理解Hsp 70系统至关重要的四个问题：（i）基于最近的晶体结构，我们想要分析由ATP与无核苷酸的Hsp 70结合以及由底物与Hsp 70结合诱导的构象变化的动力学。ATP以及它们如何被底物和辅伴侣调节。这些调查不仅应该产生一个动态模型的热休克蛋白70行动，但也对底物蛋白的能量转移的信息。(ii)为了了解JDP如何刺激Hsp 70 s的ATP酶活性，我们将尝试确定J-domain在Hsp 70 s的NBD上的相互作用位点和J-domain诱导的NBD构象变化。(iii)我们将研究JDP-底物相互作用，以阐明不同类别的JDP如何与底物结合，它们是否以及如何改变底物的构象，以及它们如何将Hsp 70靶向底物。(iv)将研究JDP和核苷酸交换因子的合作，以阐明Hsp 70分子伴侣循环过程中的协同作用。

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.