Allosteric Mechanism of Hsp70 Molecular Chaperones

描述（由申请人提供）：Hsp 70分子伴侣存在于所有生物体和基本上所有细胞区室中。在它们广泛的基本细胞功能中，它们促进新合成的蛋白质的折叠;保护细胞免受损伤，例如在应激条件下可能发生的聚集;帮助将蛋白质靶向细胞质外位置;以及促进大分子复合物的组装和分解。所有这些功能都依赖于Hsp 70结合蛋白质底物的未折叠区域的能力，以及在ATP的变构结合后释放其底物的能力。该研究主要集中在Hsp 70变构的基本分子机制上。这项工作建立在令人兴奋的最新结果之上：我们在上一个项目期间表明，范例性E. coli Hsp 70 DnaK在ATP结合后发生了主要的构象变化，我们对这些结构域的变构重构有了更深入的了解。我们的研究结果使我们建立了DnaK中结构域间变构通信的模型，该模型已被Hendrickson实验室最近的一种相关伴侣Sse 1（酵母Hsp 110）结构所验证。Liu和W. A. Hendrickson，Cell 131，106-1202007）]。我们的具体目标是：完善目前基于Sse 1的ATP结合DnaK同源模型，并利用该模型，结合我们对DnaK ADP结合状态的了解，阐明Hsp 70分子伴侣中的变构域间通讯机制，评估DnaK结果的普遍性，并发展关于Hsp 70变构功能的一般原则;探索DnaK中的变构构象变化如何通过与辅伴侣DnaJ和GrpE的相互作用来调节。我们将利用适用于大分子的新的NMR策略，以分析热休克蛋白70结合其配体和共分子伴侣后的变构构象转变的结构和动力学方面。补充数据将提供时间分辨荧光能量转移和电子自旋共振方法，以及计算方法的基础上的序列分析，正常模式的计算，和基于整体的热力学解剖配体介导的能量。热休克蛋白70构成相对简单的变构机器。详细研究它们的变构域间通讯将揭示蛋白质如何利用配体结合能来远距离调节结合和催化功能的更广泛的难题。Hsp 70分子伴侣在正常生理条件下发挥关键的细胞作用，使细胞能够承受热休克等应激。已知Hsp 70在肿瘤中是抗凋亡的和上调的;具有讽刺意味的是，它们的上调对由蛋白质错误折叠引起的神经变性疾病具有保护作用。Hsp 70在正常和疾病状态下的密切参与导致其作为可能的治疗靶点出现，但在治疗能力中使用热休克蛋白需要我们充分了解其作用机制，包括核苷酸如何调节底物结合以及与辅助分子伴侣的相互作用如何调节Hsp 70变构。

DESCRIPTION (provided by applicant): Hsp70 chaperones occur in all organisms and essentially all cellular compartments. Among their wide array of essential cellular functions, they facilitate folding of newly synthesized proteins; protect cells from damage such as aggregation that can occur under stress conditions; help to target proteins to extra- cytoplasmic locations; and facilitate assembly and disassembly of macromolecular complexes. All of these functions rely on the ability of Hsp70s to bind unfolded regions of a protein substrate, and to release their substrates upon allosteric binding of ATP. The research proposed focuses on the fundamental molecular mechanism of Hsp70 allostery. The work proposed builds on exciting recent results: We showed in the last project period that both the ATPase domain and the substrate-binding domain (SBD) of the paradigmatic E. coli Hsp70 DnaK undergo major conformational changes upon ATP binding, and we gained understanding of the allosteric remodeling of these domains. Our results led us to a model for interdomain allosteric communication in DnaK that has been validated by a recent structure from the Hendrickson lab of a related chaperone, Sse1, the yeast Hsp110 [Q. Liu and W. A. Hendrickson, Cell 131, 106-1202007)]. Our specific aims are: to refine the current Sse1-based homology model of ATP-bound DnaK and to use this model, together with our knowledge about the ADP-bound state of DnaK, to elucidate the mechanism of allosteric interdomain communication in this Hsp70 molecular chaperone; to assess the generality of results on DnaK and develop general principles about Hsp70 allosteric function; to explore how the allosteric conformational changes in DnaK are modulated by interaction with co-chaperones DnaJ and GrpE. We will utilize new NMR strategies applicable to large molecules in order to analyze both structural and dynamic aspects of the allosteric conformational transitions in Hsp70s upon binding to their ligands and co-chaperones. Complementary data will be provided by time-resolved fluorescence energy transfer and electron spin resonance methods, as well as computational approaches based on sequence analysis, normal mode calculations, and ensemble-based thermodynamic dissection of ligand-mediated energetics. Hsp70s constitute relatively simple allosteric machines. Studying in detail their allosteric interdomain communication will shed light on the broader puzzle of how proteins harness ligand-binding energy to modulate binding and catalytic functions at a distance. PUBLIC HEALTH RELEVANCE Hsp70 molecular chaperones play key cellular roles under normal physiological conditions and enable cells to withstand stress such as heat shock. Hsp70s are known to be anti-apoptotic and up- regulated in tumors; ironically, their up-regulation is protective against neurodegenerative diseases caused by protein misfolding. The intimate involvement of Hsp70s in both normal and disease states has led to their emergence as possible therapeutic targets, but using heat shock proteins in a therapeutic capacity requires that we fully understand their mechanism of action, including how nucleotide modulates substrate binding and how interactions with co-chaperones modulate Hsp70 allostery.