Allosteric Mechanism of Hsp70 Molecular Chaperones

Hsp70分子伴侣的变构机制

• 批准号: 7924926
• 负责人: LILA M GIERASCH
• 金额: $ 35.11万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7924926
• 关键词: ATPase Domain; Actins; Apoptotic; Binding; Cell physiology; Cells; Cereals; Chemicals; Communication; Complement; Coupling; Data; Deer; Disease; Dissection; Electron Spin Resonance Spectroscopy; Energy Transfer; Escherichia coli; Fluorescence; Fluorescence Resonance Energy Transfer; Heat shock proteins; Heat-Shock Proteins 70; Heat-Shock Response; Homeostasis; Homology Modeling; Individual; Knowledge; Ligand Binding; Ligands; Light; Location; Macromolecular Complexes; Mediating; Methods; Modeling; Molecular; Molecular Chaperones; Mutagenesis; Neurodegenerative Disorders; Nucleotides; Organism; Physiological; Play; Positioning Attribute; Property; Proteins; Research; Role; Sequence Analysis; Site; Spin Labels; Stress; Structure; Substrate Domain; Testing; Therapeutic; Thermodynamics; Time; Up-Regulation; Work; Yeasts; base; cell injury; comparative; conformational conversion; crosslink; fluorophore; protein misfolding; public health relevance; research study; shape analysis; therapeutic target; tumor

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.

描述（由申请人提供）：Hsp70 伴侣存在于所有生物体和基本上所有细胞区室中。在其广泛的基本细胞功能中，它们促进新合成蛋白质的折叠；保护细胞免受应激条件下可能发生的损伤，例如聚集；帮助将蛋白质靶向至细胞质外位置；并促进大分子复合物的组装和分解。所有这些功能都依赖于 Hsp70 结合蛋白质底物的未折叠区域并在 ATP 变构结合后释放其底物的能力。本研究重点关注Hsp70变构的基本分子机制。所提出的工作建立在令人兴奋的最新结果的基础上：我们在上一个项目期间表明，典型的大肠杆菌 Hsp70 DnaK 的 ATP 酶结构域和底物结合结构域 (SBD) 在 ATP 结合时经历了主要的构象变化，并且我们了解了这些结构域的变构重塑。我们的结果使我们建立了 DnaK 中域间变构通讯的模型，该模型已通过 Hendrickson 实验室的相关伴侣 Sse1（酵母 Hsp110）的最新结构得到验证。 Liu 和 W. A. Hendrickson，细胞 131, 106-1202007)]。我们的具体目标是：完善目前基于 Sse1 的 ATP 结合 DnaK 同源模型，并使用该模型，结合我们对 DnaK 的 ADP 结合状态的了解，阐明 Hsp70 分子伴侣中变构域间通信的机制；评估 DnaK 结果的普遍性并制定有关 Hsp70 变构功能的一般原则；探索如何通过与共伴侣 DnaJ 和 GrpE 的相互作用来调节 DnaK 的变构构象变化。我们将利用适用于大分子的新 NMR 策略来分析 Hsp70 在与其配体和共伴侣结合后变构构象转变的结构和动态方面。补充数据将通过时间分辨荧光能量转移和电子自旋共振方法以及基于序列分析、正常模式计算和配体介导的能量学的基于系综的热力学剖析的计算方法提供。 Hsp70 构成相对简单的变构机器。详细研究它们的变构域间通信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。公共卫生相关性 Hsp70 分子伴侣在正常生理条件下发挥关键的细胞作用，并使细胞能够承受热休克等应激。已知 Hsp70 在肿瘤中具有抗细胞凋亡和上调作用；讽刺的是，它们的上调可以预防由蛋白质错误折叠引起的神经退行性疾病。 Hsp70 在正常和疾病状态下的密切参与导致它们成为可能的治疗靶点，但在治疗中使用热休克蛋白需要我们充分了解它们的作用机制，包括核苷酸如何调节底物结合以及与共伴侣的相互作用如何调节 Hsp70 变构。