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中，基本上存在于所有细胞区室中。在它们广泛的基本细胞功能中，它们促进新合成蛋白质的折叠；保护细胞免受损伤，如在压力条件下可能发生的聚集；帮助靶向蛋白到细胞质外的位置；并促进大分子复合物的组装和拆卸。所有这些功能都依赖于hsp70结合蛋白底物未折叠区域的能力，以及在ATP变构结合时释放其底物的能力。本文拟重点研究Hsp70变构的基本分子机制。我们提出的工作建立在最近令人兴奋的结果之上：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结合时都发生了主要的构象变化，并且我们了解了这些结构域的变构重塑。我们的研究结果使我们建立了DnaK结构域间变构通信的模型，该模型已被亨德里克森实验室最近的一个相关伴侣Sse1，酵母Hsp110 [Q]的结构所验证。刘，W. A. Hendrickson, Cell, 131, 106-1202007)。我们的具体目标是：完善目前基于sse1的atp结合DnaK的同源性模型，并利用该模型，结合我们对DnaK的adp结合状态的了解，阐明Hsp70分子伴侣体内变构域间通讯的机制；评估DnaK结果的通用性，建立Hsp70变构功能的一般原则；探讨DnaK的变构变化是如何通过与共伴侣DnaJ和GrpE的相互作用来调节的。我们将利用新的适用于大分子的核磁共振策略来分析hsp70在与其配体和共伴体结合时的变构构象转变的结构和动力学方面。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、正模计算和基于配体介导能量学的集成热力学解剖的计算方法来提供。hsp70是相对简单的变构机。详细研究它们的变构结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。Hsp70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hsp70在肿瘤中具有抗凋亡和上调的作用；具有讽刺意味的是，它们的上调对蛋白质错误折叠引起的神经退行性疾病具有保护作用。Hsp70在正常和疾病状态中的密切参与导致它们作为可能的治疗靶点出现，但是在治疗能力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变体。