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(申请人提供)：HSP70分子伴侣存在于所有生物体中，基本上存在于所有细胞室中。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；保护细胞免受应激条件下可能发生的聚集等损害；帮助将蛋白质靶向细胞质外位置；以及促进大分子复合体的组装和拆解。所有这些功能都依赖于Hsp70结合蛋白质底物未折叠区域的能力，以及在变构结合ATP时释放底物的能力。提出的研究重点是Hsp70变构的基本分子机制。这项工作建立在令人兴奋的最新结果基础上：我们在上一个项目期表明，聚合的E.ColiHsp70DNAK的ATPase结构域和底物结合结构域(SBD)在ATP结合时都经历了主要的构象变化，并了解了这些结构域的变构重塑。我们的结果导致了DNAK中域间变构通信的模型，该模型已经被Hendrickson实验室最近来自Hendrickson实验室的相关伴侣Sse1，酵母Hsp110的结构所验证[Q.Liu和W.A.Hendrickson，Cell131,106-1202007]。我们的具体目标是：完善目前基于Sse1的ATP结合的DNAK的同源模型，并结合我们对DNAK的ADP结合状态的了解，阐明这种Hsp70分子伴侣的变构结构域间通讯的机制；评估关于DNAK的结果的普遍性，并发展关于Hsp70变构功能的一般原理；探索DNAK中的变构构象变化是如何通过与共伴侣DNAJ和GRPE的相互作用来调节的。我们将利用适用于大分子的新的核磁共振策略来分析Hsp70与其配体和共伴侣结合时的变构构象转变的结构和动力学方面。补充数据将由时间分辨荧光能量转移和电子自旋共振方法提供，以及基于序列分析、简正模计算和配体介导的能量学的系综热力学剖析的计算方法。Hsp70构成了相对简单的变构机器。详细研究它们的变构结构域间通讯将有助于揭示蛋白质如何利用配体结合能量来调节远距离的结合和催化功能这一更广泛的谜题。与公共健康相关的Hsp70分子伴侣在正常生理条件下发挥着关键的细胞作用，使细胞能够承受热休克等应激。众所周知，Hsp70在肿瘤中具有抗细胞凋亡和上调表达的作用；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在正常和疾病状态下的密切参与使其成为可能的治疗靶点，但使用热休克蛋白的治疗能力要求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辅助伴侣如何相互作用调节Hsp70变构。