Structure and Regulation of hsp90 Chaperones

hsp90 分子伴侣的结构和调控

• 批准号: 6921220
• 负责人: DANIEL T GEWIRTH
• 金额: $ 11.1万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2005-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6921220
• 关键词: X ray crystallography; conformation; endoplasmic reticulum; heat shock proteins; ligands; molecular chaperones; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; protein binding; protein protein interaction; protein structure function; site directed mutagenesis; small molecule; stereochemistry

DESCRIPTION (provided by applicant): The hsp90 chaperones participate in the maturation of proteins involved in diverse cellular activities ranging from signaling to bacterial recognition. They are also key players in the response to cell stress events. Cytoplasmic Hsp90s guide the maturation of steroid hormone receptors and proto-oncogenic kinases, and hsp90 inhibitors disrupt this maturation and display potent anti-cancer activity. GRP94, the ER hsp90 paralog, chaperones proteins destined for transport to the cell surface, including the Toll-like receptors. Cells deficient in GRP94 are unresponsive to microbial stimuli. GRP94 has also been identified as a tumor rejection antigen and elicits suppression of tumor growth and metastasis. The activity of hsp90 chaperones is regulated by small molecule ligands such as ATP that bind to the N-terminal domain, as well as by their conformational state. Key questions that remain unanswered include how ligands exert their inhibitory or activation effects, how different hsp90 paralogs are selectively regulated, how changes in conformation lead to altered activity, and how client proteins interact with the chaperone. An understanding of the stereochemistry that underlies hsp90 biology and hsp90-targeted therapies requires the structural analysis of the relevant macromolecular species. Using X-ray crystallography, NMR spectroscopy, and biochemical- and cell-based assays, the experiments in this proposal will address how GRP94 is regulated by small molecule ligands and interacts with client proteins. We will solve and analyze the structure of the N-terminal domain of GRP94 alone and in complex with activators, broad-spectrum antagonists, and selective inhibitors. Novel selective inhibitors of GRP94 will be designed and synthesized based on these structures. We also propose to examine the interaction of GRP94 with peptides, building on recent work that identified a peptide-binding site in the N-domain. We also plan to investigate the structural basis for N-domain dimerization and oligomerization by constructing GRP94 and Hsp90 molecules lacking candidate interacting elements and determining their functional properties. Finally, the structures of multi-domain GRP94 constructs, including full-length variants, alone or in complex with client proteins will be determined in order to understand the interplay between the domains and the mechanism of substrate interaction. Structural findings will be correlated with measurements of ligand affinity and chaperone activity in both wild-type and mutationally altered molecules.

描述（由申请人提供）：hsp 90分子伴侣参与蛋白质的成熟，所述蛋白质涉及从信号传导到细菌识别的多种细胞活性。它们也是细胞应激反应的关键参与者。细胞质Hsp 90指导类固醇激素受体和原癌基因激酶的成熟，并且Hsp 90抑制剂破坏这种成熟并显示出有效的抗癌活性。GRP 94，ER hsp 90伴侣，注定要运输到细胞表面的伴侣蛋白，包括Toll样受体。缺乏GRP 94的细胞对微生物刺激无反应。GRP 94还被鉴定为肿瘤排斥抗原，并可抑制肿瘤生长和转移。 hsp 90分子伴侣的活性受小分子配体如结合到N-末端结构域的ATP以及它们的构象状态调节。关键的问题仍然没有答案，包括配体如何发挥其抑制或激活作用，如何不同的热休克蛋白90旁系同源物的选择性调节，构象的变化如何导致改变的活动，以及客户端蛋白如何与伴侣相互作用。 对hsp 90生物学和hsp 90靶向治疗的立体化学的理解需要相关大分子物质的结构分析。使用X射线晶体学，NMR光谱学以及基于生物化学和细胞的测定，本提案中的实验将解决GRP 94如何受小分子配体调节并与客户蛋白相互作用。我们将解决和分析的N-末端结构域的GRP 94单独和复杂的激活剂，广谱拮抗剂和选择性抑制剂。基于这些结构将设计和合成新型的GRP 94选择性抑制剂。我们还建议研究GRP 94与肽的相互作用，建立在最近的工作中，确定了N-结构域中的肽结合位点。我们还计划通过构建缺乏候选相互作用元件的GRP 94和Hsp 90分子并确定其功能特性来研究N结构域二聚化和寡聚化的结构基础。最后，将确定多结构域GRP 94构建体（包括全长变体）单独或与客户蛋白复合的结构，以了解结构域之间的相互作用和底物相互作用的机制。结构研究结果将与野生型和突变改变的分子中的配体亲和力和伴侣活性的测量相关。