Structure and Regulation of hsp90 Chaperones

hsp90 分子伴侣的结构和调控

• 批准号: 7173120
• 负责人: DANIEL T GEWIRTH
• 金额: $ 18.81万
• 依托单位: HAUPTMAN-WOODWARD MEDICAL RESEARCH INST
• 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 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7173120
• 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）：hsp90 伴侣参与涉及从信号传导到细菌识别等多种细胞活动的蛋白质的成熟。它们也是应对细胞应激事件的关键参与者。细胞质 Hsp90 引导类固醇激素受体和原癌激酶的成熟，而 hsp90 抑制剂会破坏这种成熟并表现出有效的抗癌活性。 GRP94 是 ER hsp90 旁系同源物，是转运至细胞表面的伴侣蛋白，包括 Toll 样受体。缺乏 GRP94 的细胞对微生物刺激没有反应。 GRP94 还被鉴定为肿瘤排斥抗原，可抑制肿瘤生长和转移。 hsp90 分子伴侣的活性受到小分子配体（例如与 N 末端结构域结合的 ATP）及其构象状态的调节。仍未得到解答的关键问题包括配体如何发挥其抑制或激活作用、如何选择性地调节不同的 hsp90 旁系同源物、构象变化如何导致活性改变以及客户蛋白如何与伴侣相互作用。 要了解 hsp90 生物学和 hsp90 靶向疗法的立体化学，需要对相关大分子种类进行结构分析。该提案中的实验将使用 X 射线晶体学、核磁共振波谱以及生化和细胞分析来解决 GRP94 如何受小分子配体调节以及如何与客户蛋白相互作用。我们将解析和分析GRP94单独的N端结构域以及与激活剂、广谱拮抗剂和选择性抑制剂复合物的结构。基于这些结构将设计和合成新型GRP94选择性抑制剂。我们还建议以最近确定 N 结构域中肽结合位点的工作为基础，研究 GRP94 与肽的相互作用。我们还计划通过构建缺乏候选相互作用元件的 GRP94 和 Hsp90 分子并确定其功能特性来研究 N 结构域二聚和寡聚的结构基础。最后，将确定多结构域 GRP94 构建体的结构，包括全长变体，单独或与客户蛋白复合，以了解结构域之间的相互作用和底物相互作用的机制。结构发现将与野生型和突变分子中配体亲和力和伴侣活性的测量相关。