Structure and Regulation of hsp90 Chaperones

描述(由申请人提供)：HSP90伴侣参与蛋白质的成熟，涉及从信号到细菌识别的各种细胞活动。它们也是应对细胞应激事件的关键参与者。胞质中的HSP90可引导类固醇激素受体和原癌蛋白激酶的成熟，而HSP90抑制剂可阻断这一成熟过程，并显示出强大的抗癌活性。GRP94，ER HSP90 Paralog，伴侣蛋白，目的是运输到细胞表面，包括Toll样受体。缺乏GRP94的细胞对微生物刺激没有反应。GRP94也被认为是一种肿瘤排斥抗原，可以抑制肿瘤的生长和转移。 HSP90分子伴侣的活性受与N-末端结构域结合的小分子配体(如ATP)及其构象状态的调节。尚未解答的关键问题包括配体如何发挥其抑制或激活作用，不同的HSP90辅基如何被选择性地调控，构象的变化如何导致活性的改变，以及客户蛋白如何与伴侣蛋白相互作用。 要理解HSP90生物学和HSP90靶向治疗所依据的立体化学，需要对相关的大分子物种进行结构分析。利用X射线结晶学、核磁共振光谱以及生化和细胞分析，本提案中的实验将研究GRP94如何受小分子配体调节并与客户蛋白质相互作用。我们将单独解决和分析GRP94的N-末端结构域的结构，以及与激活剂、广谱拮抗剂和选择性抑制剂的复合体。基于这些结构，我们将设计和合成新型的GRP94选择性抑制剂。我们还建议在最近的工作基础上，研究GRP94与多肽的相互作用，该工作确定了N-结构域中的一个多肽结合位点。我们还计划通过构建缺乏候选相互作用元件的GRP94和Hsp90分子并确定它们的功能性质来研究N-结构域二聚和寡聚的结构基础。最后，将确定多结构域GRP94结构的结构，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联。

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.