Structural Basis of Protease-Activated Receptor Function

描述（由申请人提供）：蛋白酶激活受体（PAR）是G蛋白偶联受体（GPCR），允许凝血酶和其他细胞外蛋白酶调节细胞行为。与凝血级联反应一起，PAR将组织损伤与细胞反应联系起来，有助于协调止血和血栓形成、炎症、细胞保护、修复和疼痛感知。考虑到这些和其他作用，PAR是潜在的药物靶点。现有证据支持这样一种模型，其中凝血酶通过在特定位点切割受体的N-末端外域以产生新的N-末端来激活原型PAR，PAR 1，所述新的N-末端然后作为拴系肽激动剂起作用，分子内结合至受体的七螺旋束以影响受体激活。缺乏测试该模型并揭示栓系配体如何结合以及这种结合如何驱动跨膜结构域（TM）移动和G蛋白活化的结构，也缺乏支持靶向PAR的药物开发的结构。我们最近的晶体结构的非活性状态PAR 1与拮抗剂伏拉帕沙复合的基础上，我们提出的研究，阐明PAR的激活，信号和拮抗作用的机制在结构水平上。我们将1）解析凝血酶活化的PAR 1与Gi和Gq或G12/13复合的晶体结构。2)确定vorapaxar对PAR 1的特异性超过密切相关受体的基础和vorapaxar进入的途径（从质膜或细胞外空间），以及3）解析PAR 2拮抗剂复合物的晶体结构。将采用尖端晶体学方法，包括使用稳定纳米抗体和单颗粒EM来评估复合物。分子动力学模拟将有助于突变研究的设计和解释。我们对PAR 1的研究将揭示PAR 1栓系激动剂结合并触发TM运动和G蛋白活化的机制、PAR 1与多种G蛋白亚型（Gi、Gq和G12/13）混杂偶联的结构基础、拮抗剂进入的新途径以及进入途径对特异性的重要性。PAR 1-vorapaxar结构和PAR 2晶体结构的详细研究将为基于结构的发现和优化更好的PAR拮抗剂提供入口，这些受体需要探索这些受体在人类疾病中的作用。我们的研究将提供肽激动剂- GPCR-G蛋白复合物的第一个结构，以及对单个GPCR的不同构象是否识别不同G蛋白的第一个结构见解。

DESCRIPTION (provided by applicant): Protease-activated receptors (PARs) are G protein-coupled receptors (GPCRs) that permit thrombin and other extracellular proteases to regulate cellular behaviors. Together with the coagulation cascade, PARs link tissue injury to cellular responses that help orchestrate hemostasis and thrombosis, inflammation, cytoprotection, repair, and pain perception. Given these and other roles, PARs are potential drug targets. Available evidence supports a model in which thrombin activates the prototypical PAR, PAR1, by cleaving the N-terminal exodomain of the receptor at a specific site to generate a new N-terminus that then functions as a tethered peptide agonist, binding intramolecularly to the receptor's heptahelical bundle to effect receptor activation. Structures that test this model and reveal how the tethered ligand binds and how such binding drives transmembrane domain (TM) movement and G protein activation are lacking, as are structures to support development of pharmaceuticals targeting PARs. Building on our recent crystal structure of inactive-state PAR1 complexed with the antagonist vorapaxar, we propose studies to illuminate the mechanism of PAR activation, signaling and antagonism at a structural level. We will 1) Solve crystal structures of thrombin- activated PAR1 in complex with Gi and either Gq or G12/13. 2) Determine the basis for vorapaxar's specificity for PAR1 over closely related receptors and the route of vorapaxar entry (from the plasma membrane or the extracellular space), and 3) Solve the crystal structure of a PAR2-antagonist complex. Cutting edge crystallographic approaches, including use of stabilizing nanobodies and single particle EM to assess complexes, will be employed. Molecular dynamics simulations will aid design and interpretation of mutational studies. Our studies of PAR1 will reveal the mechanism by which the PAR1 tethered agonist binds and triggers TM movement and G protein activation, the structural basis for PAR1's promiscuous coupling to multiple G protein subtypes (Gi, Gq, and G12/13), a novel route of antagonist entry and the importance of entry route for specificity. Detailed studies of the PAR1-vorapaxar structure and the PAR2 crystal structure will provide an entry to structure-based discovery and optimization of better PAR antagonists needed to explore the role of these receptors in human disease. Our studies will provide the first structure of a peptide agonist- GPCR-G protein complex and the first structural insight into whether distinct conformers of individual GPCRs recognize different G proteins.