Structural Basis of Protease-Activated Receptor Function

描述(申请人提供)：蛋白酶激活受体(PARs)是G蛋白偶联受体(GPCRs)，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，PARS将组织损伤与细胞反应联系起来，帮助协调止血和血栓形成、炎症、细胞保护、修复和痛觉。考虑到这些和其他角色，PAR是潜在的药物靶标。现有证据支持一种模型，即凝血酶通过在特定位点裂解受体的N-末端外区来激活原型PAR，从而产生一个新的N-末端，然后作为一种拴系的多肽激动剂，与受体的七螺旋束分子内结合，从而影响受体的激活。缺乏测试这一模型并揭示拴系配体如何结合以及这种结合如何驱动跨膜域(TM)移动和G蛋白激活的结构，以及支持靶向PARS药物开发的结构。基于我们最近合成的非活化态PAR1与拮抗剂沃拉帕沙的复合晶体结构，我们提出了从结构水平上阐明PAR的激活、信号传递和拮抗机制的研究。我们将1)解决凝血酶激活的PAR1与GI或GQ或G12/13形成的复合体中的晶体结构。2)确定Vorapaxar通过密切相关的受体特异性PAR1的基础和Vorapaxar进入(从质膜或细胞外空间)的途径，以及3)解决PAR2拮抗剂复合体的晶体结构。将使用尖端结晶学方法，包括使用稳定的纳米体和单粒子EM来评估络合物。分子动力学模拟将有助于突变研究的设计和解释。我们对PAR1的研究将揭示PAR1结合并触发TM运动和G蛋白激活的机制、PAR1‘S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于特异性的重要性。对PAR1-vorapaxar结构和PAR2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，这些拮抗剂是探索这些受体在人类疾病中的作用所需的。我们的研究将提供一个肽激动剂-GPCRG蛋白复合体的第一个结构，以及第一个关于单个GPCRs的不同构象是否识别不同的G蛋白的结构洞察力。

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.