Structural enzymology of protein C

摘要 拟议的研究项目继续和扩大我们的调查凝血酶的相互作用与 抗凝血蛋白C负责凝血反应的关键反馈调节。项目 采用创新的结构性方法和计划解决外地未解决的问题，以填补现有差距 在关于蛋白C作为凝血酶-血栓调节蛋白复合物的底物和活化的基础知识中， 蛋白C作为使因子Va失活的酶。揭示涉及的多领域因素的体系结构 血液凝固、补体和纤维蛋白溶解仍然是一项具有挑战性的任务，因为难以获得 高分辨率结构。当考虑到涉及这些因素的复合物时，这种局限性甚至更加严重 以及它们的大分子底物或活化剂。我们的方法直接用冷冻EM解决了这一挑战， 生物大分子结构研究的新黄金标准。基于我们最近 成功解决了人凝血因子V和Va的结构，提出了研究项目计划 以一种与其他多结构域蛋白相关的方式彻底改变蛋白C的结构酶学， 它们的复合物在血液凝固级联反应中的作用。为此，我们已经获得了初步的冷冻电镜 蛋白C的游离结构和与凝血酶-血栓调节蛋白复合物的结合。一旦按计划完全完善 根据目标1，这些前所未有的结构将揭示蛋白C激活的机制，并测试蛋白C的功能。 假设血栓调节蛋白通过提供一种支架， 改变了它们的构象并消除了静电冲突。此外，我们还获得了初步的低温- 游离活化蛋白C和制备的与因子Va结合的活化蛋白C的稳定颗粒的EM结构 和蛋白质S用于冷冻EM数据采集。这些研究的进展将阐明， 活化的蛋白C与其酶原形式相比，将进一步完善蛋白C的机制 activation.此外，关于因子Va的识别表位的直接信息将使结构- 具有改变的特异性的活化蛋白C的变体的基于工程的潜在治疗应用。

Abstract The proposed research project continues and expands our investigation of the interaction of thrombin with the anticoagulant protein C responsible for a key feedback regulation of the coagulation response. The project addresses unresolved issues in the field using an innovative structural approach and plans to fill existing gaps in basic knowledge about protein C as a substrate of the thrombin-thrombomodulin complex and activated protein C as an enzyme that inactivates factor Va. Unraveling the architecture of multidomain factors involved in blood coagulation, complement and fibrinolysis remains a challenging task because of the difficulty of obtaining high resolution structures. This limitation is even more acute when considering complexes involving these factors and their macromolecular substrates or activators. Our approach addresses this challenge directly with cryo-EM, the new gold standard for the structural investigation of biological macromolecules. Building on our recent success in solving the structures of human coagulation factors V and Va, the proposed research project plans to revolutionize the structural enzymology of protein C in a way that is relevant to other multidomain proteins and their complexes in the blood coagulation cascade. Toward this end, we have obtained preliminary cryo-EM structures of protein C free and bound to the thrombin-thrombomodulin complex. Once fully refined as planned under aim 1, these unprecedented structures will unravel the mechanism of protein C activation and test the hypothesis that thrombomodulin promotes the interaction of thrombin with protein C by offering a scaffold that changes their conformation and alleviates electrostatic clash. In addition, we have obtained a preliminary cryo- EM structure of activated protein C free and prepared stable particles of activated protein C bound to factor Va and protein S for cryo-EM data acquisition. Progress from these studies will elucidate how the structure of activated protein C compares to that of its zymogen form and will further refine the mechanism of protein C activation. Furthermore, direct information on the epitopes of recognition of factor Va will enable a structure- based engineering of variants of activated protein C with altered specificity for potential therapeutic applications.