Structural enzymology of protein C

摘要 拟议的研究项目继续并扩大了我们对凝血酶与血管内皮细胞之间相互作用的研究。 抗凝蛋白C负责凝血反应的关键反馈调节。该项目 使用创新的结构方法解决外地未解决的问题，并计划填补现有差距 了解蛋白C作为凝血酶-血栓调节蛋白复合体的底物并被激活的基本知识 蛋白C是一种使Va因子失活的酶。拆解涉及的多领域因素的体系结构 凝血、补体和纤溶仍然是一个具有挑战性的任务，因为很难获得 高分辨率结构。当考虑到涉及这些因素的复杂情况时，这种限制就更加严重 以及它们的大分子底物或激活剂。我们的方法直接通过低温EM解决了这一挑战， 生物大分子结构研究的新金标准。在我们最近的基础上 成功解决人凝血因子V和Va的结构，建议的研究项目计划 以一种与其他多结构域蛋白相关的方式革命性地改变蛋白C的结构酶学，并 它们的复合体在血液凝固过程中级联。为此，我们已经获得了初步的低温电磁 蛋白C与凝血酶-血栓调节蛋白复合体结合的结构。一旦按计划完全提炼 在目标1下，这些前所未有的结构将解开蛋白C激活的机制，并测试 血栓调节蛋白通过提供支架促进凝血酶与蛋白C相互作用的假说 改变了它们的构象，减轻了静电碰撞。此外，我们还获得了一个初步的低温- 活化蛋白C的EM结构与凝血因子Va结合制备稳定的活化蛋白C颗粒 和蛋白质S，用于低温电磁数据采集。这些研究的进展将阐明 活化的蛋白C与其酶原形式相比较，将进一步完善蛋白C的作用机制 激活。此外，关于因子Va识别表位的直接信息将使结构- 基于改变特异性的激活蛋白C变体的工程，以用于潜在的治疗应用。

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.