Structural enzymology of protein C

蛋白 C 的结构酶学

• 批准号: 10617783
• 负责人: Enrico Di Cera
• 金额: $ 53.98万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10617783
• 关键词: Active Sites; Acute; Address; Anticoagulants; Architecture; Area; Binding; Biological; Biology; Blood Coagulation Factor; Blood coagulation; Coagulation Process; Complement; Complex; Cryoelectron Microscopy; Development; Disease; Docking; EGF gene; Electrostatics; Engineering; Enzymatic Biochemistry; Enzyme Precursors; Enzymes; Epitopes; Exposure to; Factor V; Factor Va; Factor Xa; Feedback; Fibrinolysis; Fluorescence Resonance Energy Transfer; Functional disorder; Human; Investigation; Kinetics; Knowledge; Length; Maps; Molecular; Molecular Conformation; Peptide Hydrolases; Peptides; Positioning Attribute; Protease Domain; Protein C; Protein S; Proteins; Prothrombin; Regulation; Research; Research Project Grants; Resolution; Shapes; Side; Site; Solvents; Specificity; Structure; Techniques; Tertiary Protein Structure; Testing; Therapeutic; Thrombin; Thrombomodulin; Time; Variant; X-Ray Crystallography; activated Protein C; biophysical techniques; cofactor; data acquisition; expectation; innovation; interest; macromolecule; particle; protein function; protein structure; prothrombinase complex; response; scaffold; single molecule; success

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.

抽象的 拟议的研究项目继续并扩大了我们对凝血酶与凝血酶相互作用的研究 抗凝蛋白 C 负责凝血反应的关键反馈调节。项目 使用创新的结构方法解决该领域未解决的问题，并计划填补现有空白 关于蛋白 C 作为凝血酶-血栓调节蛋白复合物的底物并被激活的基础知识 蛋白 C 作为一种使因子 Va 失活的酶。揭示参与其中的多域因子的结构 凝血、补体和纤溶仍然是一项具有挑战性的任务，因为很难获得 高分辨率结构。当考虑涉及这些因素的复杂情况时，这种限制甚至更加严重 及其大分子底物或活化剂。我们的方法通过冷冻电镜直接解决这一挑战， 生物大分子结构研究的新金标准。以我们最近的 成功解决人类凝血因子V和Va的结构，提出的研究项目计划 以与其他多域蛋白相关的方式彻底改变蛋白 C 的结构酶学， 它们在凝血级联中的复合物。为此，我们初步获得了冷冻电镜 蛋白质 C 的结构游离并与凝血酶-血栓调节蛋白复合物结合。一旦按计划全面完善 在目标 1 下，这些前所未有的结构将揭示蛋白 C 激活机制并测试 假设血栓调节蛋白通过提供支架来促进凝血酶与蛋白 C 的相互作用 改变它们的构象并减轻静电冲突。此外，我们还获得了初步的冷冻 游离的活化蛋白C和制备的与因子Va结合的活化蛋白C的稳定颗粒的电镜结构 和蛋白质 S 用于冷冻电镜数据采集。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 激活。此外，有关识别因子 Va 的表位的直接信息将使结构成为可能。 基于活化蛋白 C 变体的工程改造，具有改变的特异性，可用于潜在的治疗应用。