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展了我们对凝血酶与凝血酶相互作用的研究 抗凝蛋白C负责对凝血反应的关键反馈调节。项目 通过创新的结构方法解决现场未解决的问题，并计划填补现有空白 关于蛋白C作为凝血酶 - 肉毒杆菌蛋白复合蛋白复合物的底物的基本知识，并激活了 蛋白C作为灭活因子VA的酶。阐明涉及的多域因子的结构 血液凝血，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。在考虑涉及这些因素的复合物时，这种限制更加敏锐 及其大分子底物或激活剂。我们的方法直接解决了Cryo-Em的挑战， 生物大分子结构研究的新金标准。建立在我们最近的 在解决人类凝血因素V和VA的结构方面的成功，拟议的研究项目计划 以与其他多域蛋白质相关的方式彻底改变蛋白质C的结构酶学 它们在血液凝结级联中的复合物。为此，我们获得了初步的冷冻EM 蛋白C的结构不含蛋白质，并结合到凝血酶 - 肉毒杆菌蛋白复合物。一旦按计划进行了完整完整 在AIM 1下，这些前所未有的结构将揭示蛋白质C激活的机制，并测试 假设血小板蛋白可以通过提供脚手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，我们获得了初步的冷冻 活化蛋白C的EM结构无活化蛋白C的稳定颗粒与因子VA结合 和用于冷冻EM数据采集的蛋白质。这些研究的进展将阐明 活化的蛋白C与其酶原形式的蛋白C相比，将进一步完善蛋白C的机制 激活。此外，有关因子识别因子识别的表位的直接信息将使结构 - 基于对潜在治疗应用的特异性改变的活化蛋白C变体的工程。