Structural enzymology of factor V activation

V 因子激活的结构酶学

• 批准号: 10654432
• 负责人: Enrico Di Cera
• 金额: $ 54.88万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10654432
• 关键词: Active Sites; Address; Architecture; Area; Autolysis; Binding; Biological; Blood Coagulation Factor; Blood coagulation; C2 Domain; Coagulation Process; Complement; Complex; Cryoelectron Microscopy; Development; Disease; Docking; Enzymatic Biochemistry; Enzyme Interaction; Enzyme Precursors; Enzymes; Epitopes; Equilibrium; Factor V; Factor Va; Factor Xa; Functional disorder; Goals; Hemostatic function; Human; Investigation; Knowledge; Kringles; Length; Maps; Membrane; Molecular; Molecular Conformation; Mutagenesis; Mutation; Pathway interactions; Peptide Hydrolases; Phase; Physiological; Positioning Attribute; Protease Domain; Proteins; Prothrombin; Research Project Grants; Resolution; Site; Site-Directed Mutagenesis; Structure; Surface; System; Tertiary Protein Structure; Testing; Therapeutic; Thrombin; Thrombosis; Time; Visualization; Work; X-Ray Crystallography; activated Protein C; cofactor; enzyme structure; macromolecule; meizothrombin; novel therapeutic intervention; novel therapeutics; prothrombinase complex; response; structural biology; structural determinants; success

Abstract Work under previous support has broadened our understanding of the factors that influence the catalytic activity of thrombin and set the stage for a structure-based characterization of how this enzyme interacts with physiological substrates. Unraveling the architecture of factors involved in blood coagulation remains a challenging task because of the difficulty of obtaining high resolution structures for proteins containing multiple domains. We have recently shown how to address this challenge by using cryo-EM, the new gold standard for the structural investigation of biological macromolecules. The proposed research project is a segue to our pioneering cryo-EM structural work on human coagulation factors V and Va free and bound to factor Xa in the prothrombinase complex and the structure of this complex bound to prothrombin. Specifically, we plan to solve the cryo-EM structures of factor V in complex with thrombin (aim 1) and its active precursor meizothrombin (aim 2) with the goal of revealing the molecular basis of a key step of the initiation phase of the coagulation response that leads to assembly of the prothrombinase complex. The proposal is supported by exciting preliminary cryo- EM maps currently refined at 6.6 Å resolution for the thrombin-factor V complex (aim 1) and 3.8 Å resolution for the meizothrombin-fV complex (aim 2). Once fully refined, these structures will reveal the distinct modes of binding of thrombin and meizothrombin at preferred sites of activation and the full architecture of meizothrombin for the first time. Underlying epitopes will be validated independently by mutagenesis of specific residues of thrombin, meizothrombin and factor V. In addition, the structures will test the hypothesis that binding of thrombin and meizothrombin to factor V rigidifies the disordered B domain and visualizes the structural determinants that keep factor V in its inactive state. Success of the proposed studies will significantly advance our basic knowledge on factor V and its activation in ways that are directly relevant to other multidomain proteins in the blood coagulation cascade and that may benefit the development of new therapeutic strategies.

抽象的 在先前支持下的工作拓宽了我们对影响催化活性的因素的理解 凝血酶的结构，并为该酶如何与凝血酶相互作用的基于结构的表征奠定基础 阐明参与血液凝固的因素的结构仍然是一个难题。 由于很难获得含有多个蛋白质的高分辨率结构，因此这项任务具有挑战性 我们最近展示了如何通过使用冷冻电镜（新的黄金标准）来应对这一挑战。 生物大分子的结构研究是我们的后续研究项目。 开创性的冷冻电镜结构工作，研究人体凝血因子 V 和 Va 游离并与因子 Xa 结合 具体而言，我们计划解决凝血酶原复合物以及该复合物与凝血酶原结合的结构。 因子 V 与凝血酶复合物（目标 1）及其活性前体甲凝血酶（目标 2）目的是揭示凝血反应起始阶段关键步骤的分子基础 该提议得到了令人兴奋的初步冷冻的支持。 目前，凝血酶因子 V 复合物（目标 1）的 EM 图分辨率为 6.6 Å，凝血酶因子 V 复合物的分辨率为 3.8 Å。 一旦完全完善，这些结构将揭示酶促凝血酶-fV复合物的不同模式。 凝血酶和联用凝血酶在首选激活位点的结合以及联用凝血酶的完整结构 首次通过特定残基的诱变来独立验证基础表位。 此外，该结构将检验凝血酶结合的假设 和凝血酶 V 使无序的 B 结构域变得僵化，并使结构决定因素可视化 保持因子 V 处于非活性状态。拟议研究的成功将显着提高我们的基础知识。 因子 V 及其激活方式与血液中其他多结构域蛋白直接相关 凝血级联反应可能有利于新治疗策略的开发。