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.

摘要 在以前的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，并为基于结构的表征这种酶如何与 生理底物解开凝血因子的结构仍然是一个重要的问题。 这是一项具有挑战性的任务，因为很难获得含有多个蛋白质的高分辨率结构， 域.我们最近展示了如何通过使用cryo-EM来应对这一挑战，cryo-EM是新的黄金标准， 对生物大分子结构的研究。提议中的研究项目是我们 关于人类凝血因子V和Va游离并与Xa因子结合的开创性cryo-EM结构工作， 凝血酶原酶复合物和结合凝血酶原的该复合物的结构。具体来说，我们计划解决 凝血因子V与凝血酶（aim 1）及其活性前体美佐凝血酶（aim）复合物冷冻电镜结构 2)目的是揭示凝血反应起始阶段关键步骤的分子基础 导致凝血酶原酶复合物的组装。该提案得到了令人兴奋的初步低温- 目前，凝血酶-因子V复合物（aim 1）的EM图分辨率为6.6 nm，凝血酶-因子V复合物（aim 1）的EM图分辨率为3.8 nm。 甲藻血红蛋白-fV复合物（目的2）。一旦完全完善，这些结构将揭示不同的模式， 凝血酶和甲藻凝血酶在优选活化位点的结合和甲藻凝血酶的完整结构 第一次基础表位将通过诱变以下抗原的特定残基来独立验证： 此外，结构将测试凝血酶的结合 和甲唑凝血酶与因子V结合，使无序的B结构域刚性化，并使结构决定因素可视化， 保持因子V处于非活动状态。拟议研究的成功将大大提高我们的基础知识 对因子V及其活化的影响，与血液中的其他多结构域蛋白直接相关 凝血级联反应，这可能有利于新的治疗策略的发展。