Structural enzymology of factor V activation

摘要 在以前的支持下进行的工作拓宽了我们对影响催化活性的因素的理解 并为基于结构的表征这种酶是如何与 生理底物。解开参与凝血的因子的结构仍然是一个 一项具有挑战性的任务，因为难以获得含有多个 域名。我们最近展示了如何通过使用Cryo-EM来应对这一挑战，这是 生物大分子的结构研究。拟议的研究项目是我们 冷冻-EM在人凝血因子V和Va上的开创性结构工作 凝血酶原酶复合体及该复合体的结构与凝血酶原结合。具体地说，我们计划解决 凝血酶-凝血酶复合体(AIM-1)及其活性前体甲硫唑凝血酶(AIM)中凝血因子V的冷冻-电子显微镜结构 2)目的是揭示凝血反应启动阶段的关键步骤的分子基础 这会导致凝血酶原酶复合体的组装。这项提议得到了令人兴奋的初步低温的支持- EM图目前针对凝血酶-因子V复合体(AIM 1)以6.6？分辨率改进，对于 甲硝唑-凝血酶-抗体复合体(目标2)。一旦完全细化，这些结构将揭示出 凝血酶和甲硫咪唑凝血酶在优先激活部位的结合及甲硫咪唑凝血酶的完整结构 这是第一次。潜在的表位将通过对特定残基的突变来独立验证 此外，这些结构将检验凝血酶结合凝血酶的假说。 而因子V的甲硫托凝血酶使无序的B结构域刚性，并使结构决定因素可视化 保持因子V处于非活性状态。拟议研究的成功将大大提高我们的基础知识 因子V及其与血液中其他多结构域蛋白直接相关的激活方式 凝血级联，这可能有助于开发新的治疗策略。

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.