Structural basis of von Willebrand factor biology and physics

冯维勒布兰德因子生物学和物理学的结构基础

• 批准号: 10198035
• 负责人: TIMOTHY A SPRINGER
• 金额: $ 67.37万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10198035
• 关键词: Address; Affinity; Anabolism; Binding; Binding Sites; Biology; Blood Coagulation Disorders; Blood Coagulation Factor; Blood Platelets; Blood coagulation; C-terminal; Calorimetry; Coagulation Process; Complement Factor D; Complex; Cryoelectron Microscopy; Crystallization; Crystallography; Cysteine; Development; Dimerization; Disease; Disulfides; Endothelial Cells; Engineering; F8 gene; Factor VIII; Glycoproteins; Goals; Half-Life; Hemophilia A; Hemorrhage; Hemostatic Agents; Hemostatic function; Heritability; In Vitro; Inherited; Interferometry; Lead; Length; Link; Mediating; Modeling; Molecular Conformation; Mosaicism; Mucins; Mutation; N-terminal; Pattern; Pharmaceutical Preparations; Physics; Plasma Proteins; Platelet Glycoproteins; Play; Polysaccharides; Population; Production; Proprotein Convertases; Protein Engineering; Proteins; Replacement Therapy; Resolution; Ristocetin; Role; Site; Stroke; Structure; Testing; Therapeutic; Thrombosis; Weibel-Palade Bodies; crosslink; design; dimer; disulfide bond; experimental study; gain of function mutation; glycosylation; hydrodynamic flow; improved; monomer; reconstitution; structural biology; von Willebrand Disease; von Willebrand Factor

von Willebrand factor (VWF) is a multi-domain plasma protein secreted by endothelial cells. In hemostasis, VWF binds and crosslinks platelets to one another and the vessel wall to form the platelet plug. VWF also binds to and stabilizes factor VIII (FVIII) in the coagulation cascade. VWF mutations cause the most common heritable bleeding disorders called von Willebrand disease (VWD). The D1, D2, and D´D3 assemblies in VWF are specialized domains that enable biosynthesis of VWF into ultralong concatemers that are stored as helical tubules in Weibel-Palade bodies (WPBs). D´D3 also binds FVIII. Long length enables VWF to sense flow. Changes in flow at sites of bleeding activate VWF by 1) elongating coiled VWF concatemers into a thread-like conformation that exposes previously buried A1 domains and 2) activating a high-affinity state of VWF A1 domains that bind platelet glycoprotein Ibα (GPIbα) for platelet plug formation. High-resolution structures of D assemblies and the high-affinity state of A1 are lacking. In Aim 1, we will determine the structure of the high- affinity state of A1. Unfolding studies show that VWF A2 and A3 domains have two states, whereas A1 has three: native, intermediate, and unfolded. Preliminary studies show that truncating the O-glycosylated linkers N- and C-terminal destabilizes the native state of A1 and increases affinity for GPIbα. We propose that the intermediate state corresponds to the high-affinity state of A1. We test the hypothesis that further truncation of the linkers flanking A1, gain-of-function mutations (e.g. activating VWD mutations), and the allosteric activator ristocetin all increase A1 affinity for GPIbα by stabilizing the intermediate state over the native state. We will use combinations of truncations, mutations, and ristocetin to stabilize A1 in the intermediate state and to determine the crystal structure of the putative high-affinity state of A1 and its complex with GPIbα. Aim 2 will determine structures of D´D3 and the D´D3 dimer. Our preliminary crystal structure of the D´D3 monomer shows how the C8, TIL, and E modules pack around the VWD module to form the D3 assembly. D´ protrudes from the D3 assembly. The two cysteines that have been proposed to form the inter-dimer disulfide bonds are buried. We will solve the structure of a D´D3 dimer (D´D3)2 or a D3 dimer with the protruding D´ removed to define the structural rearrangements required for D´D3 dimerization. Proposed disulfide rearrangement that precedes dimerization will be verified by mutation and in vitro reconstitution. As backup, we will pursue a cryo- EM structure of VWF helical tubules to determine the structure of (D´D3)2 and how D assemblies enable formation of highly ordered tubules. Aim 3 uses crystallography to understand how D’D3 binds FVIII, which has the potential through protein engineering to revolutionize replacement FVIII therapy in hemophilia A. As an alternative strategy, we will determine a cryoEM structure of a D’D3 complex with FVIII. Better structural understanding of VWF D assemblies and the high-affinity state of A1 has important therapeutic implications for stroke, thrombosis, VWD, and hemophilia A.

血管性血友病因子（VWF）是内皮细胞分泌的多结构域血浆蛋白。在止血方面， VWF 将血小板彼此以及血管壁结合并交联，形成血小板栓塞。 VWF也 结合并稳定凝血级联中的因子 VIII (FVIII)。 VWF 突变导致最常见的 遗传性出血性疾病称为冯维勒布兰德病 (VWD)。 VWF 中的 D1、D2 和 D´D3 组件 是特殊的域，可将 VWF 生物合成为超长串联体，并以螺旋形式存储 Weibel-Palade 小体 (WPB) 中的小管。 D´D3 还结合 FVIII。长长度使 VWF 能够感测流量。 出血部位流量的变化通过以下方式激活 VWF：1) 将卷曲的 VWF 串联体拉长成线状 暴露先前埋藏的 A1 结构域的构象以及 2) 激活 VWF A1 的高亲和力状态 结合血小板糖蛋白 Ibα（GPIbα）以形成血小板栓塞的结构域。 D 的高分辨率结构 缺乏 A1 的组装和高亲和力状态。在目标 1 中，我们将确定高端的结构 A1的亲和状态。展开研究表明 VWF A2 和 A3 结构域有两种状态，而 A1 具有 三：原生、中间、展开。初步研究表明，截断 O-糖基化接头 N 端和 C 端破坏 A1 天然状态的稳定性并增加对 GPIbα 的亲和力。我们建议 中间状态对应于A1的高亲和力状态。我们检验进一步截断的假设 A1 侧翼的接头、功能获得突变（例如激活 VWD 突变）和变构激活剂 ristocetin 均通过稳定中间状态而非天然状态来增加 A1 对 GPIbα 的亲和力。我们将 使用截断、突变和瑞斯托菌素的组合将 A1 稳定在中间状态并 确定 A1 及其与 GPIbα 的复合物的假定高亲和力状态的晶体结构。目标2将 确定 D´D3 和 D´D3 二聚体的结构。我们的 D´D3 单体的初步晶体结构 显示 C8、TIL 和 E 模块如何包装在 VWD 模块周围以形成 D3 组件。 D´ 突出 来自 D3 组件。已提出形成二聚体间二硫键的两个半胱氨酸是 埋葬了。我们将解析 D´D3 二聚体 (D´D3)2 或删除了突出的 D´ 的 D3 二聚体的结构 定义 D´D3 二聚化所需的结构重排。提议的二硫键重排 二聚化之前的情况将通过突变和体外重建来验证。作为备份，我们将追求冷冻 VWF 螺旋管的 EM 结构以确定 (D´D3)2 的结构以及 D 组件如何实现 形成高度有序的小管。 Aim 3 使用晶体学来了解 D’D3 如何结合 FVIII，其中 通过蛋白质工程彻底改变血友病 A 的 FVIII 替代疗法的潜力。 另一种策略是，我们将确定 D’D3 与 FVIII 复合物的冷冻电镜结构。更好的结构 了解 VWF D 组装体和 A1 的高亲和力状态对于以下疾病具有重要的治疗意义： 中风、血栓形成、VWD 和 A 型血友病。