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: 10434710
• 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/10434710
• 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（vonWillebrand factor，VWF）是由内皮细胞分泌的多结构域血浆蛋白。在止血方面， VWF结合并交联血小板彼此和血管壁以形成血小板栓。VWF也 在凝血级联中结合并稳定因子VIII（FVIII）。VWF突变导致最常见的 遗传性出血性疾病称为血管性血友病（VWD）。VWF中的D1、D2和D'D3组装体 是使VWF生物合成为超长串联体的专门结构域， 韦伯-帕拉德小体（Webel-Palade body，WPB）D'D3也结合FVIII。长的长度使VWF能够感测流量。 出血部位血流的变化通过以下方式激活VWF：1）将卷曲的VWF多联体延长成线状 暴露先前被掩埋的A1结构域的构象和2）激活VWF A1的高亲和力状态 结合血小板糖蛋白Ib α（GPIb α）形成血小板栓的结构域。D的高分辨率结构 缺乏A1的高亲和力状态。在目标1中，我们将确定高- A1的亲和力状态。解折叠研究表明，VWF A2和A3结构域有两种状态，而A1结构域有两种状态， 三种：天然的、中间的和未折叠的。初步研究表明，截短O-糖基化接头 N末端和C末端使A1的天然状态不稳定，并增加对GPib α的亲和力。我们建议 中间状态对应于A1的高亲和力状态。我们测试的假设，进一步截断 侧接A1的接头、功能获得性突变（例如激活VWD突变）和变构激活剂 Ristoclavin通过稳定中间态而增加A1对GPIb α的亲和力。我们将 使用截短、突变和逆转录酶的组合以使A1稳定在中间状态， 确定A1及其与GPIb α复合物的推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。我们对D'D3单体的初步晶体结构 显示了C8、TIL和E模块如何围绕VWD模块封装以形成D3组件。D ′突出 从D3组件。已经提出形成二聚体间二硫键的两个半胱氨酸是 埋了我们将解析D ′ D3二聚体（D ′ D3）2或去除突出D ′的D3二聚体的结构， 定义D'D3二聚化所需的结构重排。提出的二硫键重排， 将通过突变和体外重构来验证在二聚化之前发生的突变。作为后援，我们要进行低温- VWF螺旋小管的EM结构，以确定（D ′ D3）2的结构以及D组装如何使 形成高度有序的小管。目的3使用晶体学来了解D'D3如何结合FVIII，其具有 通过蛋白质工程改造血友病A的FVIII替代疗法的潜力。作为 作为替代策略，我们将确定D'D3与FVIII复合物的cryoEM结构。更好的结构 了解VWF D组装和A1的高亲和力状态对治疗具有重要意义， 中风、血栓形成、VWD和血友病A。

项目成果

Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker.

• DOI: 10.7554/elife.75760
• 发表时间: 2022-05-09
• 期刊: ELIFE
• 影响因子: 7.7
• 作者: Bonazza, Klaus;Iacob, Roxana E.;Hudson, Nathan E.;Li, Jing;Lu, Chafen;Engen, John R.;Springer, Timothy A.
• 通讯作者: Springer, Timothy A.

Monomeric prefusion structure of an extremophile gamete fusogen and stepwise formation of the postfusion trimeric state.

• DOI: 10.1038/s41467-022-31744-z
• 发表时间: 2022-07-13
• 期刊: Nature communications
• 影响因子: 16.6