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(VWF)是一种由内皮细胞分泌的多结构域血浆蛋白。在止血方面， VWF将血小板相互结合，并使其与血管壁发生交联性，形成血小板堵塞。VWF还 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von Willebrand病(VWD)。VWF中的D1、D2和D‘D3部件 是能够将VWF生物合成成以螺旋形式存储的超长串联分子的专门化结构域 Webel-Palade小体(WPB)内的小管。D‘D3还与FVIII结合。较长的长度使VWF能够感测流量。 出血部位的流动变化通过1)将盘绕的VWF串联器拉长成线状来激活VWF 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 与血小板膜糖蛋白Ibα(GPIBα)结合的结构域，用于形成血小板堵塞。D的高分辨率结构 缺乏组装和A1的高亲和力状态。在目标1中，我们将确定高压的结构- A1的亲和力状态。展开研究表明，VWF A2和A3结构域有两种状态，而A1有两种状态 三：原生的、中级的和展开的。初步研究表明，截断O-糖基化接头 N-端和C-端破坏了A1的天然状态，并增加了与GPIBα的亲和力。我们建议， 中间态对应于A1的高亲和力状态。我们检验了进一步截断的假设 A1两侧的连接子、功能增益突变(例如，激活VWD突变)和变构激活剂 瑞斯托菌素都通过稳定中间状态而不是原生状态来增加对GPIBα的A1亲和力。我们会 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的配合物的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。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模块形成D3组件。D‘突起 从D3组件中。已提出的形成二聚体间二硫键的两种半胱氨酸是 被埋了。我们将解决D‘D3二聚体(D’D3)2或D3二聚体的结构，其中突出的D‘去掉 定义D‘D3二聚化所需的结构重排。建议的二硫键重排 在二聚化之前，将通过突变和体外重组来证实。作为后援，我们将寻找一个冷冻机- VWF螺旋管的EM结构以确定(D‘D3)2的结构以及D组件如何使 形成高度有序的小管。AIM 3使用结晶学来了解D‘D3是如何与FVIII结合的，FVIII具有 通过蛋白质工程革命血友病A的FVIII替代疗法的潜力 作为替代策略，我们将确定D‘D3与FVIII的配合物的低温EM结构。更好的结构 了解VWF D组件和A1的高亲和力状态对治疗糖尿病具有重要意义 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