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

基本信息

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
项目类别：
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

项目摘要

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，具有止血作用。 VWF 使血小板彼此结合并交联，并与血管壁形成血小板栓塞。与凝血级联中的因子 VIII (FVIII) 结合并使其稳定是 VWF 突变最常见的原因。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 组合。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) 中的小管也能结合 FVIII，较长的长度使 VWF 能够感知血流。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) 的结构域，形成 D 的高分辨率结构。缺乏组件和 A1 的高亲和力状态在目标 1 中，我们将确定高亲和力状态的结构。 A1 的亲和状态研究表明 VWF A2 和 A3 结构域有两种状态，而 A1 有三：天然的、中间体和未折叠的。初步研究表明，截断O-糖基化接头。 N 端和 C 端破坏 A1 的天然状态并增加对 GPIbα 的亲和力。中间状态对应于 A1 的高亲和力状态，我们测试进一步截断的假设。 A1 侧翼的接头、功能获得突变（例如激活 VWD 突变）和变构激活剂瑞斯托菌素都通过稳定中间状态而不是天然状态来增加 A1 对 GPIbα 的亲和力。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 Aim 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。被提议形成二聚体间二硫键的两个半胱氨酸是我们将解析 D´D3 二聚体 (D´D3)2 或除去突出的 D´ 的 D3 二聚体的结构。定义 D´D3 二聚化所需的结构重排。在二聚化之前将通过突变和体外重建进行验证作为备份，我们将进行冷冻。 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 Aim 3 使用晶体学来了解 D’D3 如何结合 FVIII，而 FVIII 具有高度有序的结构。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。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。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Disulfide exchange in multimerization of von Willebrand factor and gel-forming mucins.

冯维勒布兰德因子和凝胶形成粘蛋白多聚化中的二硫键交换。

DOI：
发表时间：
2021
期刊：
影响因子：
20.3
作者：
Dong, Xianchi;Springer, Timothy A
通讯作者：
Springer, Timothy A

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

Von Willebrand 因子 A1 结构域稳定性和 GPIbα 亲和力受其 O 糖基化 N 和 C 连接子的差异调节。

DOI：
发表时间：
2022-05-09
期刊：
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.

极端微生物配子融合剂的单体预融合结构和融合后三聚体状态的逐步形成。