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.

von Willebrand因子（VWF）是一种由内皮细胞分泌的多域血浆蛋白。在止血中， VWF将血小板结合并彼此结合，并将血管壁形成血小板塞。 VWF也是如此结合并稳定凝血级联因子VIII（FVIII）。 VWF突变导致最常见可遗传的流血疾病称为von Willebrand疾病（VWD）。 VWF中的D1，D2和D´D3组件是将VWF生物合成的专业域，将其存储为螺旋形的超龙串。微花甲状动物体（WPB）中的小管。 d´d3也绑定了FVIII。长长的长度使VWF能够感知流动。通过1）将盘绕的vwf confeTemer延长到螺纹状的位置上的流量变化。暴露先前埋葬A1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IBα（GPIBα）以形成血小板的结构域。 D的高分辨率结构缺乏组装和高亲和力状态。在AIM 1中，我们将确定高位的结构 A1的亲和力状态。展开的研究表明，VWF A2和A3域具有两个状态，而A1具有三：本地，中间和展开。初步研究表明，将O-糖基化接头截断 N-和C末端破坏了A1的本地状态，并增加了对GPIBα的亲和力。我们建议中间状态对应于A1的高亲和力状态。我们检验了一个假设，即进一步截断 A1的链接器，功能获得突变（例如激活VWD突变）和变构激活剂 ristocetin均通过稳定本地状态的中间状态来增加对GPIBα的A1亲和力。我们将使用截断，突变和风险赛的组合来稳定中间状态的A1 确定A1的假定高亲和力状态的晶体结构及其与GPIBα的复合物。 AIM 2意志确定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模块如何在VWD模块周围包装以形成D3组件。 d´突出来自D3组件。提议形成二聚体间二硫键的两个半胱氨酸是被埋葬。我们将解决D´D3二聚体（D´D3）2或D3二聚体的结构，该二聚体的突出d´删除至定义D´D3二聚化所需的结构重排。拟议的二硫键重排先前的二聚化将通过突变和体外重构进行验证。作为备份，我们将追求一个冷冻 VWF螺旋管的EM结构，以确定（d´d3）2的结构以及d组件如何启用高度有序的管的形成。 AIM 3使用晶体学来了解D'D3如何绑定FVIII 通过蛋白质工程的潜力彻底改变了血友病的替代FVIII治疗。替代策略，我们将确定具有FVIII的D'D3复合物的冷冻结构。更好的结构对大众组装和A1的高亲和力状态的理解具有重要的治疗意义中风，血栓形成，VWD和血友病。

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）

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.