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:
- 金额:$ 67.37万
- 依托单位:
- 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:
- 关键词:AddressAffinityAnabolismBindingBinding SitesBiologyBlood Coagulation DisordersBlood Coagulation FactorBlood PlateletsBlood coagulationC-terminalCalorimetryCoagulation ProcessComplement Factor DComplexCryoelectron MicroscopyCrystallizationCrystallographyCysteineDevelopmentDimerizationDiseaseDisulfidesEndothelial CellsEngineeringF8 geneFactor VIIIGlycoproteinsGoalsHalf-LifeHemophilia AHemorrhageHemostatic AgentsHemostatic functionHeritabilityIn VitroInheritedInterferometryLeadLengthLinkMediatingModelingMolecular ConformationMosaicismMucinsMutationN-terminalPatternPharmaceutical PreparationsPhysicsPlasma ProteinsPlatelet GlycoproteinsPlayPolysaccharidesPopulationProductionProprotein ConvertasesProtein EngineeringProteinsReplacement TherapyResolutionRistocetinRoleSiteStrokeStructureTestingTherapeuticThrombosisWeibel-Palade Bodiescrosslinkdesigndimerdisulfide bondexperimental studygain of function mutationglycosylationhydrodynamic flowimprovedmonomerreconstitutionstructural biologyvon Willebrand Diseasevon 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。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
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:
- 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:
- 影响因子:16.6
- 作者:
- 通讯作者:
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
数据更新时间:{{ journalArticles.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ monograph.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ sciAawards.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ conferencePapers.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ patent.updateTime }}
TIMOTHY A SPRINGER其他文献
TIMOTHY A SPRINGER的其他文献
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
{{ truncateString('TIMOTHY A SPRINGER', 18)}}的其他基金
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 - 财政年份:2019
- 资助金额:
$ 67.37万 - 项目类别:
Structures and Conformational Equilibria of Integrin alpha5 beta1
整合素α5β1的结构和构象平衡
- 批准号:
9079774 - 财政年份:2016
- 资助金额:
$ 67.37万 - 项目类别:
Structures and Conformational Equilibria of Integrin alpha5 beta1
整合素α5β1的结构和构象平衡
- 批准号:
9265127 - 财政年份:2016
- 资助金额:
$ 67.37万 - 项目类别:
Structural mechanisms underlying latency and activation of GDF8
GDF8 潜伏期和激活的结构机制
- 批准号:
9302311 - 财政年份:2016
- 资助金额:
$ 67.37万 - 项目类别:
Structural mechanisms underlying latency and activation of GDF8
GDF8 潜伏期和激活的结构机制
- 批准号:
9175103 - 财政年份:2016
- 资助金额:
$ 67.37万 - 项目类别:
相似海外基金
Construction of affinity sensors using high-speed oscillation of nanomaterials
利用纳米材料高速振荡构建亲和传感器
- 批准号:
23H01982 - 财政年份:2023
- 资助金额:
$ 67.37万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
Affinity evaluation for development of polymer nanocomposites with high thermal conductivity and interfacial molecular design
高导热率聚合物纳米复合材料开发和界面分子设计的亲和力评估
- 批准号:
23KJ0116 - 财政年份:2023
- 资助金额:
$ 67.37万 - 项目类别:
Grant-in-Aid for JSPS Fellows
Platform for the High Throughput Generation and Validation of Affinity Reagents
用于高通量生成和亲和试剂验证的平台
- 批准号:
10598276 - 财政年份:2023
- 资助金额:
$ 67.37万 - 项目类别:
Development of High-Affinity and Selective Ligands as a Pharmacological Tool for the Dopamine D4 Receptor (D4R) Subtype Variants
开发高亲和力和选择性配体作为多巴胺 D4 受体 (D4R) 亚型变体的药理学工具
- 批准号:
10682794 - 财政年份:2023
- 资助金额:
$ 67.37万 - 项目类别:
Collaborative Research: DESIGN: Co-creation of affinity groups to facilitate diverse & inclusive ornithological societies
合作研究:设计:共同创建亲和团体以促进多元化
- 批准号:
2233343 - 财政年份:2023
- 资助金额:
$ 67.37万 - 项目类别:
Standard Grant
Collaborative Research: DESIGN: Co-creation of affinity groups to facilitate diverse & inclusive ornithological societies
合作研究:设计:共同创建亲和团体以促进多元化
- 批准号:
2233342 - 财政年份:2023
- 资助金额:
$ 67.37万 - 项目类别:
Standard Grant
Molecular mechanisms underlying high-affinity and isotype switched antibody responses
高亲和力和同种型转换抗体反应的分子机制
- 批准号:
479363 - 财政年份:2023
- 资助金额:
$ 67.37万 - 项目类别:
Operating Grants
Deconstructed T cell antigen recognition: Separation of affinity from bond lifetime
解构 T 细胞抗原识别:亲和力与键寿命的分离
- 批准号:
10681989 - 财政年份:2023
- 资助金额:
$ 67.37万 - 项目类别:
CAREER: Engineered Affinity-Based Biomaterials for Harnessing the Stem Cell Secretome
职业:基于亲和力的工程生物材料用于利用干细胞分泌组
- 批准号:
2237240 - 财政年份:2023
- 资助金额:
$ 67.37万 - 项目类别:
Continuing Grant
ADVANCE Partnership: Leveraging Intersectionality and Engineering Affinity groups in Industrial Engineering and Operations Research (LINEAGE)
ADVANCE 合作伙伴关系:利用工业工程和运筹学 (LINEAGE) 领域的交叉性和工程亲和力团体
- 批准号:
2305592 - 财政年份:2023
- 资助金额:
$ 67.37万 - 项目类别:
Continuing Grant