Structure and Function of the VWF Helical Tubule Required for Hemostasis

项目概要/摘要 血管性血友病（VWD）是世界上最常见的出血性疾病，是由血管内皮生长因子（VEGF）基因突变引起的。 Willebrand Factor（VWF）是一种大型多结构域蛋白。在血液中，VWF作为头部的长多聚体循环- - 成熟VWF的头二硫键连接的二聚体。这些长的多聚体对于VWF功能是至关重要的，因为它们提供 循环VWF多价性，用于在内皮损伤部位活化和结合血小板，形成止血剂 用塞子止血另外，长VWF多聚体稳定血液中的凝血因子VIII（FVIII）。 为了形成这些对正常止血至关重要的长多聚体，VWF在晚期低pH下形成螺旋小管。 高尔基体和韦伯-帕拉德体（WPB）。螺旋小管作为模板形成二硫键 通过将D3结构域定位在非常接近的位置形成长的多聚体。同时，VWF的前结构域是 切割，产生结合血液中FVIII的成熟VWF。这些成熟步骤中的异常， VWF突变导致几种VWD亚型。尽管螺旋小管对VWF的重要性 虽然多聚化的螺旋小管的高分辨率结构是未知的。本研究金提案旨在 确定VWF螺旋小管在三个成熟阶段的结构，测试2A型VWD突变， 引起短小管，并询问前结构域切割对FVIII-VWF小管结合的影响。 在目的1中，使用C-末端截短的VWF构建体，在转染前的VWF小管的高分辨率结构， 头对头二硫键形成后，将使用冷冻电子显微镜（cryo-EM）测定， 螺旋重建使用冷冻电子断层扫描（cryo-ET）和子断层图像平均， 将确定原位VWF小管的三维重建以测试VWF螺旋的紧密堆积是否 天然WPB环境内的小管对小管中VWF的分子结构具有影响。 在这种结构洞察的指导下，将测试VWD突变的子集对稳健小管的影响。 形成和正常VWF多聚体长度。目的2将确定VWF中的结构重排， 前结构域切割并测试切割的小管是否可以结合FVIII。这项研究将阐明 VWF头对头二硫键形成的机制，VWF多聚化和正常 止血。VWF小管的结构表征将导致对VWD引起的分子理解。 低效率的多聚化。FVIII-VWF小管结合的鉴定将提供一个新的背景来理解 它们的关联并告知在分泌到血液中之前调节FVIII-VWF结合的治疗努力。一 初步的VWF小管重建表明，额外的数据收集将允许原子模型的建立。 这项研究将在Timothy Springer博士的赞助下进行，他在结构方面经验丰富， VWF的表征，和艾伦布朗博士，在冷冻EM专家，创造了一个强大的培训环境， 博士前的医学科学家培训。

PROJECT SUMMARY/ABSTRACT Von Willebrand Disease (VWD), the most common bleeding disorder worldwide, is caused by mutations in von Willebrand Factor (VWF), a large multidomain protein. In the blood, VWF circulates as a long multimer of head- to-head disulfide linked dimers of mature VWF. These long multimers are critical for VWF function as they give circulating VWF polyvalency for activating and binding platelets at sites of endothelial injury, forming a hemostatic plug to staunch bleeding. Additionally, long VWF multimers stabilize coagulation factor VIII (FVIII) in the blood. To form these long multimers, crucial for normal hemostasis, VWF forms helical tubules in the low pH of the late- Golgi and Weibel-Palade bodies (WPB). The helical tubule templates the disulfide bond formation needed to form long multimers by positioning D3 domains in close proximity. At the same time, VWF’s prodomain is cleaved, generating the mature VWF that binds FVIII in the blood. Aberrancy in these maturation steps due to VWF mutations causes several VWD subtypes. Despite the importance of the helical tubule for VWF multimerization, the high-resolution structure of the helical tubule is not known. This fellowship proposal aims to determine structures of VWF helical tubules at three stages of maturation, test Type 2A VWD mutations for causing short tubules, and interrogate the implications of prodomain cleavage for FVIII-VWF tubule association. In Aim 1, using a C-terminally truncated VWF construct, a high-resolution structure of the VWF tubule before and after head-to-head disulfide bonds form will be determined using cryo-electron microscopy (cryo-EM) and helical reconstruction. Using cryo-electron tomography (cryo-ET) and subtomogram averaging, a three- dimensional reconstruction of the in situ VWF tubule will be determined to test if the close packing of VWF helical tubules inside the native WPB environment has consequences for the molecular structure of VWF in the tubule. Guided by this structural insight, a subset of VWD mutations will be tested for their effect on robust tubule formation and normal VWF multimer length. Aim 2 will determine the structural rearrangements in VWF upon prodomain cleavage and test if the cleaved tubule can bind FVIII. This research will elucidate the molecular mechanism of VWF head-to-head disulfide bond formation, necessary for VWF multimerization and normal hemostasis. Structural characterization of the VWF tubule will lead to a molecular understanding of VWD caused by inefficient multimerization. Identification of FVIII-VWF tubule binding will provide a novel context to understand their association and inform therapeutic efforts to modulate FVIII-VWF binding before secretion into the blood. A preliminary VWF tubule reconstruction indicates that additional data collection will allow atomic model building. This research will be carried out under the sponsorship of Dr. Timothy Springer, experienced in structural characterization of VWF, and Dr. Alan Brown, an expert in cryo-EM, creating a strong training environment for predoctoral physician-scientist training.