Interrogation of structure-function relationships in network formation of von Willebrand Factor domains by X-ray crystallography

血管性血友病因子（VWF）是一种多功能蛋白质，具有多个结构域，含有胶原蛋白、血小板糖蛋白受体和其他VWF配体的结合位点。虽然已经产生了丰富的数据对这个复杂的蛋白质，有相对较少的高分辨率的结构数据，以指导我们的理解的高度调节，机械敏感的功能的个人或集群的域。在这个项目中，我们将重点了解特定VWF结构域的三维结构与其在多聚化和网络形成中的功能之间的联系。为此，我们将使用高分辨率X射线晶体学来确定单个结构域和结构域阵列的结构，使我们能够产生解释特定突变对VWF病理生理学影响的假设。我们的专业知识将利用计算和其他实验方法，我们的第一个研究目标是针对VWF的C末端茎区，该区域在整合素结合形成蛋白质网络以及维持和潜在调节蛋白质的整体结构中具有关键作用在不同的细胞环境条件下。我们的目标是使用结构生物学来解决的重要问题是什么功能的C结构域是在介导网络的形成和特定的氨基酸多态性的C结构域阵列（A1/Schneppenheim）干扰VWF蛋白的正常功能？我们的第二个研究目标集中在VWF的A结构域区域，这是VWF最广泛表征的区域，并且已经确定了许多高分辨率结构。尽管如此，关于A1结构域如何产生针对不同范围的蛋白质靶标的特异性仍然存在未回答的问题，所述蛋白质靶标已经被提出与诸如GPib、胶原亚型和在自动调节机制中的A2结构域相互作用。我们的目标是定量和结构特征的A1-A2相互作用，使用生物物理方法和X射线晶体学。我们期望这些数据将解释A结构域和这些结构域中的突变之间的相互作用如何影响GPib和胶原结合的调节。我们相信，高分辨率的结构信息将有助于SHENC合作伙伴在第一个资助期内产生的现有假设，并揭示参与VWF功能的关键因素。

Von Willebrand factor (VWF) is a multi-functional protein with multiple domains harboring binding sites for collagen, platelet glycoprotein receptors and other VWF ligands. Although a wealth of data has been generated on this complex protein, there is relatively little high-resolution structural data available to guide our understanding of the highly regulated, mechano-sensitive functions of the individual or clusters of domains. ln this project we will focus on understanding the link between the three-dimensional structure of specific VWF domains and their function in multimerization and network formation. To this end we will use high-resolution X-ray crystallography to determine structures of individual domains and domain arrays, allowing us to generate hypotheses explaining the effect of specific mutations on the pathophysiology of VWF. Our expertise will complement those of others in the SHENC initiative, using computational and other experimental methods, aiming to have a significant impact in moving forward mechanistic explanations in the field of VWF research.Our first research aim addresses the C terminal stem region of VWF which has a crucial role in both protein network formation by integrin binding and in maintaining and potentially regulating the global architecture of the protein under different cellular environmental conditions. We aim using structural biology to address the important question of what the functional roles of the C domains are in mediating network formation and how do specific amino acid polymorphisms in the C domain array (A1/Schneppenheim) perturb the normal function of VWF proteins? Our secend research aim focuses on the A domain region of VWF that is the most extensively characterized region of VWF and a number of high-resolution structures have been determined. Despite this, there are still unanswered questions as to how the A 1-domain generates specificity for the distinct range of protein targets it has been proposed to interact with such as GPib, collagen subtypes and in an auto-regulatory mechanism, the A2-domain. We aim to quantitatively and structurally characterize this A 1-A2 interaction using biophysical methods and X-ray crystallography. We expect that these data will explain how the interaction between A domains and mutations in these domains influences the regulation of GPib and collagen binding. We believe that high resolution structural information will contribute to existing hypotheses generated by SHENC partners in the first funding period and reveal crucial factors involved in VWF function.