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

点击翻译按钮获取中文摘要

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.