Mechanical Regulation of Binding and Cleavage of VWF by ADAMTS-13

项目总结 我们将使用生物物理方法和诱变相结合的方法来研究结构基础和 调节von Willebrand因子与VWF分子相互作用的生物物理机制 VWF裂解金属蛋白酶ADAMTS-13(一种具有凝血酶反应蛋白类型的去整合素和金属蛋白酶 1个基序)。我们将重点介绍VWF结构域的展开、构象变化、结合和 ADAMTS-13在单分子或单对分子水平上的蛋白水解性切割。目标是 是阐明力学是如何调节这些分子过程的，以便了解它们的功能 是由循环中的血液流动调节的。这些研究分为两个具体目标。目标1是 阐明ADAMTS-13/VWF结合的调控机制和结构基础。我们的假设 包括：ADAMTS-13的Cub结构域和间隔区与A域上的不同位点结合 VWF。初始结合涉及Cub结构域或间隔区，并受分离调节 距离。第二位点的后续结合由第一位点的结合诱导，并由 施加的力。我们将确定距离如何调节形成，以及力如何调节解离， ADAMTS-13/VWF键，测量结构变化对距离相关地层的影响，以及 依赖于力的ADAMTS-13/VWF键的解离，并形成多位多态结合 ADAMTS-13/VWF相互作用的模型。目标2是研究VWF的结构稳定性，其结构 决定因素，其与ADAMTS-13结合的调节，以及其对ADAMTS-13蛋白降解的调节。我们的 假设包括：FORCE通过破坏非共价相互作用调节ADAMTS-13对VWF的切割 在A域之间和/或在A域内。这会破坏蛋白质结构的稳定性，并导致灾难性的 结构变化，这暴露了A2结构域中的隐蔽切割位点，允许蛋白质通过 ADAMTS-13。强迫诱导的A结构域的结构变化也可能受结合的调节 ADAMTS-13到A结构域，这可能改变A结构域的构象。我们将确定动力学 力诱导的A结构域的结构变化，ADAMTS-13结合对其的调节，以及其对 ADAMTS-13蛋白水解酶。我们还将测量结构变化对A的强迫失稳的影响 结构域以及ADAMTS-13对VWF蛋白水解性切割的力调节作用。这个项目将阐明如何 力学调节ADAMTS-13结合和切割VWF的化学以满足严格的 在血液循环的应激环境中执行其生物功能的要求 快速流动的血液。破译分子结构如何决定这些调节机制将提供 对血管生理学和病理学的重要见解。这样获得的信息也将有助于开发新的 血栓形成期间抑制病理性血小板黏附和/或干预的治疗方法 血栓性血小板减少性紫癜(TTP)和/或出血性疾病血管性血友病(VWD)。

PROJECT SUMMARY We will employ a biophysical approach combined with mutagenesis to study the structural bases and biophysical mechanisms that regulate the molecular interaction between von Willebrand factor (VWF) and VWF-cleaving metalloprotease ADAMTS-13 (A Disintegrin And Metalloprotease with a ThromboSpondin type 1 motifs). We will focus on the processes of VWF domain unfolding, conformational changes, binding to and proteolytic cleavage by ADAMTS-13 at the level of single molecules or single pairs of molecules. The objective is to elucidate how mechanics regulate these molecular processes in order to understand how their functions are regulated by the blood flow in the circulation. These studies are organized into two specific aims. Aim 1 is to elucidate the regulatory mechanisms and structural bases of ADAMTS-13/VWF binding. Our hypotheses include: The CUB domains and the spacer domain of ADAMTS-13 bind to separate sites on the A domains of VWF. Initial binding involves either the CUB domains or the spacer domain and is regulated by separation distance. Subsequent binding of the second site is induced by the binding of the first site and regulated by applied force. We will determine how distance regulates formation, and how force regulates dissociation, of ADAMTS-13/VWF bonds, measure the effects of structural variations on distance-dependent formation and force-dependent dissociation of ADAMTS-13/VWF bonds, and develop a multi-site and multi-state binding model for the ADAMTS-13/VWF interaction. Aim 2 is to investigate the structural stability of VWF, its structural determinants, its regulation by ADAMTS-13 binding, and its regulation of ADAMTS-13 proteolysis. Our hypotheses include: Force regulates VWF-cleavage by ADAMTS-13 via disrupting noncovalent interactions between and/or within the A domains. This destabilizes the protein structure and induces catastrophic structural changes, which exposes the cryptic cleavage site in the A2 domain, allowing proteolysis by ADAMTS-13. The forced-induced structural changes in the A domains may also be regulated by binding of ADAMTS-13 to the A domains, which may change the A domain conformations. We will determine the kinetics of force-induced structural changes in A domains, its regulation by ADAMTS-13 binding, and its regulation of ADAMTS-13 proteolysis. We will also measure the effects of structural variations on forced-destabilization of A domains and on the force-regulated VWF proteolytic cleavage by ADAMTS-13. This project will clarify how mechanics regulates the chemistry of binding and cleavage of VWF by ADAMTS-13 to meet the stringent requirements for them to carry out their biological functions in the stressful environment of the circulation of rapidly flowing blood. Decoding how molecular structures determine these regulatory mechanisms will provide crucial insights into vascular physiology and pathology. Information thus obtained will also help develop new therapeutic approaches to inhibiting pathological platelet adhesion during thrombosis and/or intervention to thrombotic thrombocytopenic purpura (TTP) and/or the bleeding disorder von Willebrand diseases (VWD).