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

描述（由申请人提供）：我们将采用与诱变相结合的生物物理方法来研究调节血管性血友病因子（VWF）和切割VWF的金属蛋白酶ADAMTS-13（一种具有血栓形成蛋白1型基序的去整合素和金属蛋白酶）之间分子相互作用的结构基础和生物物理机制。我们将着重于VWF结构域的解折叠，构象变化，结合和蛋白水解切割ADAMTS-13在单分子或单对分子水平的过程。目的是阐明力学如何调节这些分子过程，以了解它们的功能是如何通过血液循环中的血流调节的。这些研究分为两个具体目标。目的1阐明ADAMTS-13与VWF结合的调控机制和结构基础。我们的假设包括：ADAMTS-13的CUB结构域和间隔结构域结合到VWF的A结构域上的不同位点。初始结合涉及CUB结构域或间隔区结构域，并受间隔距离调节。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调节。我们将确定距离如何调节ADAMTS-13/VWF键的形成，以及力如何调节ADAMTS-13/VWF键的解离，测量结构变化对ADAMTS-13/VWF键的距离依赖性形成和力依赖性解离的影响，并为ADAMTS-13/VWF相互作用开发多位点和多状态结合模型。目的二是研究VWF的结构稳定性、结构决定因素、ADAMTS-13对VWF结构的调节以及对ADAMTS-13蛋白水解的调节。我们的假设包括：力通过破坏A结构域之间和/或内部的非共价相互作用来调节ADAMTS-13的VWF切割。这使蛋白质结构不稳定并诱导灾难性的结构变化，这暴露了A2结构域中的隐蔽切割位点，允许ADAMTS-13进行蛋白水解。在A结构域中的强制诱导的结构变化也可以通过ADAMTS-13与A结构域的结合来调节，这可以改变A结构域的构象。我们将确定力诱导的A结构域的结构变化的动力学，其通过ADAMTS-13结合的调节，以及其对ADAMTS-13蛋白水解的调节。我们还将测量结构变化对A结构域的强制不稳定化和ADAMTS-13对力调节的VWF蛋白水解裂解的影响。该项目将阐明力学如何调节ADAMTS-13结合和切割VWF的化学反应，以满足它们在快速流动血液循环的应激环境中发挥生物学功能的严格要求。解码分子结构如何决定这些调节机制将为血管生理学和病理学提供重要的见解。由此获得的信息还将有助于开发新的治疗方法，以抑制血栓形成期间的病理性血小板粘附和/或干预血栓性血小板减少性紫癜（TTP）和/或出血性血管性血友病（VWD）。公共卫生相关性：我们建议研究金属蛋白酶ADAMTS-13对血管性血友病因子的结合和切割，该蛋白酶通过调节其大小来调节血小板与血管性血友病因子的粘附。这种调节是至关重要的，因为不充分的粘附不能止血以维持止血，但过度的粘附会导致血栓形成。这些数据可能提供新的治疗方法，以抑制病理性血小板粘附在血栓形成和/或干预血栓性血小板减少性紫癜和/或出血性疾病血管性血友病。

DESCRIPTION (provided by applicant): 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). PUBLIC HEALTH RELEVANCE: We propose to study binding and cleaving of von Willebrand factor by metalloprotease ADAMTS-13, which regulates platelet adhesion to von Villebrand factor by regulating its size. This regulation is crucial because insufficient adhesion cannot stop bleeding to maintain hemostasis but excessive adhesion results in thrombosis. The data may offer new therapeutic approaches to inhibiting pathological platelet adhesion during thrombosis and/or intervention to thrombotic thrombocytopenic purpura and/or to the bleeding disorder von Willebrand diseases.