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

描述(申请人提供)：我们将采用生物物理方法和突变相结合的方法来研究调控von Willebrand因子(VWF)和VWF裂解金属蛋白酶ADAMTS-13(具有血栓反应蛋白1型基序的去整合素和金属蛋白酶)之间的分子相互作用的结构基础和生物物理机制。我们将集中讨论ADAMTS-13在单分子或单对分子水平上的VWF结构域的展开、构象变化、与ADAMTS-13的结合和蛋白水解性切割过程。其目的是阐明机械如何调节这些分子过程，以便了解它们的功能是如何被循环中的血液流动调节的。这些研究分为两个具体目标。目的1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VWF的A结构域上的不同位点结合。初始结合涉及Cub结构域或间隔区，并受分离距离调节。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键的距离依赖型形成和力依赖型解离的影响，并建立ADAMTS-13/VWF相互作用的多位多态结合模型。目的2研究VWF的结构稳定性、结构决定因素、受ADAMTS-13结合的调控以及对ADAMTS-13蛋白降解的调控。我们的假设包括：FORCE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对VWF的切割。这会破坏蛋白质结构的稳定性，并导致灾难性的结构变化，从而暴露出A2结构域中的隐蔽切割位点，从而允许ADAMTS-13对蛋白质进行分解。强迫诱导的A结构域的结构变化也可能通过ADAMTS-13与A结构域的结合来调节，这可能会改变A结构域的构象。我们将确定力诱导A结构域结构变化的动力学，它被ADAMTS-13结合调节，以及它对ADAMTS-13蛋白降解的调节。我们还将测量结构变化对A结构域的强迫失稳和ADAMTS-13对VWF蛋白水解性切割的影响。本项目将阐明力学如何调节ADAMTS-13结合和切割VWF的化学，以满足它们在快速流动的血液循环的压力环境中履行其生物学功能的严格要求。破译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至关重要的见解。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黏附和/或干预血栓性血小板减少性紫癜(TTP)和/或出血性疾病von Willebrand疾病(VWD)。公共卫生相关性：我们建议研究金属蛋白酶ADAMTS-13与von Willebrand因子的结合和切割，它通过调节其大小来调节血小板与von Villebrand因子的黏附。这一调节是至关重要的，因为粘合不足不能止血以维持止血，但粘合过多会导致血栓形成。这些数据可能为抑制血栓形成过程中病理性血小板黏附和/或干预血栓性血小板减少性紫癜和/或出血性疾病von Willebrand疾病提供新的治疗方法。

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.