Allosteric equilibria of thrombin and its precursors

凝血酶及其前体的变构平衡

• 批准号: 9789457
• 负责人: Enrico Di Cera
• 金额: $ 37.88万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9789457
• 关键词: Active Sites; Address; Area; Autolysis; Basic Science; Binding; Biology; Blood Coagulation Factor; Blood coagulation; Catalysis; Clinical Treatment; Complement; Core Protein; Deposition; Development; Engineering; Enzyme Precursors; Enzymes; Equilibrium; Family; Fibrinolysis; Goals; Hydrogen Bonding; Immune response; Inflammation; Investigation; Kinetics; Knowledge; Ligand Binding; Link; Measurement; Measures; Molecular Conformation; Peptide Hydrolases; Process; Property; Protein C; Proteins; Regulation; Research Project Grants; Roentgen Rays; Role; Side; Structure; Testing; Thrombin; Time; Trypsin; Work; base; biophysical techniques; catalyst; data warehouse; member; mutant; phase I trial; prethrombins; synergism

The proposed research project focuses a recently uncovered, paradigm-shifting structure-function link relevant to the entire family of trypsin-like enzymes to which thrombin belongs. A pre-existing, allosteric equilibrium between ensembles of closed (E*) and open (E) conformations of the active site influences the level of activity and mechanism of binding in the protease. The equilibrium also exists in the zymogen and explains the spontaneous autoactivation observed with several proteins involved in blood coagulation, immune response, fibrinolysis and development. Observational evidence of the E*-E equilibrium comes from a large body of structures currently deposited in the Protein Data Bank. Additional independent evidence comes from rapid kinetics measurements of ligand binding to the active site of protease and zymogen that support conformational selection as a general mechanism of recognition in the trypsin fold. Studies under specific aim 1 will test the hypothesis that protease and zymogen undergo the E*-E equilibrium in solution and that the relative distribution of E* and E influences activity in the protease and the mechanism of activation in the zymogen. A significant component of these studies will involve pioneering NMR (2D and 19F) measurements of thrombin and prethrombin-2 with the goal of elucidating, for the first time, the structure and dynamics of their free conformation(s) in solution. We will focus on the likely structural determinants of the E*-E equilibrium and critical residues that decorate the entrance to the active site region in the 215-217 segment (W215, G216, E217), the 60-loop (W60d), the autolysis loop (W148) and the 190-193 corridor (E192). The functional role of these residues will be tested by rapid kinetics measurements of ligand binding to the active site of wild-type and mutants of thrombin and its direct zymogen precursor prethrombin-2. These studies will advance our understanding of a basic structure-function link of the trypsin fold and will provide background for studies to be carried out under specific aim 2. Members of the trypsin family of proteases, to which thrombin belongs, are expressed as inactive zymogens and irreversibly converted to the mature protease by proteolytic cleavage at R15 in the activation domain. The cleavage generates a new N-terminus that inserts into the protein core and H-bonds to the side chain of residue D194. Elucidating how the Huber-Bode mechanism of zymogen activation described above is linked to the allosteric E*-E equilibrium will be center stage in our investigation. We will perturb the critical I16- D194 H-bond with several substitutions that weaken or abolish the interaction. Each mutant will be studied by rapid kinetics to directly measure the E*-E distribution in solution. Additionally, key mutants such as D914A will be characterized structurally for the first time by X-ray and NMR to complement studies of prethrombin-2 and thrombin under specific aim 1. Developments from this specific aim will elucidate the linkage between two critical features of the trypsin fold, i.e., the allosteric E*-E equilibrium and the Huber-Bode mechanism, in ways that will advance our basic knowledge of one the largest families of proteases.

拟议的研究项目集中于最近发现的，范式转移结构 - 功能函数链接相关 凝血酶所属的整个胰蛋白酶状酶。现有的变构平衡 在封闭（E*）的集合和活动位点的开放构象之间影响活动水平 和蛋白酶结合的机制。平衡也存在于Zymogen中，并解释了 与几种参与血液凝血，免疫反应，自发自身活化观察到 纤维蛋白溶解和发育。 E*-e平衡的观察性证据来自很大的机构 目前沉积在蛋白质数据库中的结构。其他独立证据来自快速 配体与蛋白酶和Zymogen的活性位点结合的动力学测量值支持构象 选择是胰蛋白酶折叠中识别的一般机制。在特定目标1下的研究将测试 假设蛋白酶和Zymogen在溶液中经历E*-e平衡，并且相对分布 E*和E影响蛋白酶的活性以及Zymogen中激活的机理。重要的 这些研究的组成部分将涉及凝血酶和 预凝蛋白-2的目标是首次阐明他们自由的结构和动态 溶液中的构象。我们将重点关注E*-E平衡和关键的可能结构决定因素 在215-217段（W215，G216，E217）中装饰有活性位点区域入口的残留物， 60循环（W60D），自动溶解环（W148）和190-193走廊（E192）。这些残基的功能作用 将通过将配体与野生型和突变体的活性位点结合的快速动力学测量进行测试 凝血酶及其直接的酶原前体凝血酶-2。这些研究将提高我们对 胰蛋白酶折叠的基本结构功能链路，将为研究提供背景 特定目的2。胰蛋白酶的胰蛋白酶成员（凝血酶所属的蛋白酶家族）表示为无活性 Zymogens和不可逆地通过R15处的蛋白水解裂解转化为成熟的蛋白酶 领域。裂解产生了一种新的N末端，将蛋白质核心插入并honds插入侧面 残留链D194。阐明上述酶原激活的Huber-Bode机制如何 与变构E*-e平衡有关的链接将是我们调查的中心阶段。我们将扰动关键的I16-- D194 H键，几种替代可以削弱或废除相互作用。每个突变体将通过 快速动力学直接测量溶液中的E*-e分布。此外，诸如D914A之类的关键突变体将 首次通过X射线和NMR在结构上进行表征，以补充凝血酶素-2和 在特定目标下的凝血酶1。从这个特定目标的发展将阐明两个关键 胰蛋白酶折叠的特征，即变构E*-e平衡和Huber-Bode机构的特征 促进我们对最大蛋白酶家族的基本知识。