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.

拟议的研究项目重点关注最近发现的、范式转变的结构-功能链接相关 凝血酶所属的整个胰蛋白酶样酶家族。预先存在的变构平衡 活性位点的闭合 (E*) 和开放 (E) 构象集合之间的相互作用影响活性水平 以及蛋白酶的结合机制。平衡也存在于酶原中并解释了 观察到与凝血、免疫反应、 纤维蛋白溶解和发育。 E*-E 平衡的观察证据来自大量 目前存放在蛋白质数据库中的结构。额外的独立证据来自快速 配体与支持构象的蛋白酶和酶原活性位点结合的动力学测量 选择作为胰蛋白酶折叠中的一般识别机制。具体目标 1 下的研究将测试 假设蛋白酶和酶原在溶液中经历 E*-E 平衡，并且相对分布 E* 和 E 的变化影响蛋白酶的活性和酶原的激活机制。一个重要的 这些研究的组成部分将涉及凝血酶和凝血酶的开创性 NMR（2D 和 19F）测量 prethrombin-2 的目标是首次阐明其游离的结构和动态 溶液中的构象。我们将重点关注 E*-E 平衡的可能结构决定因素和关键因素 装饰 215-217 片段（W215、G216、E217）中活性位点区域入口的残基， 60 环路 (W60d)、自溶环路 (W148) 和 190-193 走廊 (E192)。这些残基的功能作用 将通过配体与野生型和突变体活性位点结合的快速动力学测量进行测试 凝血酶及其直接酶原前体凝血酶原-2。这些研究将加深我们对 胰蛋白酶折叠的基本结构-功能联系，将为将要进行的研究提供背景 具体目标 2. 凝血酶所属的胰蛋白酶家族成员被表示为无活性 酶原并通过激活过程中 R15 处的蛋白水解切割不可逆地转化为成熟蛋白酶 领域。裂解产生一个新的 N 末端，插入蛋白质核心并在侧面形成氢键 残基D194的链。阐明上述酶原激活的 Huber-Bode 机制是如何进行的 与变构 E*-E 平衡相关的问题将成为我们研究的中心阶段。我们将扰乱关键的 I16- D194 氢键具有多个削弱或消除相互作用的取代基。每个突变体都将被研究 快速动力学直接测量溶液中的 E*-E 分布。此外，D914A 等关键突变体将 首次通过 X 射线和 NMR 进行结构表征，以补充凝血酶前体 2 和 特定目标下的凝血酶 1。该特定目标的发展将阐明两个关键之间的联系 胰蛋白酶折叠的特征，即变构 E*-E 平衡和 Huber-Bode 机制，其方式将 增进我们对最大的蛋白酶家族之一的基本了解。