Allosteric equilibria of thrombin and its precursors

拟议的研究项目集中在一个最近发现的，范式转移的结构-功能联系相关 凝血酶所属的整个胰蛋白酶样酶家族。一种预先存在的变构平衡 活性位点的闭合（E *）和开放（E）构象的集合之间的差异影响活性水平 和蛋白酶中的结合机制。平衡也存在于酶原中，并解释了 观察到与血液凝固，免疫应答， 纤维蛋白溶解和发育。E *-E平衡的观测证据来自于大量的 目前存放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识别的一般机制。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， E * 和E的不同影响蛋白酶的活性和酶原的活化机制。显著 这些研究的一部分将涉及凝血酶和凝血酶的开创性NMR（2D和19F）测量， 凝血酶前体-2的目的是阐明，第一次，他们的自由的结构和动力学， 溶液中的构象。我们将重点关注E *-E均衡的可能结构决定因素和关键因素。 修饰215 - 217区段中活性位点区域入口的残基（W215、G216、E217）， 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， 凝血酶及其直接酶原前体凝血酶原-2。这些研究将促进我们对 胰蛋白酶折叠的基本结构-功能联系，并将为在以下条件下进行的研究提供背景 具体目标2.凝血酶所属的蛋白酶胰蛋白酶家族的成员被表示为无活性的 在活化过程中，通过在R15处的蛋白水解切割， 域切割产生一个新的N-末端插入蛋白质核心和H-键的侧面 残基D194的链。阐明了上述酶原激活的Huber-Bode机制是如何 与变构E *-E平衡的联系将是我们研究的中心。我们将扰动临界的I16- D194 H-键与几个取代，削弱或消除相互作用。每一个突变体都将由 快速动力学直接测量溶液中的E *-E分布。此外，关键突变体如D914A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的研究， 凝血酶在特定目标下1.这一具体目标的发展将阐明两个关键目标之间的联系。 胰蛋白酶折叠的特征，即，变构E *-E平衡和Huber-Bode机制， 推进我们对蛋白酶最大家族之一的基础知识。

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.