Allosteric equilibria of thrombin and its precursors

拟议的研究项目侧重于最近发现的、范式转换的结构-功能联系 凝血酶所属的胰酶样酶的整个家族。预先存在的变构平衡 活性中心的闭合构象(E*)和开放构象(E)之间的系综影响活性水平 和蛋白水解酶的结合机制。这种平衡也存在于酶原中，并解释了 观察到几种参与凝血、免疫反应、 纤溶和发育。E*-E平衡的观测证据来自一大群 目前存放在蛋白质数据库中的结构。其他独立证据来自RAPID 与支持构象的蛋白酶和酶原活性部位结合的配体的动力学测定 选择作为在胰酶折叠中识别的一般机制。特定目标1下的研究将测试 假设蛋白酶和酶原在溶液中经历E*-E平衡及其相对分布 E*和E的浓度影响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包括开创性的凝血酶和19F的核磁共振测量 凝血酶原-2，目的是首次阐明其游离的结构和动力学 溶液中的构象(S)。我们将集中讨论E*-E平衡的可能结构决定因素和临界 修饰215-217片段(W215，G216，E217)活性位点区域入口的残基， 60环(W60d)、自溶环(W148)和190-193走廊(E192)。这些残基的功能作用 将通过与野生型和突变体活性部位的配基结合的快速动力学测量来进行测试 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 胰酶折叠的基本结构-功能联系，并将为将在 特定目的2.凝血酶所属的胰酶家族的成员被表达为不活跃 酶原在R15处被蛋白水解酶不可逆地转化为成熟的蛋白水解酶 域。裂解产生一个新的N末端，插入到蛋白质核心和侧边的H键中 残基链D194。阐明上述酶原激活的Huber-Bode机制是如何 与变构相关的E*-E平衡将是我们研究的中心阶段。我们将扰乱关键的I16- 带有几个取代基的D194氢键，可减弱或取消相互作用。每个突变体都将被研究 快速动力学直接测量E*-E在溶液中的分布。此外，D914A等关键突变体将 首次用X-射线和核磁共振对凝血酶原-2和凝血酶原-2的结构进行表征 特定目的下的凝血酶1.这一特定目的的进展将阐明两个关键因素之间的联系 胰酶折叠的特征，即变构E*-E平衡和Huber-Bode机制，其方式将 提高我们对最大的蛋白水解酶家族之一的基本知识。

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.