Functional Dynamics of Thrombin

 描述（由申请方提供）：拟定的研究将提供与催化功能和变构控制相关的凝血蛋白酶动力学的定量和预测性理解。丝氨酸蛋白酶的级联是所有肽酶家族中最大的，控制凝血。凝血和抗凝蛋白酶的功能和调节的控制很可能涉及动态变构，然而，没有研究测量凝血蛋白酶的动力学。凝血酶提供凝血和抗凝之间的转换，并通过血栓调节蛋白（TM）结合进行变构调节。在动态变构中，蛋白质存在于快速相互转换的状态的集合中，并且当变构效应物结合时选择状态的亚群。核磁共振动力学实验是观察变构蛋白质相互转换亚态的唯一方法。因此，我们提出了核磁共振骨架和侧链动力学实验结合增强采样分子动力学（aMD）模拟，充分描述动态运动在apo-thrombin，PPACK-凝血酶，两个W215突变体的凝血酶和凝血酶-TM 456复杂。该项目的目标是获得定量的动态数据，将用于校准aMD模拟。一旦校准，模拟就可以预测参与凝血的其他蛋白酶的动态变构，这些蛋白酶不适合NMR。该项目的三个相辅相成的目标是：目标1。确定凝血酶的动力学运动对催化功能很重要。将进行加速MD（aMD）模拟和NMR实验，以探测apo和PPACK结合形式的人α-凝血酶在ns至ms时间尺度上的运动。我们的假设是，通过比较载脂蛋白和PPACK-凝血酶的结果，我们将能够发现那些运动的底物结合后的变化，因此可能是重要的催化活性。目标2.确定对变构控制重要的凝血酶的动态运动。扩展aMD模拟将有助于解释实验结果，相反，NMR结果将提供有关相关主链运动速率的定量信息，以验证模拟。将研究凝血酶的两种W215突变形式以及凝血酶-TM 456复合物。我们发表的10 ns aMD模拟凝血酶-TM 456表明，TM导致凝血酶的不同运动合并成相关的运动。我们的假设是，不同变构形式的凝血酶之间的动力学差异将揭示凝血酶和其他丝氨酸蛋白酶的动态变构机制。目标3。发展侧链动力学实验，探讨效应子结合位点与凝血酶活性位点之间的动态变构。侧链动力学结果将根据扩展的aMD模拟进行迭代解释。假设：侧链动力学，已被证明在构象熵中起关键作用，也在凝血酶变构中起关键作用。

 DESCRIPTION (provided by applicant): The proposed studies will provide a quantitative and predictive understanding of coagulation protease dynamics in relation to catalytic function and allosteric control. Cascades of serine proteases, the largest of all of the peptidase families, control coagulation. Control of function and regulation of the coagulation and anticoagulation proteases very likely involves dynamic allostery, however, no studies have measured the dynamics of coagulation proteases. Thrombin provides the switch between coagulation and anti-coagulation, and is allosterically regulated by thrombomodulin (TM) binding. In dynamic allostery, the protein exists in an ensemble of states that rapidly interconvert, and sub-populations of states are selected when an allosteric effector binds. NMR dynamics experiments are the only way to observe interconverting sub-states in allosteric proteins. We therefore propose NMR backbone and side chain dynamics experiments combined with enhanced sampling molecular dynamics (aMD) simulations to fully describe dynamic motions in apo-thrombin, PPACK-thrombin, two W215 mutants of thrombin and the thrombin-TM456 complex. The goal of the project is to obtain quantitative dynamic data that will be used to calibrate aMD simulations. Once calibrated, the simulations could predict dynamic allostery in other proteases involved in coagulation that are not amenable to NMR. The three complementary aims of the project are: Aim 1. Determine the dynamic motions in thrombin that are important for catalytic function. Accelerated MD (aMD) simulations and NMR experiments will be performed to probe motions on time scales from ns to ms in the apo, and PPACK-bound forms of human α-thrombin. Our hypothesis is that by comparing results from apo and PPACK-thrombin we will be able to discover those motions which change upon substrate binding, and are therefore likely to be important for catalytic activity. Aim 2. Determine the dynamic motions in thrombin that are important for allosteric control. Extended aMD simulations will help interpret the experimental results and conversely the NMR results will provide quantitative information about rates of correlated backbone motions with which to validate the simulations. Two W215 mutant forms of thrombin and also the thrombin-TM456 complex will be studied. Our published 10 ns aMD simulations on thrombin-TM456 show that TM causes the disparate motions in thrombin to coalesce into correlated motions. Our hypothesis is that differences in dynamics between different allosteric forms of thrombin will reveal the mechanism of dynamic allostery in thrombin and other serine proteases. Aim 3. Develop side chain dynamics experiments to probe the dynamic allostery between effector-binding sites and the active site of thrombin. The side chain dynamics results will be iteratively interpreted in light of extended aMD simulations. Hypothesis: That side chain dynamics, which have been shown to play a critical role in conformational entropy, also play a critical role in thrombin allostery.