Functional Dynamics of Thrombin

凝血酶的功能动力学

• 批准号: 9204854
• 负责人: ELIZABETH A. KOMIVES
• 金额: $ 36.37万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-15 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9204854
• 关键词: Active Sites; Anticoagulation; Binding; Binding Sites; Blood coagulation; Catalysis; Cleaved cell; Coagulation Process; Complex; Computer Simulation; Data; Entropy; Family; Fibrin; Fibrinogen; Goals; Human; Light; Measures; Modeling; Molecular Conformation; Motion; Nuclear Magnetic Resonance; Peptide Hydrolases; Play; Protease Domain; Proteins; Protocols documentation; Publishing; Regulation; Relaxation; Research; Role; Sampling; Serine Protease; Side; Substrate Specificity; Thrombin; Thrombomodulin; Time; Tube; Validation; Vertebral column; Work; alpha-Thrombin; base; experimental study; molecular dynamics; mutant; public health relevance; simulation

 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.

 描述（由申请人提供）：拟议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级联（所有肽酶家族中最大的）控制凝血功能。凝血和抗凝血蛋白酶的调节很可能涉及动态变构，然而，没有研究测量凝血蛋白酶的动态。凝血酶提供凝血和抗凝血之间的转换，并通过血栓调节蛋白 (TM) 结合进行变构调节。在动态变构中，该蛋白质以快速相互转换的状态集合存在，并且当变构效应子时选择状态亚群。 NMR 动力学实验是观察变构蛋白中相互转换亚状态的唯一方法，因此我们建议将 NMR 主链和侧链动力学实验与增强采样分子动力学相结合。 (aMD) 模拟，以充分描述 apo-凝血酶、PPACK-凝血酶、凝血酶的两种 W215 突变体和凝血酶-TM456 复合物的动态运动。该项目的目标是获得用于校准 aMD 模拟的定量动态数据。经过校准后，模拟可以预测参与凝血的其他蛋白酶的动态变构，而这些蛋白酶不适合 NMR 该项目的三个互补目标。目标 1. 确定凝血酶中对催化功能很重要的动态运动，将进行加速 MD (aMD) 模拟和 NMR 实验，以探测 apo 中从 ns 到 ms 的时间尺度上的运动，以及 PPACK 结合形式。我们的假设是，通过比较 apo 和 PPACK 凝血酶的结果，我们将能够发现那些在底物结合时发生变化的运动，因此很可能是目标 2. 确定对变构控制很重要的凝血酶动态运动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，以验证相关骨架运动的速率。我们将研究两种 W215 突变形式的凝血酶以及凝血酶-TM456 复合物的 10 ns aMD 模拟。 thrombin-TM456 表明 TM 导致凝血酶中的不同运动合并为相关运动。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中的动态变构机制。链动力学实验探索效应子结合位点和凝血酶活性位点之间的动态变构，侧链动力学结果将被迭代地解释。假设：侧链动力学已被证明在构象熵中发挥着关键作用，在凝血酶变构中也发挥着关键作用。