Conformational State Distributions and Catalytically Relevant Dynamics of a Hinge-Bending Enzyme Studied by Single-Molecule FRET and a Coarse-Grained Simulation
Conformational State Distributions and Catalytically Relevant Dynamics of a Hinge-Bending Enzyme Studied by Single-Molecule FRET and a Coarse-Grained Simulation
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DOI：
10.1016/j.bpj.2014.08.016
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发表时间：
2014-10-21
影响因子：
3.4
通讯作者：
Fitter, Joerg
中科院分区：
文献类型：
--
作者：
Gabba, Matteo;Poblete, Simon;Fitter, Joerg
在过去的几十年里，一种观点表明，多结构域酶是一种生物机器，它可以利用溶剂颗粒的随机踢来进行高度定向的功能运动。这些内在运动在结构上被编码，大自然利用它们通过配体诱导的构象变化和状态重分配来催化化学反应。这种机制将反应基团排列成有效的化学反应，并稳定最擅长催化的构象。通过将单分子福斯特共振能量转移测量与正态分析和粗粒度介观模拟相结合，我们获得了一种铰链弯曲酶，即磷酸甘油酸激酶（PGK）的结果，支持并扩展了这些想法。从单分子Forster共振能量转移中，我们了解了结构域的构象态分布和动力学性质。模拟可以表征PGK致密态的域间运动。数据表明，PGK本质上是一个高度动态的系统，在从纳秒到毫秒甚至更高的时间尺度上采样丰富的构象。折叠中编码的功能运动是由已经处于无配体形式的PGK结构域执行的，并且不需要底物结合来实现它们。与其他多结构域蛋白相比，这些运动相当快，在酶促反应中可能没有速率限制。配体结合略微调整了结构域的方向，并可行地锁定了蛋白质沿着优先方向的运动。此外，功能相关的致密态由基底稳定，并通过布朗运动作为达到主动构象的前提。
Over the last few decades, a view has emerged showing that multidomain enzymes are biological machines evolved to harness stochastic kicks of solvent particles into highly directional functional motions. These intrinsic motions are structurally encoded, and Nature makes use of them to catalyze chemical reactions by means of ligand-induced conformational changes and states redistribution. Such mechanisms align reactive groups for efficient chemistry and stabilize conformers most proficient for catalysis. By combining single-molecule Forster resonance energy transfer measurements with normal mode analysis and coarse-grained mesoscopic simulations, we obtained results for a hinge-bending enzyme, namely phosphoglycerate kinase (PGK), which support and extend these ideas. From single-molecule Forster resonance energy transfer, we obtained insight into the distribution of conformational states and the dynamical properties of the domains. The simulations allowed for the characterization of interdomain motions of a compact state of PGK. The data show that PGK is intrinsically a highly dynamic system sampling a wealth of conformations on timescales ranging from nanoseconds to milliseconds and above. Functional motions encoded in the fold are performed by the PGK domains already in its ligand-free form, and substrate binding is not required to enable them. Compared to other multidomain proteins, these motions are rather fast and presumably not rate-limiting in the enzymatic reaction. Ligand binding slightly readjusts the orientation of the domains and feasibly locks the protein motions along a preferential direction. In addition, the functionally relevant compact state is stabilized by the substrates, and acts as a prestate to reach active conformations by means of Brownian motions.