Roccatano D, Mark A E, Hayward S
BIOSON Research Institute, Laboratory of Biophysical Chemistry, University of Groningen, The Netherlands.
J Mol Biol. 2001 Jul 27;310(5):1039-53. doi: 10.1006/jmbi.2001.4808.
Six, 2 ns molecular dynamics simulations have been performed on the homodimeric enzyme citrate synthase. In three, both monomers were started from the open, unliganded X-ray conformation. In the remaining three, both monomers started from a closed, liganded X-ray conformation, with the ligands removed. Projecting the motion from the simulations onto the experimental domain motion revealed that the free-energy profile is rather flat around the open conformation, with steep sides. The most closed conformations correspond to hinge-bending angles of 12-14 compared to the 20 degrees that occurs upon the binding of oxaloacetate. It is also found that the open, unliganded X-ray conformation is situated at the edge of the steep rise in free energy, although conformations that are about 5 degrees more open were sampled. A rigid-body essential dynamics analysis of the combined open trajectories has shown that domain motions in the direction of the closed X-ray conformation are compatible with the natural domain motion of the unliganded protein, which has just two main degrees of freedom. The simulations starting from the closed conformation suggest a free-energy profile with a small barrier in going from the closed to open conformation. A combined essential dynamics and hinge-bending analysis of a trajectory that spontaneously converts from the closed to open state shows an almost exact correspondence to the experimental transition that occurs upon ligand binding. The simulations support the conclusion from an earlier analysis of the experimental transition that the beta-hairpin acts as a mechanical hinge by attaching the small domain to the large domain through a conserved main-chain hydrogen bond and salt-bridges, and allowing rotation to occur via its two flexible termini. The results point to a mechanism of domain closure in citrate synthase that has analogy to the process of closing a door.
我们对同二聚体酶柠檬酸合酶进行了六次2纳秒的分子动力学模拟。其中三次模拟中，两个单体均从开放的、未结合配体的X射线构象开始。在其余三次模拟中，两个单体从封闭的、结合配体的X射线构象开始，且配体已去除。将模拟中的运动投影到实验结构域运动上，结果表明，在开放构象周围自由能分布相当平坦，两侧陡峭。与草酰乙酸结合时出现的20度相比，最封闭的构象对应的铰链弯曲角度为12 - 14度。还发现，开放的、未结合配体的X射线构象位于自由能急剧上升的边缘，不过也采样到了比其开放约5度的构象。对组合的开放轨迹进行刚体主成分动力学分析表明，向封闭X射线构象方向的结构域运动与未结合配体蛋白质的天然结构域运动兼容，该蛋白质只有两个主要自由度。从封闭构象开始的模拟表明，从封闭构象转变为开放构象时自由能分布存在一个小障碍。对从封闭状态自发转变为开放状态的轨迹进行主成分动力学和铰链弯曲分析的组合，结果显示与配体结合时发生的实验转变几乎完全对应。这些模拟支持了早期对实验转变分析得出的结论，即β - 发夹通过一个保守的主链氢键和盐桥将小结构域连接到大结构域，并通过其两个柔性末端允许旋转，从而起到机械铰链的作用。结果指向了柠檬酸合酶中结构域闭合的一种机制，该机制类似于关门的过程。
