Lee Tai-Sung, Silva López Carlos, Giambasu George M, Martick Monika, Scott William G, York Darrin M
Consortium for Bioinformatics and Computational Biology, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, USA.
J Am Chem Soc. 2008 Mar 12;130(10):3053-64. doi: 10.1021/ja076529e. Epub 2008 Feb 14.
Molecular dynamics simulations have been performed to investigate the role of Mg2+ in the full-length hammerhead ribozyme cleavage reaction. In particular, the aim of this work is to characterize the binding mode and conformational events that give rise to catalytically active conformations and stabilization of the transition state. Toward this end, a series of eight 12 ns molecular dynamics simulations have been performed with different divalent metal binding occupations for the reactant, early and late transition state using recently developed force field parameters for metal ions and reactive intermediates in RNA catalysis. In addition, hybrid QM/MM calculations of the early and late transition state were performed to study the proton-transfer step in general acid catalysis that is facilitated by the catalytic Mg2+ ion. The simulations suggest that Mg2+ is profoundly involved in the hammerhead ribozyme mechanism both at structural and catalytic levels. Binding of Mg2+ in the active site plays a key structural role in the stabilization of stem I and II and to facilitate formation of near attack conformations and interactions between the nucleophile and G12, the implicated general base catalyst. In the transition state, Mg2+ binds in a bridging position where it stabilizes the accumulated charge of the leaving group while interacting with the 2'OH of G8, the implicated general acid catalyst. The QM/MM simulations provide support that, in the late transition state, the 2'OH of G8 can transfer a proton to the leaving group while directly coordinating the bridging Mg2+ ion. The present study provides evidence for the role of Mg2+ in hammerhead ribozyme catalysis. The proposed simulation model reconciles the interpretation of available experimental structural and biochemical data, and provides a starting point for more detailed investigation of the chemical reaction path with combined QM/MM methods.
已进行分子动力学模拟以研究Mg2+在全长锤头状核酶切割反应中的作用。具体而言，这项工作的目的是表征导致催化活性构象和过渡态稳定的结合模式和构象事件。为此，使用最近开发的用于RNA催化中金属离子和反应中间体的力场参数，对反应物、早期和晚期过渡态进行了一系列八个12纳秒的分子动力学模拟，模拟中具有不同的二价金属结合占据情况。此外，对早期和晚期过渡态进行了混合QM/MM计算，以研究由催化性Mg2+离子促进的一般酸催化中的质子转移步骤。模拟结果表明，Mg2+在结构和催化水平上都深度参与了锤头状核酶机制。活性位点中Mg2+的结合在茎I和茎II的稳定中起关键结构作用，并有助于形成接近攻击构象以及亲核试剂与G12（可能的一般碱催化剂）之间的相互作用。在过渡态中，Mg2+以桥连位置结合，在该位置它稳定离去基团积累的电荷，同时与G8的2'OH（可能的一般酸催化剂）相互作用。QM/MM模拟提供了支持，即在晚期过渡态中，G8的2'OH可以将质子转移到离去基团，同时直接配位桥连的Mg2+离子。本研究为Mg2+在锤头状核酶催化中的作用提供了证据。所提出的模拟模型协调了对现有实验结构和生化数据的解释，并为使用组合QM/MM方法更详细地研究化学反应路径提供了一个起点。
