Molecular dynamics suggest multifunctionality of an adenine imino group in acid-base catalysis of the hairpin ribozyme
Molecular dynamics suggest multifunctionality of an adenine imino group in acid-base catalysis of the hairpin ribozyme
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DOI：
10.1261/rna.1416709
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发表时间：
2009-04-01
期刊：
影响因子：
4.5
通讯作者：
Walter, Nils G.
中科院分区：
文献类型：
--
作者：
Ditzler, Mark A.;Sponer, Jiri;Walter, Nils G.
尽管进行了大量的结构和生物化学研究，但发夹状核酶自切割的催化机制仍然不清楚。为了深入了解活性中心动力学与这种小催化RNA活性的耦合，我们分析了总共300 ns的分子动力学(MD)模拟。我们的模拟预测了一个体外选择的“功能增益”突变的全局稳定性得到了改善，这一点通过天然凝胶电泳迁移率变化分析得到了验证。我们观察到，活性中心碱基和水分子通过动态氢键网络稳定了有利于催化的几何构型。在未质子化的模拟中，A38的N1移动到活性中心2‘-OH的附近，表明A38在切割过程中可能起到一般碱基的作用，这一作用通常被忽视，因为在涉及失活底物类似物的晶体结构中观察到的距离较长。相比之下，A38的N1被质子化的模拟将N1放置在5‘-氧离开基团附近，这支持了A38作为通用酸的提议。类似于蛋白质酶，我们讨论了一种合理的机制，在该机制中，A38具有双功能，并将质子直接从2‘-OH穿梭到5’-氧。此外，我们的模拟表明，A38的N1质子化在促进类似于在过渡态模拟晶体结构中观察到的有利几何结构方面发挥了重要作用，并支持了先前提出的A38、G8和长驻留水分子在过渡态稳定中的作用。
Despite numerous structural and biochemical investigations, the catalytic mechanism of hairpin ribozyme self-cleavage remains elusive. To gain insight into the coupling of active site dynamics with activity of this small catalytic RNA, we analyzed a total of similar to 300 ns of molecular dynamics ( MD) simulations. Our simulations predict improved global stability for an in vitro selected "gain of function'' mutation, which is validated by native gel electrophoretic mobility shift assay. We observe that active site nucleobases and water molecules stabilize a geometry favorable to catalysis through a dynamic hydrogen bonding network. Simulations in which A38 is unprotonated show its N1 move into close proximity of the active site 2'-OH, indicating that A38 may act as a general base during cleavage, a role that has generally been discounted due to the longer distances observed in crystal structures involving inactivating substrate analogs. By contrast, simulations in which N1 of A38 is protonated place N1 in close proximity to the 5'-oxygen leaving group, which supports the proposal that A38 serves as a general acid. In analogy to protein enzymes, we discuss a plausible mechanism in which A38 acts bifunctionally and shuttles a proton directly from the 2'-OH to the 5'-oxygen. Furthermore, our simulations suggest an important role for protonation of N1 of A38 in promoting a favorable geometry similar to that observed in transition-state analog crystal structures, and support previously proposed roles of A38, G8, and long residency water molecules in transition-state stabilization.