Divalent Metal Ion Activation of a Guanine General Base in the Hammerhead Ribozyme: Insights from Molecular Simulations.
Divalent Metal Ion Activation of a Guanine General Base in the Hammerhead Ribozyme: Insights from Molecular Simulations.
复制标题
DOI：
10.1021/acs.biochem.6b01192
复制
发表时间：
2017-06-20
期刊：
影响因子：
2.9
通讯作者：
York DM
中科院分区：
文献类型：
--
作者：
Chen H;Giese TJ;Golden BL;York DM
锤头状核酶是一种研究广泛的核酶，能催化RNA磷酸二酯骨架的自切割。尽管有实验和理论上的努力，但关于一般碱鸟嘌呤(G12)激活亲核试剂的机制细节，仍然存在关键问题。对测量的活性-pH数据的直接解释表明，G12核苷酸碱基的pKa值明显受到核酶环境的影响。最近的结晶学和生化研究发现，G12的N7/O6位上的二价金属离子与pH有关，这一结合模式可能会导致G12的PKA向中性转变。我们给出了计算结果，统一了对现有结构和生化数据的解释，并描绘了反应的一般基本步骤的详细机理图。电子结构计算量化了几种镁-鸟嘌呤络合物中由镁离子结合引起的pKa移动的大小。利用分子动力学和新开发的二价金属离子与核酸结合的12-6-4参数的自由能模拟，表征了核酶活性中心环境，并评估了G12在有无镁离子结合的HHR中的pKa。结果表明，Mg~(2+)能使G12的pKa下移1.2个单位，这与由活度-pH测定得到的表观pKa值一致。此外，还用从头算量子力学/分子力学模拟方法研究了在有无镁离子存在的情况下，一般基步的自由能分布。综上所述，这些结果与现有的实验数据在数量上是一致的，并支持一种合作机制，即在N7/O6位置上的镁离子结合有助于稳定G12的功能，去质子化形式可以在反应的一般碱基步骤中从亲核试剂中提取质子。在这种情况下，特定部位的镁离子结合作为开关来激活HHR中的一般碱基。最后，对G12的突变和挽救作用进行了实验可检验的预测，这将为进一步深入了解其催化机理提供依据。这些结果有助于我们越来越多地了解二价金属离子在RNA催化中的潜在作用。
The hammerhead ribozyme is a well-studied nucleolytic ribozyme that catalyzes the self-cleavage of the RNA phosphodiester backbone. Despite experimental and theoretical efforts, there remain key questions about details of the mechanism with regard to the activation of the nucleophile by the putative general base guanine (G12). Straight-forward interpretation of the measured activity-pH data implies the pKa value of the G12 nucleobase is significantly shifted by the ribozyme environment. Recent crystallographic and biochemical work has identified pH-dependent divalent metal ion binding at the N7/O6 position of G12, leading to the hypothesis that this binding mode could induce a pKa shift of G12 towards neutrality. We present computational results that unify the interpretation of available structural and biochemical data, and paint a detailed mechanistic picture of the general base step of the reaction. Electronic structure calculations quantify the magnitude of predicted pKa shifts induced by Mg2+ binding in several Mg2+-guanine complexes. Molecular dynamics and free energy simulations using newly developed 12-6-4 parameters for divalent metal ion binding to nucleic acids are used to characterize the ribozyme active site environment and evaluate the pKa of G12 in HHR with and without the Mg2+ ion bound. The results suggest that Mg2+ is able to down-shift the pKa of G12 by −1.2 units in accord with the apparent pKa value determined from activity-pH measurements. In addition, ab initio quantum mechanical/molecular mechanical simulations are performed to explore the free energy profile for the general base step in the presence and absence of Mg2+. Taken together, these results are in quantitative agreement with available experimental data, and support a cooperative mechanism whereby Mg2+ binding at the N7/O6 position serves to stabilize G12 in the functional, deprotonated form that can abstract a proton from the nucleophile in the general base step of the reaction. In this scenario, site-specific Mg2+ ion binding acts as a switch to activate the general base in HHR. Finally, experimentally-testable predictions are made on the mutational and rescue effects on G12, which will give further insights into the catalytic mechanism. These results contribute to our growing knowledge of the potential roles of divalent metal ions in RNA catalysis.
登录
查看更多内容
影响因子：
2.9
作者：
Horton, TE;DeRose, VJ
通讯作者：
DeRose, VJ
影响因子：
5.6
作者：
Anderson, Michael;Schultz, Eric P.;Martick, Monika;Scott, William G.
通讯作者：
Scott, William G.
影响因子：
5.5
作者：
Giese TJ;York DM
通讯作者：
York DM
影响因子：
2.9
作者：
DAHM, SC;UHLENBECK, OC
通讯作者：
UHLENBECK, OC
影响因子：
4.5
作者：
Curtis, EA;Bartel, DP
通讯作者：
Bartel, DP