Krasovska Maryna V, Sefcikova Jana, Spacková Nad'a, Sponer Jirí, Walter Nils G
National Center for Biomolecular Research, Faculty of Science, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic.
J Mol Biol. 2005 Aug 26;351(4):731-48. doi: 10.1016/j.jmb.2005.06.016.
The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA enzyme involved in the replication of a human pathogen, the hepatitis delta virus. Recent crystal structures of the precursor and product of self-cleavage, together with detailed kinetic analyses, have led to hypotheses on the catalytic strategies employed by the HDV ribozyme. We report molecular dynamics (MD) simulations (approximately 120 ns total simulation time) to test the plausibility that specific conformational rearrangements are involved in catalysis. Site-specific self-cleavage requires cytidine in position 75 (C75). A precursor simulation with unprotonated C75 reveals a rather weak dynamic binding of C75 in the catalytic pocket with spontaneous, transient formation of a H-bond between U-1(O2') and C75(N3). This H-bond would be required for C75 to act as the general base. Upon protonation in the precursor, C75H+ has a tendency to move towards its product location and establish a firm H-bonding network within the catalytic pocket. However, a C75H+(N3)-G1(O5') H-bond, which would be expected if C75 acted as a general acid catalyst, is not observed on the present simulation timescale. The adjacent loop L3 is relatively dynamic and may serve as a flexible structural element, possibly gated by the closing U20.G25 base-pair, to facilitate a conformational switch induced by a protonated C75H+. L3 also controls the electrostatic environment of the catalytic core, which in turn may modulate C75 base strength and metal ion binding. We find that a distant RNA tertiary interaction involving a protonated cytidine (C41) becomes unstable when left unprotonated, leading to disruptive conformational rearrangements adjacent to the catalytic core. A Na ion temporarily compensates for the loss of the protonated hydrogen bond, which is strikingly consistent with the experimentally observed synergy between low pH and high Na+ concentrations in mediating residual self-cleavage of the HDV ribozyme in the absence of divalents.
丁型肝炎病毒（HDV）核酶是一种参与人类病原体丁型肝炎病毒复制的自我切割RNA酶。最近，自我切割前体和产物的晶体结构，以及详细的动力学分析，催生了关于HDV核酶所采用催化策略的假说。我们报告了分子动力学（MD）模拟（总模拟时间约120纳秒），以检验特定构象重排参与催化作用的合理性。位点特异性自我切割需要75位的胞嘧啶（C75）。对未质子化C75的前体模拟显示，C75在催化口袋中的动态结合较弱，U-1（O2'）与C75（N3）之间自发形成瞬态氢键。这种氢键是C75作为通用碱所必需的。在前体中质子化后，C75H+倾向于向其产物位置移动，并在催化口袋内建立牢固的氢键网络。然而，在当前模拟时间尺度上未观察到如果C75作为通用酸催化剂所预期的C75H+（N3）-G1（O5'）氢键。相邻的环L3相对动态，可能作为一个灵活的结构元件，可能由封闭的U20.G25碱基对门控，以促进质子化的C75H+诱导的构象转换。L3还控制催化核心的静电环境，进而可能调节C75碱基强度和金属离子结合。我们发现，涉及质子化胞嘧啶（C41）的远距离RNA三级相互作用在未质子化时变得不稳定，导致催化核心附近的构象重排中断。一个钠离子暂时补偿了质子化氢键的损失，这与实验观察到的在没有二价离子的情况下低pH和高Na+浓度在介导HDV核酶残余自我切割中的协同作用惊人地一致。
