Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic.
J Phys Chem B. 2010 Aug 19;114(32):10581-93. doi: 10.1021/jp102572k.
Kink-turns (K-turns) are recurrent elbow-like RNA motifs that participate in protein-assisted RNA folding and contribute to RNA dynamics. We carried out a set of molecular dynamics (MD) simulations using parm99 and parmbsc0 force fields to investigate structural dynamics of the box C/D RNA and its complexes with two proteins: native archaeal L7ae protein and human 15.5 kDa protein, originally bound to very similar structure of U4 snRNA. The box C/D RNA forms K-turn with A-minor 0 tertiary interaction between its canonical (C) and noncanonical (NC) stems. The local K-turn architecture is thus different from the previously studied ribosomal K-turns 38 and 42 having A-minor I interaction. The simulations reveal visible structural dynamics of this tertiary interaction involving altogether six substates which substantially contribute to the elbow-like flexibility of the K-turn. The interaction can even temporarily shift to the A-minor I type pattern; however, this is associated with distortion of the G/A base pair in the NC-stem of the K-turn. The simulations show reduction of the K-turn flexibility upon protein binding. The protein interacts with the apex of the K-turn and with the NC-stem. The protein-RNA interface includes long-residency hydration sites. We have also found long-residency hydration sites and major ion-binding sites associated with the K-turn itself. The overall topology of the K-turn remains stable in all simulations. However, in simulations of free K-turn, we observed instability of the key C16(O2')-A7(N1) H-bond, which is a signature interaction of K-turns and which was visibly more stable in simulations of K-turns possessing A-minor I interaction. It may reflect either some imbalance of the force field or it may be a correct indication of early stages of unfolding since this K-turn requires protein binding for its stabilization. Interestingly, the 16(O2')-7(N1) H- bond is usually not fully lost since it is replaced by a water bridge with a tightly bound water, which is adenine-specific similarly as the original interaction. The 16(O2')-7(N1) H-bond is stabilized by protein binding. The stabilizing effect is more visible with the human 15.5 kDa protein, which is attributed to valine to arginine substitution in the binding site. The behavior of the A-minor interaction is force-field-dependent because the parmbsc0 force field attenuates the A-minor fluctuations compared to parm99 simulations. Behavior of other regions of the box C/D RNA is not sensitive to the force field choice. Simulation with net-neutralizing Na(+) and 0.2 M excess salt conditions appear in all aspects equivalent. The simulations show loss of a hairpin tetraloop, which is not part of the K-turn. This was attributed to force field limitations.
发夹环（K-turns）是一种反复出现的类似肘状的 RNA 基序，参与蛋白质辅助的 RNA 折叠，并有助于 RNA 的动态变化。我们使用 parm99 和 parmbsc0 力场进行了一系列分子动力学（MD）模拟，以研究框 C/D RNA 及其与两种蛋白质的复合物的结构动力学：天然古菌 L7ae 蛋白和最初与 U4 snRNA 非常相似结构结合的人 15.5 kDa 蛋白。框 C/D RNA 形成 K- 转角，其规范（C）和非规范（NC）茎之间存在 A- 次要 0 三级相互作用。因此，局部 K- 转角结构与先前研究的核糖体 K- 转角 38 和 42 的 A- 次要 I 相互作用不同。模拟显示，这种三级相互作用具有可见的结构动力学，总共涉及六个亚状态，这极大地促进了 K- 转角的肘状灵活性。该相互作用甚至可以暂时转变为 A- 次要 I 型模式；然而，这与 K- 转角的 NC- 茎中的 G/A 碱基对的扭曲有关。模拟表明，蛋白质结合会降低 K- 转角的灵活性。蛋白质与 K- 转角的顶点和 NC- 茎相互作用。蛋白质-RNA 界面包括长期居留的水合位点。我们还发现了与 K- 转角本身相关的长期居留水合位点和主要离子结合位点。在所有模拟中，K- 转角的整体拓扑结构保持稳定。然而，在自由 K- 转角的模拟中，我们观察到关键的 C16（O2'）-A7（N1）氢键的不稳定性，这是 K- 转角的特征相互作用，在具有 A- 次要 I 相互作用的 K- 转角的模拟中，该氢键明显更稳定。这可能反映了力场的某种不平衡，或者它可能是展开早期阶段的正确指示，因为这种 K- 转角需要蛋白质结合才能稳定。有趣的是，16（O2'）-7（N1）氢键通常不会完全丢失，因为它被一个与紧密结合的水形成的水桥取代，该水桥与腺嘌呤特异性结合，与原始相互作用相似。16（O2'）-7（N1）氢键通过蛋白质结合得到稳定。与人 15.5 kDa 蛋白的结合作用更明显，这归因于结合位点中缬氨酸到精氨酸的取代。框 C/D RNA 的其他区域的行为不受力场选择的影响。带有中和 Na(+)和 0.2 M 过量盐条件的模拟在各个方面都表现出等效性。模拟显示发夹环四肽的丢失，该环不是 K- 转角的一部分。这归因于力场的限制。
