Protein-RNA interactions are crucially important for numerous cellular processes and often involve coupled folding and binding of peptide segments upon association. The Nut-utilization site (N)-protein of bacteriophages contains an N-terminal arginine-rich motif that undergoes such a folding transition upon binding to the boxB RNA hairpin loop target structure. Molecular dynamics free energy simulations were used to calculate the absolute binding free energy of the N-peptide of bacteriophage P22 in complex with the boxB RNA hairpin motif at different salt concentrations and using two different water force field models. We obtained good agreement with experiment also at different salt concentrations for the TIP4P-D water model that has a stabilizing effect on unfolded protein structures. It allowed us to estimate the free energy contribution resulting from restricting the molecules’ spatial and conformational freedom upon binding, which makes a large opposing contribution to binding. In a second set of umbrella sampling simulations to dissociate/associate the complex along a separation coordinate, we analyzed the onset of preorientation of the N-peptide and onset of structure formation relative to the RNA and its dependence on the salt concentration. Peptide orientation and conformational transitions are significantly coupled to the first contact formation between peptide and RNA. The initial contacts are mostly formed between peptide residues and the boxB hairpin loop nucleotides. A complete transition to an α-helical bound peptide conformation occurs only at a late stage of the binding process a few angstroms before the complexed state has been reached. However, the N-peptide orients also at distances beyond the contact distance such that the sizable positive charge points toward the RNA’s center-of-mass. Our result may have important implications for understanding protein- and peptide-RNA complex formation frequently involving coupled folding and association processes.

中文翻译：

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噬菌体 P22 N 肽与 boxB RNA 偶联折叠和结合的高级采样模拟


蛋白质-RNA 相互作用对于许多细胞过程至关重要，并且通常涉及结合时肽段的偶联折叠和结合。噬菌体的 Nut 利用位点 （N） 蛋白包含一个富含 N 末端精氨酸的基序，该基序在与 boxB RNA 发夹环靶结构结合时经历这种折叠转变。分子动力学自由能模拟用于计算噬菌体 P22 的 N 肽在不同盐浓度下与 boxB RNA 发夹基序复合物中的绝对结合自由能，并使用两种不同的水力场模型。对于对未折叠蛋白质结构具有稳定作用的 TIP4P-D 水模型，我们也在不同盐浓度下的实验获得了良好的一致性。它使我们能够估计由于限制分子在结合时的空间和构象自由度而产生的自由能贡献，这对结合有很大的相反贡献。在第二组沿分离坐标解离/关联复合物的伞式采样模拟中，我们分析了 N 肽预取向的开始和相对于 RNA 的结构形成的开始及其对盐浓度的依赖性。肽方向和构象转变与肽和 RNA 之间的第一次接触形成显著耦合。初始接触主要形成于肽残基和 boxB 发夹环核苷酸之间。向 α 螺旋结合肽构象的完全转变仅发生在结合过程的后期，即达到络合状态之前的几埃。 然而，N 肽的方向也超过了接触距离，因此相当大的正电荷指向 RNA 的质心。我们的结果可能对理解蛋白质和肽 RNA 复合物的形成具有重要意义，这些复合物通常涉及耦合折叠和关联过程。