Department of Pediatrics, Suzhou Kowloon Hospital, Shanghai Jiao Tong University School of Medicine, Suzhou 215000, China.
Department of Pediatrics, Suzhou Kowloon Hospital, Shanghai Jiao Tong University School of Medicine, Suzhou 215000, China.
Biophys Chem. 2023 Jan;292:106916. doi: 10.1016/j.bpc.2022.106916. Epub 2022 Oct 29.
Human respiratory syncytial virus (RSV) is a primary cause of lower respiratory tract infections and hospital visits during infancy and childhood. The RSV phosphoprotein (P) is a major polymerase cofactor that interacts with nucleoprotein (N) to promote the recognition of ribonucleoprotein complex (RNP) by viral RNA polymerase. The binding pocket of N protein is chemically diverse, in or around which a number of aromatic and charged amino acid residues are observed. Previously, a nonapeptide segment (P peptide, DNDLSLEDF) representing the C-terminal tail of P protein was identified to mediate the N-P interaction with a moderate affinity, in which the Phe241 at the end of P's C-terminus plays a critical role in the binding of P peptide to N protein. Here, we found that the side-chain aromatic phenyl moiety of P Phe241 residue can form short- and long-range cation-π interactions with N Arg132 and Arg150 residues, respectively, as well as T-shaped and parallel-displaced π-π stackings with N Tyr135 and His151 residues, respectively, which co-define a geometrically satisfactory π-stacking system at the complex interface of N protein with P peptide, thus largely stabilizing the complex architecture. The stacking effect was further optimized by systematically mutating the P Phe241 residue to other natural and non-natural aromatic amino acids with diverse chemical substitutions at the phenyl moiety to examine their structural and energetic effects on π-stacking system and on protein-peptide binding. The electron-donating mutations at the phenyl moiety of P Phe241 residue can effectively enhance the π-stacking system and then promote peptide binding, whereas the bulky and positively charged mutations would considerably impair the peptide potency by introducing steric hindrance and electrostatic repulsion. The [Tyr]P, [Thp]P and [Fph]P mutants were determined to have an increased affinity relative to wild-type P peptide, which could be used as self-inhibitory peptides to competitively disrupt the native interaction between N and P proteins.
人类呼吸道合胞病毒（RSV）是婴儿和儿童下呼吸道感染和住院的主要原因。RSV 磷蛋白（P）是一种主要的聚合酶辅助因子，与核蛋白（N）相互作用，促进病毒 RNA 聚合酶对核糖核蛋白复合物（RNP）的识别。N 蛋白的结合口袋在化学上是多种多样的，在这个口袋内或周围观察到许多芳香族和带电氨基酸残基。以前，鉴定出代表 P 蛋白 C 末端尾部的九肽片段（P 肽，DNDLSLEDF）介导 N-P 相互作用，具有中等亲和力，其中 P 蛋白 C 末端末端的 Phe241 在 P 肽与 N 蛋白结合中起关键作用。在这里，我们发现 P 蛋白 Phe241 侧链芳香苯基部分可以分别与 N Arg132 和 Arg150 残基形成短程和远程阳离子-π相互作用，以及与 N Tyr135 和 His151 残基形成 T 形和平行位移的π-π堆积，分别与 N 蛋白与 P 肽的复合物界面形成几何上令人满意的π堆积系统，从而大大稳定了复合物的结构。通过系统地突变 P Phe241 残基为其他天然和非天然芳香族氨基酸，并在苯基部分进行不同的化学取代，来检查其对π堆积系统和蛋白-肽结合的结构和能量影响，进一步优化了堆积效应。P Phe241 残基苯基部分的电子供体突变可以有效地增强π堆积系统，从而促进肽结合，而体积大和带正电荷的突变会通过引入空间位阻和静电排斥而严重损害肽的效力。与野生型 P 肽相比，[Tyr]P、[Thp]P 和[Fph]P 突变体具有更高的亲和力，可作为自抑制肽，竞争性破坏 N 和 P 蛋白之间的天然相互作用。
