Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

中文翻译：

---

G蛋白模拟纳米体与毒蕈碱G蛋白偶联受体结合的机制。

蛋白质-蛋白质结合是细胞信号传导过程的关键。然而，由于时间尺度有限，蛋白质-蛋白质结合的分子动力学 (MD) 模拟具有挑战性。特别是，迄今为止尚未用 MD 模拟医学上重要的 G 蛋白偶联受体 (GPCR) 与细胞内信号蛋白的结合。在这里，我们报告了使用稳健的高斯加速 MD (GaMD) 方法成功模拟了 G 蛋白模拟纳米体与 M2 毒蕈碱 GPCR 的结合。通过超过 4,500 ns 的长时间尺度 GaMD 模拟，观察到纳米体与受体细胞内 G 蛋白偶联位点结合，与 X 射线结构相比，纳米体核心域的最小 rmsd 为 2.48 Å。纳米体的结合变构封闭了正构配体结合口袋，与最近的实验结果一致。在没有纳米抗体结合的情况下，受体正构袋取样开放和完全开放的构象。GaMD 模拟揭示了纳米抗体与 M2 受体结合期间的两个低能中间状态。柔性受体细胞内环在纳米体的识别和结合中贡献了显着的静电、极性和疏水残基相互作用。这些模拟提供了对 GPCR-纳米抗体结合机制的重要见解，并证明了 GaMD 在模拟动态蛋白质-蛋白质相互作用中的适用性。GaMD 模拟揭示了纳米抗体与 M2 受体结合期间的两个低能中间状态。柔性受体细胞内环在纳米体的识别和结合中贡献了显着的静电、极性和疏水残基相互作用。这些模拟提供了对 GPCR-纳米抗体结合机制的重要见解，并证明了 GaMD 在模拟动态蛋白质-蛋白质相互作用中的适用性。GaMD 模拟揭示了纳米抗体与 M2 受体结合期间的两个低能中间状态。柔性受体细胞内环在纳米体的识别和结合中贡献了显着的静电、极性和疏水残基相互作用。这些模拟提供了对 GPCR-纳米抗体结合机制的重要见解，并证明了 GaMD 在模拟动态蛋白质-蛋白质相互作用中的适用性。