Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 115; no. 12; pp. 3036 - 3041
• Main Authors: Miao, Yinglong, McCammon, J. Andrew
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 20.03.2018; National Academy of Sciences, Washington, DC (United States)
• Subjects: Acetylcholine receptors (muscarinic); BASIC BIOLOGICAL SCIENCES; Binding; Binding sites; Biological Sciences; biomolecular recognition; Cells; Computer Simulation; Coupling (molecular); enhanced sampling; G protein-coupled receptors; GPCR signaling; Hydrophobicity; INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Intracellular; Intracellular signalling; Ligands; Models, Molecular; Molecular dynamics; Nanobodies; pathways; Physical Sciences; Protein Binding; Protein Conformation; Protein interaction; Proteins; Receptor, Muscarinic M2 - chemistry; Receptor, Muscarinic M2 - metabolism; Receptors; Receptors, G-Protein-Coupled - chemistry; Receptors, G-Protein-Coupled - metabolism; Simulation; Single-Domain Antibodies - physiology; Thermodynamics; pathways; protein binding; biomolecular recognition; enhanced sampling; GPCR signaling
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1800756115

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.