Mechanistic Insights into Specific G Protein Interactions with Adenosine Receptors

Coupling between G-protein-coupled receptors (GPCRs) and the G proteins is a key step in cellular signaling. Despite extensive experimental and computational studies, the mechanism of specific GPCR-G protein coupling remains poorly understood. This has greatly hindered effective drug design of GPCRs that are primary targets of ∼¹/₃ of currently marketed drugs. Here, we have employed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method to decipher the mechanism of the GPCR-G protein interactions. Adenosine receptors (ARs) were used as model systems based on very recently determined cryo-EM structures of the A₁AR and A_2AAR coupled with the G_i and G_s proteins, respectively. Changing the G_i protein to the G_s led to increased fluctuations in the A₁AR and agonist adenosine (ADO), while agonist 5'-N-ethylcarboxamidoadenosine (NECA) binding in the A_2AAR could be still stabilized upon changing the G_s protein to the G_i. Free energy calculations identified one stable low-energy conformation for each of the A₁AR-G_i and A_2AAR-G_s complexes as in the cryo-EM structures, similarly for the A_2AAR-G_i complex. In contrast, the ADO agonist and G_s protein sampled multiple conformations in the A₁AR-G_s system. GaMD simulations thus indicated that the A₁AR preferred to couple with the G_i protein to the G_s, while the A_2AAR could couple with both the G_s and G_i proteins, being highly consistent with experimental findings of the ARs. More importantly, detailed analysis of the atomic simulations showed that the specific AR-G protein coupling resulted from remarkably complementary residue interactions at the protein interface, involving mainly the receptor transmembrane 6 helix and the G_α α5 helix and α4-β6 loop. In summary, the GaMD simulations have provided unprecedented insights into the dynamic mechanism of specific GPCR-G protein interactions at an atomistic level.