We investigated the Gi-coupled A3 adenosine receptor (A3AR) activation mechanism by running 7.2 µs of molecular dynamics (MD) simulations. Based on homology to G protein-coupled receptor (GPCR) structures, three constitutively active mutant (CAM) and the wild-type (WT) A3ARs in the apo form were modeled. Conformational signatures associated with three different receptor states (inactive R, active R*, and bound to Gi protein mimic) were predicted by analyzing and comparing the CAMs with WT receptor and by considering site-directed mutagenesis data. Detected signatures that were correlated with receptor state included: Persistent salt-bridges involving key charged residues for activation (including a novel, putative ionic lock), rotameric state of conserved W6.48, and Na+ ions and water molecules present. Active-coupled state signatures similar to the X-ray structures of β2 adrenergic receptor-Gs protein and A2AAR-mini-Gs and the recently solved cryo-EM A1AR-Gi complexes were found. Our MD analysis suggests that constitutive activation might arise from the D1073.49-R1083.50 ionic lock destabilization in R and the D1073.49-R1113.53 ionic lock stabilization in R* that presumably lowers the energy barrier associated with an R to R* transition. This study provides new opportunities to understand the underlying interactions of different receptor states of other Gi protein-coupled GPCRs.
Keywords: Activation; Adenosine receptor; G protein-coupled receptor; Homology modeling; Molecular dynamics simulation; Purinergic signaling.