BPS2026 – The investigation of GDP/GTP exchange in activated dopamine receptor D1 in complex with Gs protein through in silico submolecular analysis

G protein-coupled receptors (GPCRs) are key mediators of cellular signaling, in which the dopamine receptor D1 (D1R) regulates neuronal processes by controlling nucleotide exchange on the Gs protein. Although structural advances have clarified D1R-G protein coupling, the precise steps of GDP release and GTP binding at the D1R-Gs interface remain unresolved. To investigate these processes, we performed extensive molecular dynamics simulations of D1R in complex with the Gsα subunit. Essential Dynamics sampling was applied to enhance conformational exploration and capture the slow exchange events. This approach enabled us to observe GDP release, GTP entry, and the intermediate states associated with local rearrangements in the binding pocket. The results revealed coordinated motions of the α5 helix and P-loop that facilitate GDP release, highlighting an allosteric pathway linking receptor activation to nucleotide cycling. We further examined the influence of the Mg 2+ ion positioned near the nucleotide-binding site. In GDP-bound simulations, Mg 2+ ion stabilized interactions within the pocket and prevented GDP from dissociating, consistent with its role in maintaining nucleotide affinity. In contrast, during GTP binding, Mg 2+ ion enhanced reassociation kinetics by promoting a favorable coordination geometry and stabilizing catalytic residues, thereby accelerating the GTP capture. These contrasting effects demonstrate a dual function for Mg 2+ in supporting high-affinity GDP binding while also facilitating efficient GTP exchange. Together, our results provide atomistic insight into the dynamic coupling between receptor activation, protein domain motions, and ion-mediated regulation of nucleotide exchange. By establishing the structural basis of D1R-Gsα conformational transitions, this work advances mechanistic understanding of GPCR-G protein signaling and underscores the utility of enhanced sampling approaches for elucidating rare molecular events.