Mapping the conformational landscape of the stimulatory heterotrimeric G protein

Heterotrimeric G proteins serve as membrane-associated signaling hubs, in concert with their cognate G-protein-coupled receptors. Fluorine nuclear magnetic resonance spectroscopy was employed to monitor the conformational equilibria of the human stimulatory G-protein α subunit (G_sα) alone, in the intact G_sαβ₁γ₂ heterotrimer or in complex with membrane-embedded human adenosine A_2A receptor (A_2AR). The results reveal a concerted equilibrium that is strongly affected by nucleotide and interactions with the βγ subunit, the lipid bilayer and A_2AR. The α1 helix of G_sα exhibits significant intermediate timescale dynamics. The α4β6 loop and α5 helix undergo membrane/receptor interactions and order-disorder transitions respectively, associated with G-protein activation. The αN helix adopts a key functional state that serves as an allosteric conduit between the βγ subunit and receptor, while a significant fraction of the ensemble remains tethered to the membrane and receptor upon activation.