Mapping the conformational landscape of the stimulatory heterotrimeric G protein

Abstract Heterotrimeric G proteins serve as key membrane-associated signaling hubs, in concert with their cognate G protein-coupled receptors (GPCRs). Using site-directed labels located at four key allosteric sites within the Ras-homology domain of the stimulatory G protein α-subunit, Gsα, fluorine nuclear magnetic resonance spectroscopy (19F NMR) was employed to monitor the conformational equilibria of Gsα by itself, in the intact Gsαβγ heterotrimer, or in complex with either membrane alone or membrane-embedded adenosine A2A receptor (A2AR). The results reveal a concerted equilibrium which is strongly affected by nucleotide and interactions with the βγ-subunit, the lipid bilayer, and A2AR. 19F NMR spectra of the α1 helix of Gsα exhibit significant intermediate timescale dynamics, while those associated with the α4β6 loop and the α5 helix reflect respective membrane/receptor interactions and order/disorder transitions associated with G protein activation. The αN helix adopts a key functional state that serves as an allosteric conduit between the βγ-subunit and the receptor, while spectra in the presence of GTPγS reveal that a significant fraction of the ensemble remains tethered to the membrane and the receptor upon activation.