Coupled Motions in β₂AR:Gαs Conformational Ensembles

G protein-coupled receptors (GPCRs) act as conduits in the plasma membrane, facilitating cellular responses to physiological events by activating intracellular signal transduction pathways. Extracellular signaling molecules can induce conformational changes in GPCR, which allow it to selectively activate intracellular protein partners such as heterotrimeric protein G. However, a major unsolved problem is how GPCRs and G proteins form complexes and how their interaction results in G protein activation. Here, we show that an inactive, agonist-free β₂AR:Gαs complex can collectively sample intermediate states of the receptor on an activation pathway. An <i>in silico</i> conformational ensemble around the inactive state manifests significant conformational coupling between structural elements implicated in G protein activation throughout the complex. While Gαs helix α₅ has received much attention as a driver for nucleotide exchange, we also observe interactions between helix α_<i>N</i> with Intra Cellular Loop 2, which can be transmitted by β₁ to facilitate nucleotide exchange by disrupting a salt bridge between the P-loop and Switch I. These interactions are moderated in an active state ensemble. Collectively, our results support an alternative view of G protein activation, in which precoupling can allosterically modulate an agonist-free receptor. Subsequent selective agonist recruitment would result in collective activation of the complex. This alternative view can help us understand how distinct extracellular binding partners result in different but interdependent signaling pathways, with broad implications for GPCR drug discovery