Structural Elements in the Gαs and Gαq C Termini That Mediate Selective G Protein-coupled Receptor (GPCR) Signaling

Although the importance of the C terminus of the subunit of the heterotrimeric G protein in G protein-coupled receptor (GPCR)-G protein pairing is well established, the structural basis of selective interactions remains unknown. Here, we combine live cell FRET-based measurements and molecular dynamics simulations of the interaction between the GPCR and a peptide derived from the C terminus of the G subunit (G peptide) to dissect the molecular mechanisms of G protein selectivity. We observe a direct link between G peptide binding and stabilization of the GPCR conformational ensemble. We find that cognate and non-cognate G peptides show deep and shallow binding, respectively, and in distinct orientations within the GPCR. Binding of the cognate G peptide stabilizes the agonist-bound GPCR conformational ensemble resulting in favorable binding energy and lower flexibility of the agonist-GPCR pair. We identify three hot spot residues (G(s)/G(q)-Gln-384/Leu-349, Gln-390/Glu-355, and Glu-392/Asn-357) that contribute to selective interactions between the 2-adrenergic receptor (2-AR)-G(s) and V-1A receptor (V1AR)-G(q). The G(s) and G(q) peptides adopt different orientations in 2-AR and V1AR, respectively. The 2-AR/G(s) peptide interface is dominated by electrostatic interactions, whereas the V1AR/G(q) peptide interactions are predominantly hydrophobic. Interestingly, our study reveals a role for both favorable and unfavorable interactions in G protein selection. Residue Glu-355 in G(q) prevents this peptide from interacting strongly with 2-AR. Mutagenesis to the G(s) counterpart (E355Q) imparts a cognate-like interaction. Overall, our study highlights the synergy in molecular dynamics and FRET-based approaches to dissect the structural basis of selective G protein interactions.