Structural elements in the Gα_s and Gβ_q C termini that mediate selective G Protein-coupled Receptor (GPCR) signaling

Although the importance of theCterminus of theα 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 theCterminus of theGα subunit (Gα peptide) to dissect the molecular mechanisms of G protein selectivity. We observe a direct link between Gα peptide binding and stabilization of the GPCR conformational ensemble. We find that cognate and non-cognate Gα peptides show deep and shallow binding, respectively, and in distinct orientations within the GPCR. Binding of the cognate Gα peptide stabilizes the agonistbound GPCR conformational ensemble resulting in favorable binding energy and lower flexibility of the agonist-GPCR pair. We identify three hot spot residues (Gα_sGβ_q-Gln-384/Leu- 349, Gln-390/Glu-355, and Glu-392/Asn-357) that contribute to selective interactions between the β2-adrenergic receptor (β2-AR)-Gα_s and V_1A receptor (V_1AR)-Gβ_q. The Gα_s and Gβ_q peptides adopt different orientations in β2-AR and V_1AR, respectively. The β2-AR/Gα_s peptide interface is dominated by electrostatic interactions, whereas the V_1AR/Gβ_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 Gβ_q prevents this peptide from interacting strongly withβ2-AR. Mutagenesis to the Gα_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.