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 (GαsGβq-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)-Gαs and V1A receptor (V1AR)-Gβq. The Gαs and Gβq peptides adopt different orientations in β2-AR and V1AR, respectively. The β2-AR/Gαs peptide interface is dominated by electrostatic interactions, whereas the V1AR/Gβq 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 Gβq prevents this peptide from interacting strongly withβ2-AR. Mutagenesis to the Gαs 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.
Bibliographical noteFunding Information:
This work was supported by American Heart Association Scientist Development Grant 13SDG14270009 and National Institutes of Health Grants 1DP2 CA186752-01 and 1-R01-GM-105646-01-A1 (to S. S.) and R01 GM097261-04 (to N. V.).
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.