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 agonistbound 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 (V 1A R)-G␣ q . The G␣ s and G␣ q peptides adopt different orientations in 2-AR and V 1A R, respectively. The 2-AR/G␣ s peptide interface is dominated by electrostatic interactions, whereas the V 1A R/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.In recent years, there has been significant progress in structural and spectroscopic studies of Class A G protein-coupled receptors (GPCR), 3 which are energizing structure-based drug discovery efforts (1-8). Although these studies clearly demonstrate ligand-dependent structural changes in the GPCR, there remains a paucity of information on how GPCR conformation translates to selective G protein activation (9). Without structural information to compare and contrast multiple GPCR/G protein interfaces, the underlying mechanisms of selection remain incompletely understood. Currently, there is only one crystal structure of the complete GPCR/G protein interface (2-AR⅐G s complex) that provides a single essential snapshot of a highly dynamic interaction (10). Hence, alternative approaches are essential to dissect the structural elements within both the GPCR and its effectors that confer signaling specificity within the cellular environment. In this study, we combine FRETbased measurements of the GPCR/G protein interface in live cells, combined with computational modeling to build a rational scalable approach to identify structural hot spots that drive effector selection.One critical and well characterized component of the GPCR/G prote...
Fӧrster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between Gα subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the β2-adrenergic receptor and the Gαs C-terminus peptide upon treatment with 100 µM isoproterenol hydrochloride as previously characterized(1). Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a Gα C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.
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