Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 291, Issue 34, Pages 17929-17940Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M116.735720
Keywords
cell signaling; fluorescence resonance energy transfer (FRET); G protein; G protein-coupled receptor (GPCR); molecular dynamics; Receptor Conformation
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Funding
- American Heart Association Scientist Development Grant [13SDG14270009]
- National Institutes of Health [1DP2 CA186752-01, 1-R01-GM-105646-01-A1, R01 GM097261-04]
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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.
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