1. Structural Elements in the Gαs and Gαq C Termini That Mediate Selective G Protein-coupled Receptor (GPCR) Signaling.
- Author
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Semack A, Sandhu M, Malik RU, Vaidehi N, and Sivaramakrishnan S
- Subjects
- Animals, Cell Line, Chromogranins genetics, Chromogranins metabolism, Enzyme Stability, Fluorescence Resonance Energy Transfer, GTP-Binding Protein alpha Subunits, Gq-G11 genetics, GTP-Binding Protein alpha Subunits, Gq-G11 metabolism, GTP-Binding Protein alpha Subunits, Gs genetics, GTP-Binding Protein alpha Subunits, Gs metabolism, Humans, Mice, Mutation, Missense, Peptides genetics, Peptides metabolism, Protein Domains, Receptors, Adrenergic, beta-2, Sus scrofa, Chromogranins chemistry, GTP-Binding Protein alpha Subunits, Gq-G11 chemistry, GTP-Binding Protein alpha Subunits, Gs chemistry, Molecular Dynamics Simulation, Peptides chemistry
- Abstract
Although the importance of the C terminus of the α 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 Gα 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 agonist-bound GPCR conformational ensemble resulting in favorable binding energy and lower flexibility of the agonist-GPCR pair. We identify three hot spot residues (Gαs/Gαq-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)-Gαq The Gαs and Gαq peptides adopt different orientations in β2-AR and V1AR, respectively. The β2-AR/Gαs peptide interface is dominated by electrostatic interactions, whereas the V1AR/Gαq 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 Gαq prevents this peptide from interacting strongly with β2-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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
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