Dataset Information

Delineating the conformational landscape of the adenosine A_2A receptor during G protein coupling.

ABSTRACT: G-protein-coupled receptors (GPCRs) represent a ubiquitous membrane protein family and are important drug targets. Their diverse signaling pathways are driven by complex pharmacology arising from a conformational ensemble rarely captured by structural methods. Here, fluorine nuclear magnetic resonance spectroscopy (¹⁹F NMR) is used to delineate key functional states of the adenosine A_2A receptor (A_2AR) complexed with heterotrimeric G protein (Gα_sβ₁γ₂) in a phospholipid membrane milieu. Analysis of A_2AR spectra as a function of ligand, G protein, and nucleotide identifies an ensemble represented by inactive states, a G-protein-bound activation intermediate, and distinct nucleotide-free states associated with either partial- or full-agonist-driven activation. The Gβγ subunit is found to be critical in facilitating ligand-dependent allosteric transmission, as shown by ¹⁹F NMR, biochemical, and computational studies. The results provide a mechanistic basis for understanding basal signaling, efficacy, precoupling, and allostery in GPCRs.

SUBMITTER: Huang SK

PROVIDER: S-EPMC10266235 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Delineating the conformational landscape of the adenosine A2A receptor during G protein coupling.

Huang Shuya Kate SK Pandey Aditya A Tran Duy Phuoc DP Villanueva Nicolas L NL Kitao Akio A Sunahara Roger K RK Sljoka Adnan A Prosser R Scott RS

Cell 20210319 7

G-protein-coupled receptors (GPCRs) represent a ubiquitous membrane protein family and are important drug targets. Their diverse signaling pathways are driven by complex pharmacology arising from a conformational ensemble rarely captured by structural methods. Here, fluorine nuclear magnetic resonance spectroscopy (19F NMR) is used to delineate key functional states of the adenosine A2A receptor (A2AR) complexed with heterotrimeric G protein (Gαsβ ...[more]

PMID: 33743210

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Project description:G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra- and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Förster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes ≥ 3 ms. Local conformational dynamics around residue 2296.31 on TM6 was measured using fluorescence correlation spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150-350 ns), intermediate (50-60 μs) and slow (200-300 μs) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y2887.53, and Y2907.55) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 2296.31 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.

Dataset Information

Delineating the conformational landscape of the adenosine A_2A receptor during G protein coupling.

Publications

Delineating the conformational landscape of the adenosine A<sub>2A</sub> receptor during G protein coupling.

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