Sub-millisecond conformational dynamics of the A_2A adenosine receptor revealed by single-molecule FRET

Abstract

The complex pharmacology of G-protein-coupled receptors (GPCRs) is defined by their multi-state conformational dynamics. Single-molecule Förster Resonance Energy Transfer (smFRET) is well-suited to quantify dynamics for individual protein molecules, however, its application to GPCRs is challenging; therefore, smFRET has been limited to studies of interreceptor interactions in cellular membranes and receptors in detergent environments. Here, we performed smFRET experiments on functionally active human A2A adenosine receptor (A2AAR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A2AAR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A2AAR, explaining the receptor’s constitutive activity. For the agonist-bound A2AAR, we detected faster (390±80 μs) ligand efficacy-dependent dynamics. This work establishes a general smFRET platform for GPCR investigations that can potentially be used for drug screening and/or mechanism-of-action studies.

Slow conformational dynamics of the human A2A adenosine receptor are temporally ordered

“…Using single-molecule total internal reflection fluorescence (TIRF) imaging, we observed relatively slower (k ex % 10 s À1 ) exchange processes in A 2A AR with a fluorophore covalently attached to helix 7 at the intracellular surface. Our data complement other A 2A AR studies that investigated relatively faster timescales using single-molecule Fo ¨rster resonance energy transfer (FRET), which measured ligand efficacy-dependent changes for sub-millisecond to $3 millisecond molecular motions of A 2A AR in detergent micelles (Fernandes et al, 2021) and in lipid nanodiscs (Maslov et al, 2020). In the present study, measurements of slower exchange processes provide an exciting window into A 2A AR function-related conformational dynamics.…”

“…In a recent report of single-molecule FRET of A 2A AR in detergent micelles containing LMNG and CHS, ligand-depending shifts and broadening of smFRET distributions were interpreted to suggest exchange between inactive and active conformations of A 2A AR that occurred on a timescale of $3 ms or k ex R $30-40 s À1 (Fernandes et al, 2021). In a separate study, Maslov et al reported observations of exchange processes on a similar timescale of sub-millisecond to low millisecond fluctuations with A 2A AR in nanodiscs containing POPC and POPG lipids (Maslov et al, 2020). Both the Fernandes et al and Maslov et al studies utilized a diffusion-based experimental design in which the observation of FRET signals depends on the rate of diffusion of the receptors in aqueous solutions.…”

Structurally similar G protein complexes with β1-adrenergic receptor active state show differential binding kinetics, mediating selectivity

“…Therefore, we used the β 1 AR-E construct to characterise the conformational properties of the active state in solution by NMR spectroscopy. We labelled β 1 AR-E with 13 C methyl methionine, facilitating examination of the NMR response to full agonist on the natively occurring methionine positions M153 34.57 (IL2), M178 4.62 (EL2), M223 5.54 (TM5), M283 6.28 (TM6), and M296 6.41 (TM6) (Fig. 2a) assigned in previous work using β 1 AR-W 20 .…”

“…2a) assigned in previous work using β 1 AR-W 20 . We assessed conformational changes of the receptor upon isoprenaline addition through 2D 1 H- 13 C HMQC methionine NMR shift correlation experiments and used peak changes in both dimensions compared to the apo state spectrum to inspect differences in conformations and dynamics (Supplementary Fig. 3).…”

“…Spectroscopic methods, including solution nuclear magnetic resonance (NMR) spectroscopy, have established GPCRs as highly plastic entities capable of sampling an equilibrium of conformations [9][10][11][12][13][14][15][16] .…”