Sub-millisecond conformational dynamics of the A2A 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 inter-receptor 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. Our work establishes a general smFRET platform for GPCR investigations that can potentially be used for drug screening and/or mechanism-of-action studies.

Distinct Activation Mechanisms of CXCR4 and ACKR3 Revealed by Single-Molecule Analysis of their Conformational Landscapes

“…In summary, the conformational energy landscape of ACKR3 is flatter and has more local minima reflecting its greater conformational heterogeneity than CXCR4 or other previously studied GPCRs 37,38,40 . The structural heterogeneity is consistent with the ability of ACKR3 to bind and be activated by different ligands via a distortion mechanism, and the fact that most ligands are activating rather than inhibiting.…”

“…For example, smFRET studies of the β2 adrenergic receptor (β2AR) revealed a dynamic equilibrium between inactive and active conformations that was responsive to agonist and G protein binding 37 . More recent smFRET studies of the glucagon receptor 38 and the A2A receptor (A2AR) 39,40 have revealed the existence of stable intermediate conformations in addition to inactive and active receptor conformations. Similar to these studies, our system allows real-time observation of the conformational fluctuations of individual receptors in a native-like lipid environment, and assessment of the differences in the conformational dynamics of CXCR4 and ACKR3 in their apo states and in response to ligands.…”

Structural Insights into Partial Activation of the Prototypic G Protein-Coupled Adenosine A2A Receptor

“…In contrast, we previously showed that the full agonist NECA does not show any binding to the A 2A -PSB1-bRIL construct (at concentrations of up to 3 mM, determined vs. the antagonist radioligand [ 3 H]MSX-2), whereas the affinity of antagonists and of the antagonist radioligand itself was unaffected. 9 It is worth noting that structures of NECA have been solved with 33,52 or without 53 G proteins. While intracellular rearrangements and especially the extent of the outward movement of helix VI are largely dependent on the presence of G proteins, the ligand binding pocket in these full agonist-bound structures is identical� independent of the presence of G proteins�and clearly distinct from the inactive receptor conformation.…”

“…Interestingly, NMR 18 and single-molecule Forster resonance energy transfer (smFRET) investigations of the A 2A AR in complex with LUF5834 revealed distinct conformations when compared to those found for NECA or the antagonist ZM241385. 53 However, the covalent β 2 -adrenergic receptor agonist FAUC50 54 as well as β 1 -adrenergic receptor agonists 55 were reported to stably bind to inactive conformations without G proteins present. Alternatively, the partial agonistic mechanism of action may also be explained by a combination of the above-mentioned principles 56 and may even be different for various GPCRs or ligands.…”

“…Therefore, the conformational selection of full A 2A AR agonists would be restricted to active states, thus resulting in greater efficacy, in contrast to partial agonists. Interestingly, NMR and single-molecule Förster resonance energy transfer (smFRET) investigations of the A 2A AR in complex with LUF5834 revealed distinct conformations when compared to those found for NECA or the antagonist ZM241385 . However, the covalent β 2 -adrenergic receptor agonist FAUC50 as well as β 1 -adrenergic receptor agonists were reported to stably bind to inactive conformations without G proteins present.…”