(L4977) Structural dynamics of the A2A adenosine receptor revealed by single-molecule FRET

Ivan Maslov (Moscow Institute of Physics and Technology, Hasselt University, KU Leuven)| Oleksandr Volkov (Forschungszentrum Jülich)| Polina Khorn (Moscow Institute of Physics and Technology)| Philipp Orekhov (Moscow Institute of Physics and Technology)| Anastasiia Gusach (Moscow Institute of Physics and Technology, MRC Laboratory of Molecular Biology)| Pavel Kuzmichev (Moscow Institute of Physics and Technology)| Andrey Gerasimov (Moscow Institute of Physics and Technology, Vyatka State University)| Aleksandra Luginina (Moscow Institute of Physics and Technology)| Quinten Coucke (KU Leuven)| Andrey Bogorodskiy (Moscow Institute of Physics and Technology)| Valentin Gordeliy (Moscow Institute of Physics and Technology, Forschungszentrum Jülich)| Simon Wanninger (Ludwig-Maximilians-Universität Munich)| Anders Barth (Ludwig-Maximilians-Universität Munich, Delft University of Technology)| Alexey Mishin (Moscow Institute of Physics and Technology)| Johan Hofkens (KU Leuven, Max Plank Institute for Polymer Research)| Vadim Cherezov (Moscow Institute of Physics and Technology, University of Southern California)| Thomas Gensch (Forschungszentrum Jülich)| Jelle Hendrix (KU Leuven, Hasselt University)| Valentin Borshchevskiy (Moscow Institute of Physics and Technology, Forschungszentrum Jülich)

G-protein-coupled receptors (GPCRs) constitute the largest superfamily of membrane proteins in humans, which mediate critical physiological processes, such as neurotransmission, homeostasis, inflammation, reproduction, olfaction, vision, taste, and others. Over 30% of all FDA-approved drugs target GPCRs.

The A2A adenosine receptor (A2AAR) is one of the best studied GPCRs. A2AAR is expressed in many organs and tissues, it regulates the cardiovascular tonus and promotes healing of inflammation-induced injuries by suppressing immune cells. In the brain, A2AAR modulates dopamine and glutamate neurotransmission. A2AAR is a promising target for drugs against insomnia, chronic pain, depression, Parkinson’s disease, and cancer. More than 50 X-Ray or CryoEM structures provide high-resolution structural snapshots of A2AAR, but current understanding of the A2AAR structure-function relationships remains critically incomplete without detailed knowledge of its conformational dynamics.

Here we applied single-molecule FRET (smFRET) to investigate the conformational dynamics of A2AAR in lipid nanodiscs. Using smFRET, we tracked the relative movements of two fluorescent dyes attached to the intracellular tip of the transmembrane helix TM6 and to the C-terminal intracellular helix H8 of A2AAR. Our measurements revealed sub-millisecond conformational dynamics of A2AAR. We propose a dynamic model of A2AAR activation that involves a slow (>2 ms) exchange between 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.