Sub-millisecond conformational dynamics of the A2A adenosine receptor revealed by single-molecule FRET
Ivan Maslov, Oleksandr Volkov, Polina Khorn, Philipp S. Orekhov, Anastasiia Gusach, Pavel Kuzmichev, А. С. Герасимов, Aleksandra Luginina, Quinten Coucke, Andrey Bogorodskiy, Valentin Gordeliy, Simon Wanninger, Anders Barth, Alexey Mishin, Johan Hofkens, Vadim Cherezov, Thomas Gensch, Jelle Hendrix, Valentin Borshchevskiy
Abstract
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 A 2A adenosine receptor (A 2A AR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A 2A AR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A 2A AR, explaining the receptor’s constitutive activity. For the agonist-bound A 2A AR, 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.