Photochemical affinity switches to resolve ligand dissociation from a G protein-coupled receptor by time-resolved serial crystallography

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors in humans. The binding and dissociation of ligands tunes the inherent conformational flexibility of these important drug targets towards distinct functional states. To trigger such protein-ligand interaction dynamics within the human adenosine A _2A receptor, we designed seven photochemical affinity switches derived from the anti-Parkinson’s drug istradefylline. In a rational approach based on UV/Vis spectroscopy, time-resolved absorption spectroscopy, differential scanning fluorimetry and cryo-crystallography, we identified compounds suitable for time-resolved serial crystallography. Our analysis of millisecond-scale dynamics revealed how trans-cis isomerization shifts selected istradefylline derivatives within the binding pocket. Depending on the chemical nature of the ligand, this disrupts interactions between extracellular loops 2 and 3, acting as a lid on the binding pocket, followed by large-scale receptor rearrangements upon ligand dissociation. This innovative approach provides insights into GPCR dynamics at the atomic level, offering potential for developing novel pharmaceutics.