Supplementary data are available at Bioinformatics online.

Highlights d An active b2AR structure was obtained by fusion with residues 381-394 of Gs (GsCT) d GsCT interacts with the b2AR differently compared with the b2AR-Gs complex structure d The structure of Gs GDP reveals the conformation of GsCT before b2AR engagement d A model for the initial stages of b2AR-Gs complex formation is proposed

Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs.