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.

Department of Inorganic Chemistry Organic Chemistry and Biochemistry Faculty of Science and Chemical Technologies and Faculty of Medicine of Ciudad Real Regional Center of Biomedical Research 13071 Ciudad Real Spain

Department of Physics Tampere University of Technology PO Box 692 FI 33101 Tampere Finland

Department of Physics Tampere University of Technology PO Box 692 FI 33101 Tampere Finland Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic CZ 16610 Prague Czech Republic

Institut für Medizinische Physik und Biophysik AG ProteiInformatics Charité Universitätsmedizin Berlin Charitéplatz 1 D 10117 Berlin Germany

Research Programme on Biomedical Informatics 08003 Barcelona Spain

Research Programme on Biomedical Informatics 08003 Barcelona Spain Institut für Medizinische Physik und Biophysik AG ProteiInformatics Charité Universitätsmedizin Berlin Charitéplatz 1 D 10117 Berlin Germany

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