Filling of a water-free void explains the allosteric regulation of the β-adrenergic receptor by cholesterol.

Recent high-pressure NMR results indicate that the preactive conformation of the β-adrenergic receptor (βAR) harbours completely empty cavities of ~100 Å volume, which disappear in the active conformation of the receptor. Here we have localized these cavities using X-ray crystallography of xenon-derivatized βAR crystals. One of the cavities is in direct contact with the cholesterol-binding pocket. Solution NMR shows that addition of the cholesterol analogue cholesteryl hemisuccinate impedes the formation of the active conformation of detergent-solubilized βAR by blocking conserved G protein-coupled receptor microswitches, concomitant with an affinity reduction of both isoprenaline and G protein-mimicking nanobody Nb80 for βAR detected by isothermal titration calorimetry. This wedge-like action explains the function of cholesterol as a negative allosteric modulator of βAR. A detailed understanding of G protein-coupled receptor regulation by cholesterol by filling of a dry void and the easy scouting for such voids by xenon may provide new routes for the development of allosteric drugs.