title: Two classes of cholesterol binding sites for the β2AR revealed by thermostability and NMR.
abstract: Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the ?2 adrenergic receptor (β2AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in β2AR. By analyzing the β2AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and β2AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites.
authors.affiliation: Institute of Pharmacology and Structural Biology - UMR 5089, CNRS and Université de Toulouse - UPS, 205 Route de Narbonne, 31077 Toulouse, France; Khalifa University of Science, Technology and Research, P.O. Box 127788, Abu Dhabi, UAE.
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Gater
authors.affiliation: Institute of Pharmacology and Structural Biology - UMR 5089, CNRS and Université de Toulouse - UPS, 205 Route de Narbonne, 31077 Toulouse, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Saurel
authors.affiliation: Institute of Pharmacology and Structural Biology - UMR 5089, CNRS and Université de Toulouse - UPS, 205 Route de Narbonne, 31077 Toulouse, France.
authors.forename: Iordan
authors.initials: I
authors.lastname: Iordanov
authors.affiliation: Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, California.
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.affiliation: Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, California. Electronic address: vcherezo@scripps.edu.
authors.forename: Vadim
authors.initials: V
authors.lastname: Cherezov
authors.affiliation: Institute of Pharmacology and Structural Biology - UMR 5089, CNRS and Université de Toulouse - UPS, 205 Route de Narbonne, 31077 Toulouse, France. Electronic address: alain.milon@ipbs.fr.
authors.forename: Alain
authors.initials: A
authors.lastname: Milon
mesh_terms: D001665:Binding Sites; D002784:Cholesterol / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D011489:Protein Denaturation; D055550:Protein Stability; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D018343:Receptors, Adrenergic, beta-2; D002784:Cholesterol