title: Molecular characterization of oxysterol binding to the Epstein-Barr virus-induced gene 2 (GPR183).
abstract: Oxysterols are oxygenated cholesterol derivates that are emerging as a physiologically important group of molecules. Although they regulate a range of cellular processes, only few oxysterol-binding effector proteins have been identified, and the knowledge of their binding mode is limited. Recently, the family of G protein-coupled seven transmembrane-spanning receptors (7TM receptors) was added to this group. Specifically, the Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) was shown to be activated by several oxysterols, most potently by 7α,25-dihydroxycholesterol (7α,25-OHC). Nothing is known about the binding mode, however. Using mutational analysis, we identify here four key residues for 7α,25-OHC binding: Arg-87 in TM-II (position II:20/2.60), Tyr-112 and Tyr-116 (positions III:09/3.33 and III:13/3.37) in TM-III, and Tyr-260 in TM-VI (position VI:16/6.51). Substituting these residues with Ala and/or Phe results in a severe decrease in agonist binding and receptor activation. Docking simulations suggest that Tyr-116 interacts with the 3β-OH group in the agonist, Tyr-260 with the 7α-OH group, and Arg-87, either directly or indirectly, with the 25-OH group, although nearby residues likely also contribute. In addition, Tyr-112 is involved in 7α,25-OHC binding but via hydrophobic interactions. Finally, we show that II:20/2.60 constitutes an important residue for ligand binding in receptors carrying a positively charged residue at this position. This group is dominated by lipid- and nucleotide-activated receptors, here exemplified by the CysLTs, P2Y12, and P2Y14. In conclusion, we present the first molecular characterization of oxysterol binding to a 7TM receptor and identify position II:20/2.60 as a generally important residue for ligand binding in certain 7TM receptors.
authors.affiliation: Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Tau
authors.initials: T
authors.lastname: Benned-Jensen
authors.affiliation: Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Christoffer
authors.initials: C
authors.lastname: Norn
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, 4056 Basel, Switzerland.
authors.forename: Stephane
authors.initials: S
authors.lastname: Laurent
authors.affiliation: Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Christian M
authors.initials: CM
authors.lastname: Madsen
authors.affiliation: Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Hjalte M
authors.initials: HM
authors.lastname: Larsen
authors.affiliation: Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Kristine N
authors.initials: KN
authors.lastname: Arfelt
authors.affiliation: Global Discovery Chemistry, Novartis Institutes for BioMedical Research, 4056 Basel, Switzerland.
authors.forename: Romain M
authors.initials: RM
authors.lastname: Wolf
authors.affiliation: Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark; Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Frimurer
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, 4056 Basel, Switzerland.
authors.forename: Andreas W
authors.initials: AW
authors.lastname: Sailer
authors.affiliation: Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. Electronic address: rosenkilde@sund.ku.dk.
authors.forename: Mette M
authors.initials: MM
authors.lastname: Rosenkilde
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D057809:HEK293 Cells; D006801:Humans; D006888:Hydroxycholesterols / Q000737:chemistry* / Q000378:metabolism; D062105:Molecular Docking Simulation; D020125:Mutation, Missense; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C080105:GPR183 protein, human; D006888:Hydroxycholesterols; D043562:Receptors, G-Protein-Coupled; C057657:7,25-dihydroxycholesterol