Molecular characterization of oxysterol binding to the Epstein-Barr virus-induced gene 2 (GPR183)

Tau Benned-Jensen; Christoffer Norn; Stephane Laurent; Christian M Madsen; Hjalte Martin Larsen; Kristine N Arfelt; Romain M Wolf; Thomas Frimurer; Andreas W Sailer; Mette M Rosenkilde

doi:10.1074/jbc.M112.387894

Molecular characterization of oxysterol binding to the Epstein-Barr virus-induced gene 2 (GPR183)

Tau Benned-Jensen, Christoffer Norn, Stephane Laurent, Christian M Madsen, Hjalte Martin Larsen, Kristine N Arfelt, Romain M Wolf, Thomas Frimurer, Andreas W Sailer, Mette M Rosenkilde

26 Citations (Scopus)

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.

Original language	English
Journal	The Journal of Biological Chemistry
Volume	287
Issue number	42
Pages (from-to)	35470-83
Number of pages	14
ISSN	0021-9258
DOIs	https://doi.org/10.1074/jbc.M112.387894
Publication status	Published - 12 Oct 2012

Keywords

Amino Acid Substitution
Catalytic Domain
HEK293 Cells
Humans
Hydroxycholesterols
Molecular Docking Simulation
Mutation, Missense
Protein Binding
Receptors, G-Protein-Coupled

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10.1074/jbc.M112.387894

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@article{4657bc83c8504a4084f8c7c75e720c34,

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.",

keywords = "Amino Acid Substitution, Catalytic Domain, HEK293 Cells, Humans, Hydroxycholesterols, Molecular Docking Simulation, Mutation, Missense, Protein Binding, Receptors, G-Protein-Coupled",

author = "Tau Benned-Jensen and Christoffer Norn and Stephane Laurent and Madsen, {Christian M} and Larsen, {Hjalte Martin} and Arfelt, {Kristine N} and Wolf, {Romain M} and Thomas Frimurer and Sailer, {Andreas W} and Rosenkilde, {Mette M}",

year = "2012",

month = oct,

day = "12",

doi = "10.1074/jbc.M112.387894",

language = "English",

volume = "287",

pages = "35470--83",

journal = "Journal of Biological Chemistry",

issn = "0021-9258",

publisher = "American Society for Biochemistry and Molecular Biology, Inc.",

number = "42",

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TY - JOUR

T1 - Molecular characterization of oxysterol binding to the Epstein-Barr virus-induced gene 2 (GPR183)

AU - Benned-Jensen, Tau

AU - Norn, Christoffer

AU - Laurent, Stephane

AU - Madsen, Christian M

AU - Larsen, Hjalte Martin

AU - Arfelt, Kristine N

AU - Wolf, Romain M

AU - Frimurer, Thomas

AU - Sailer, Andreas W

AU - Rosenkilde, Mette M

PY - 2012/10/12

Y1 - 2012/10/12

N2 - 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.

AB - 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.

KW - Amino Acid Substitution

KW - Catalytic Domain

KW - HEK293 Cells

KW - Humans

KW - Hydroxycholesterols

KW - Molecular Docking Simulation

KW - Mutation, Missense

KW - Protein Binding

KW - Receptors, G-Protein-Coupled

U2 - 10.1074/jbc.M112.387894

DO - 10.1074/jbc.M112.387894

M3 - Journal article

C2 - 22875855

SN - 0021-9258

VL - 287

SP - 35470

EP - 35483

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 42

ER -

Molecular characterization of oxysterol binding to the Epstein-Barr virus-induced gene 2 (GPR183)

Abstract

Keywords

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