title: Identification of structural motifs critical for epstein-barr virus-induced molecule 2 function and homology modeling of the ligand docking site.
abstract: Epstein-Barr virus-induced molecule 2 (EBI2) (also known as G-protein-coupled receptor 183) is a G-protein-coupled receptor (GPCR) that is best known for its role in B cell migration and localization. Our recent deorphanization effort led to the discovery of 7α,25-dihydroxycholesterol (7α,25-OHC) as the endogenous ligand for EBI2, which provides a tool for mechanistic studies of EBI2 function. Because EBI2 is the first GPCR known to bind and to be activated by an oxysterol, the goal of this study was to understand the molecular and structural bases for its ligand-dependent activation; this was achieved by identifying structural moieties in EBI2 or in 7α,25-OHC that might affect receptor-ligand interactions. By using a series of chemically related OHC analogs, we demonstrated that all three hydroxyl groups in 7α,25-OHC contributed to ligand-induced activation of the receptor. To determine the location and composition of the ligand binding domain in EBI2, we used a site-directed mutagenesis approach and generated mutant receptors with single amino acid substitutions at selected positions of interest. Biochemical and pharmacological profiling of these mutant receptors allowed for structure-function analyses and revealed critical motifs that likely interact with 7α,25-OHC. By using a hybrid β(2)-adrenergic receptor-C-X-C chemokine receptor type 4 structure as a template, we created a homology model for EBI2 and optimized the docking of 7α,25-OHC into the putative ligand binding site, so that the hydroxyl groups interact with residues Arg87, Asn114, and Glu183. This model of ligand docking yields important structural insight into the molecular mechanisms mediating EBI2 function and may facilitate future efforts to design novel therapeutic agents that target EBI2.
authors.affiliation: Janssen Pharmaceutical Research and Development, San Diego, California, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.forename: Amy Y
authors.initials: AY
authors.lastname: Shih
authors.forename: Xia V
authors.initials: XV
authors.lastname: Yang
authors.forename: Chester
authors.initials: C
authors.lastname: Kuei
authors.forename: Jiejun
authors.initials: J
authors.lastname: Wu
authors.forename: Xiaohu
authors.initials: X
authors.lastname: Deng
authors.forename: Neelakandha S
authors.initials: NS
authors.lastname: Mani
authors.forename: Taraneh
authors.initials: T
authors.lastname: Mirzadegan
authors.forename: Siquan
authors.initials: S
authors.lastname: Sun
authors.forename: Timothy W
authors.initials: TW
authors.lastname: Lovenberg
authors.forename: Changlu
authors.initials: C
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D019743:Chemokines, CXC / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D019743:Chemokines, CXC; C080105:GPR183 protein, human; D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D003545:Cysteine