title: Mapping the ligand-binding site on a G protein-coupled receptor (GPCR) using genetically encoded photocrosslinkers.
abstract: We developed a general cell-based photocrosslinking approach to investigate the binding interfaces necessary for the formation of G protein-coupled receptor (GPCR) signaling complexes. The two photoactivatable unnatural amino acids p-benzoyl-L-phenylalanine and p-azido-L-phenylalanine were incorporated by amber codon suppression technology into CXC chemokine receptor 4 (CXCR4). We then probed the ligand-binding site for the HIV-1 coreceptor blocker, T140, using a fluorescein-labeled T140 analogue. Among eight amino acid positions tested, we found a unique UV-light-dependent crosslink specifically between residue 189 and T140. These results are evaluated with molecular modeling using the crystal structure of CXCR4 bound to CVX15.
authors.affiliation: Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York, United States.
authors.forename: Amy
authors.initials: A
authors.lastname: Grunbeck
authors.forename: Thomas
authors.initials: T
authors.lastname: Huber
authors.forename: Pallavi
authors.initials: P
authors.lastname: Sachdev
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Sakmar
mesh_terms: D001386:Azides / Q000737:chemistry / Q000378:metabolism; D001577:Benzophenones / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000378:metabolism; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes; D057809:HEK293 Cells; D015497:HIV-1; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D019718:Receptors, CXCR4 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: C488060:4-benzoylphenylalanine; D001386:Azides; D001577:Benzophenones; D003432:Cross-Linking Reagents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008024:Ligands; D009842:Oligopeptides; D019718:Receptors, CXCR4; C027162:4-azidophenylalanine; D010649:Phenylalanine; C116977:T140 peptide