Mapping the Ligand-Binding Site on a G Protein-Coupled Receptor (GPCR) Using Genetically Encoded Photocrosslinkers

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 incorpora...

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Bibliographic Details
Published in:Biochemistry (Easton) 2011-05, Vol.50 (17), p.3411-3413
Main Authors: Grunbeck, Amy, Huber, Thomas, Sachdev, Pallavi, Sakmar, Thomas P
Format: Article
Language:English
Subjects:
Azides - chemistry
Azides - metabolism
Benzophenones - chemistry
Benzophenones - metabolism
Binding Sites
Cross-Linking Reagents - chemistry
Cross-Linking Reagents - metabolism
Fluoresceins - chemistry
Fluorescent Dyes
HEK293 Cells
HIV-1
Humans
Ligands
Models, Molecular
Mutation
Oligopeptides - chemistry
Oligopeptides - metabolism
Phenylalanine - analogs & derivatives
Phenylalanine - chemistry
Phenylalanine - metabolism
Protein Binding
Receptors, CXCR4 - chemistry
Receptors, CXCR4 - genetics
Receptors, CXCR4 - metabolism
Ultraviolet Rays
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Summary: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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi200214r