Identification of a Small Molecule-Ligand-Binding Pocket in a G Protein-Coupled Receptor using Genetically-Encoded Photocrosslinkers

Abstract

The G protein-coupled receptor (GPCR) CC chemokine receptor 5 (CCR5) is the primary co-receptor required for HIV-1 cellular entry and the molecular target for the HIV-1 cellular entry inhibitor maraviroc. Despite the fact that maraviroc obtained FDA approval for therapeutic use in 2007, the precise mode of receptor-drug interaction and its mechanism of action has not been directly defined. Using unnatural amino acid mutagenesis and targeted photocrosslinking, we have identified amino acids in CCR5 that are within 3-5 A of the bound ligand. We introduced p-benzoyl-L-phenylalanine (BzF) and p-azido-L-phenylalanine (AzF) at multiple specific sites in engineered CCR5 expressed in HEK-293T cells. Photocrosslinking experiments were performed in live cells in the presence of [3H]maraviroc. Site-specific crosslinks were detected by scintillation counting of Western Blot membranes of cell extracts. The pattern of BzF and AzF crosslinks was compared based on existed models of CCR5. This method is an extension of earlier work in which ligand analogues with fluorescent tags were employed (1). Targeted photocrosslinking using isotopically-labelled GPCR ligands allows the direct mapping of a drug-binding site with chemical precision and can be used to discriminate among various models of receptor-drug interaction. To our knowledge, this is the first demonstration of a direct chemical crosslink between a GPCR and a native small-molecule ligand. 1. Grunbeck, et al. (2011) Biochemistry 50, 3411-3413.