Peptide epitope tags offer a valuable means for detection and manipulation of protein targets for which high quality detection reagents are not available. Most commonly used epitope tags are bound by conventional, full-size antibodies (Abs). The complex architecture of Abs complicates their application in protein engineering and intracellular applications. To address these shortcomings, single domain antibodies (nanobodies, Nbs) that recognize short peptide epitopes have become increasingly prized. Here we characterize the interaction between a Nb (Nb6E) and a 14-mer peptide epitope. We identify residues in the peptide epitope essential for high affinity binding. Using this information in combination with computational modeling we propose a mode of interaction between Nb6E and this epitope. We apply this nanobody-epitope pair to augment the potency of a ligand at an engineered adenosine A2A receptor. This characterization of the nanobody-epitope pair opens the door to diverse applications including mechanistic studies of G protein-coupled receptor function.
