Structural basis of μ-opioid receptor targeting by a nanobody antagonist

The mu-opioid receptor (mu OR), a prototypical G protein-coupled receptor (GPCR), is the target of opioid analgesics such as morphine and fentanyl. Due to the severe side effects of current opioid drugs, there is considerable interest in developing novel modulators of mu OR function. Most GPCR ligands today are small molecules, however biologics, including antibodies and nanobodies, represent alternative therapeutics with clear advantages such as affinity and target selectivity. Here, we describe the nanobody NbE, which selectively binds to the mu OR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded mu OR ligand and uncover the molecular basis for mu OR antagonism by determining the cryo-EM structure of the NbE-mu OR complex. NbE displays a unique ligand binding mode and achieves mu OR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a beta-hairpin loop formed by NbE that deeply protrudes into the mu OR, we design linear and cyclic peptide analogs that recapitulate NbE's antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes lower molecular weight mu OR ligands that can serve as a basis for therapeutic developments. The mu -opioid receptor is a key clinical target. Here, the authors describe nanobody NbE, a selective and high affinity antagonist, which is downsized to small cyclic peptides. The work enables unique receptor targeting based on nanobody interaction.