Structural Basis of μ-Opioid Receptor-Targeting by a Nanobody Antagonist

The micro-opioid receptor (microOR), a prototypical member of the G protein-coupled receptor (GPCR) family, is the molecular target of opioid analgesics such as morphine and fentanyl. Due to the limitations and severe side effects of currently available opioid drugs, there is considerable interest in developing novel modulators of microOR function. Most GPCR ligands today are small molecules, however biologics, including antibodies and nanobodies, are emerging as alternative therapeutics with clear advantages such as affinity and target selectivity. Here, we describe the nanobody NbE, which selectively binds to the microOR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded microOR ligand and uncover the molecular basis for microOR antagonism by solving the cryo-EM structure of the NbE-microOR complex. NbE displays a unique ligand binding mode and achieves microOR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a β-hairpin loop formed by NbE that deeply inserts into the microOR and centers most binding contacts, we design short peptide analogues that retain microOR antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes novel microOR ligands that can serve as a basis for therapeutic developments.