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

Abstract

The -opioid receptor (OR), 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 OR 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 OR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded {micro}OR ligand and uncover the molecular basis for {micro}OR antagonism by solving the cryo-EM structure of the NbE-{micro}OR complex. NbE displays a unique ligand binding mode and achieves {micro}OR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a {beta}-hairpin loop formed by NbE that deeply inserts into the {micro}OR and centers most binding contacts, we design short peptide analogues that retain {micro}OR antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes novel OR ligands that can serve as a basis for therapeutic developments.