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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 \u03bcOR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded \u00b5OR ligand and uncover the molecular basis for \u00b5OR antagonism by solving the cryo-EM structure of the NbE-\u00b5OR complex. NbE displays a unique ligand binding mode and achieves \u00b5OR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a \u03b2-hairpin loop formed by NbE that deeply inserts into the \u00b5OR and centers most binding contacts, we design short peptide analogues that retain \u00b5OR antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes novel \u03bcOR ligands that can serve as a basis for therapeutic developments.<\/jats:p>","DOI":"10.1101\/2023.12.06.570395","type":"posted-content","created":{"date-parts":[[2023,12,7]],"date-time":"2023-12-07T22:35:34Z","timestamp":1701988534000},"source":"Crossref","is-referenced-by-count":5,"title":["Structural Basis of \u03bc-Opioid Receptor-Targeting by a Nanobody Antagonist"],"prefix":"10.64898","author":[{"given":"Jun","family":"Yu","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Amit","family":"Kumar","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xuefeng","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Charlotte","family":"Martin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pierre","family":"Raia","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Antoine","family":"Koehl","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Toon","family":"Laeremans","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jan","family":"Steyaert","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aashish","family":"Manglik","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Steven","family":"Ballet","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andreas","family":"Boland","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Miriam","family":"Stoeber","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2023121122003727000_2023.12.06.570395v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/nrd.2016.230"},{"key":"2023121122003727000_2023.12.06.570395v1.2","doi-asserted-by":"publisher","DOI":"10.1124\/mol.117.111062"},{"key":"2023121122003727000_2023.12.06.570395v1.3","doi-asserted-by":"publisher","DOI":"10.1038\/nrd.2017.178"},{"key":"2023121122003727000_2023.12.06.570395v1.4","doi-asserted-by":"crossref","unstructured":"C. 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