title: Propagation of conformational changes during μ-opioid receptor activation.
abstract: µ-Opioid receptors (µORs) are G-protein-coupled receptors that are activated by a structurally diverse spectrum of natural and synthetic agonists including endogenous endorphin peptides, morphine and methadone. The recent structures of the μOR in inactive and agonist-induced active states (Huang et al., ref. 2) provide snapshots of the receptor at the beginning and end of a signalling event, but little is known about the dynamic sequence of events that span these two states. Here we use solution-state NMR to examine the process of μOR activation using a purified receptor (mouse sequence) preparation in an amphiphile membrane-like environment. We obtain spectra of the μOR in the absence of ligand, and in the presence of the high-affinity agonist BU72 alone, or with BU72 and a G protein mimetic nanobody. Our results show that conformational changes in transmembrane segments 5 and 6 (TM5 and TM6), which are required for the full engagement of a G protein, are almost completely dependent on the presence of both the agonist and the G protein mimetic nanobody, revealing a weak allosteric coupling between the agonist-binding pocket and the G-protein-coupling interface (TM5 and TM6), similar to that observed for the β2-adrenergic receptor. Unexpectedly, in the presence of agonist alone, we find larger spectral changes involving intracellular loop 1 and helix 8 compared to changes in TM5 and TM6. These results suggest that one or both of these domains may play a role in the initial interaction with the G protein, and that TM5 and TM6 are only engaged later in the process of complex formation. The initial interactions between the G protein and intracellular loop 1 and/or helix 8 may be involved in G-protein coupling specificity, as has been suggested for other family A G-protein-coupled receptors.
authors.affiliation: Institut de Genomique Fonctionnelle, CNRS UMR-5203 INSERM U1191, University of Montpellier, F-34000 Montpellier, France.
authors.forename: Rémy
authors.initials: R
authors.lastname: Sounier
authors.affiliation: Institut de Genomique Fonctionnelle, CNRS UMR-5203 INSERM U1191, University of Montpellier, F-34000 Montpellier, France.
authors.forename: Camille
authors.initials: C
authors.lastname: Mas
authors.affiliation: Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium.|Structural Biology Research Center, VIB, Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Jan
authors.initials: J
authors.lastname: Steyaert
authors.affiliation: Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium.|Structural Biology Research Center, VIB, Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Toon
authors.initials: T
authors.lastname: Laeremans
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Aashish
authors.initials: A
authors.lastname: Manglik
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Weijiao
authors.initials: W
authors.lastname: Huang
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Brian K
authors.initials: BK
authors.lastname: Kobilka
authors.affiliation: Centre de Biochimie Structurale, CNRS UMR 5048-INSERM 1054- University of Montpellier, 29 rue de Navacelles, 34090 Montpellier Cedex, France.
authors.forename: Héléne
authors.initials: H
authors.lastname: Déméné
authors.affiliation: Institut de Genomique Fonctionnelle, CNRS UMR-5203 INSERM U1191, University of Montpellier, F-34000 Montpellier, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Granier
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D009019:Morphinans / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011758:Pyrroles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry; D017450:Receptors, Opioid, mu / Q000737:chemistry* / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: C496287:BU72 compound; D009019:Morphinans; D011758:Pyrroles; D018343:Receptors, Adrenergic, beta-2; D017450:Receptors, Opioid, mu; D057127:Single-Chain Antibodies; D020962:Heterotrimeric GTP-Binding Proteins; D008239:Lysine