Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for μOR activation, here we report a 2.1 Å X-ray crystal structure of the murine μOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2-adrenergic receptor (β2AR) and the M2 muscarinic receptor. Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.
- Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the µ-opioid-receptor gene. Nature 383, 819–823 (1996) et al.
- A brief history of opiates, opioid peptides, and opioid receptors. Proc. Natl Acad. Sci. USA 90, 5391–5393 (1993)
- 2015) , & (McGraw-Hill Medical,
- Morphine side effects in β-arrestin 2 knockout mice. J. Pharmacol. Exp. Ther. 314, 1195–1201 (2005) , &
- μ-Opioid receptor desensitization by β-arrestin-2 determines morphine tolerance but not dependence. Nature 408, 720–723 (2000) , , , &
- Enhanced morphine analgesia in mice lacking β-arrestin 2. Science 286, 2495–2498 (1999) et al.
- Mu opioids and their receptors: evolution of a concept. Pharmacol. Rev. 65, 1257–1317 (2013) &
- Specific receptor for the opioid peptide dynorphin: structure–activity relationships. Proc. Natl Acad. Sci. USA 78, 6543–6547 (1981) &
- Crystal structure of the μ-opioid receptor bound to a morphinan antagonist. Nature 485, 321–326 (2012) et al.
- Structure of the δ-opioid receptor bound to naltrindole. Nature 485, 400–404 (2012) et al.
- Molecular control of δ-opioid receptor signalling. Nature 506, 191–196 (2014) et al.
- Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469, 175–180 (2011) et al.
- Adrenaline-activated structure of β2-adrenoceptor stabilized by an engineered nanobody. Nature 502, 575–579 (2013) et al.
- High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science 318, 1258–1265 (2007) et al.
- GPCR engineering yields high-resolution structural insights into β2-adrenergic receptor function. Science 318, 1266–1273 (2007) et al.
- Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist. Nature 482, 547–551 (2012) et al.
- Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101–106 (2013) et al.
- Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289, 739–745 (2000) et al.
- Crystal structure of metarhodopsin II. Nature 471, 651–655 (2011) et al.
- Structure and function of an irreversible agonist-β2 adrenoceptor complex. Nature 469, 236–240 (2011) et al.
- The dynamic process of β2-adrenergic receptor activation. Cell 152, 532–542 (2013) et al.
- The role of protein dynamics in GPCR function: insights from the β2AR and rhodopsin. Curr. Opin. Cell Biol. 27, 136–143 (2014) &
- Structural insights into the dynamic process of β2-adrenergic receptor signaling. Cell 161, 1101–1111 (2015) et al.
- A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled β-adrenergic receptor. J. Biol. Chem. 255, 7108–7117 (1980) , &
- Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011) et al.
- Synthesis and in vitro opioid activity profiles of DALDA analogues. Eur. J. Med. Chem. 35, 895–901 (2000) et al.
- Characterization of the complex morphinan derivative BU72 as a high efficacy, long-lasting mu-opioid receptor agonist. Eur. J. Pharmacol. 499, 107–116 (2004) et al.
- Crystallizing membrane proteins for structure determination: use of lipidic mesophases. Annu. Rev. Biophys. 38, 29–51 (2009)
- Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. Methods Neurosci. 25, 366–428 (1995) &
- Structure of the human κ-opioid receptor in complex with JDTic. Nature 485, 327–332 (2012) et al.
- Propagation of conformational changes during μ-opioid receptor activation. Nature http://dx.doi.org/10.1038/nature14680 (2015) et al.
- μ Opioid receptor: role for the amino terminus as a determinant of ligand binding affinity. Brain Res. Mol. Brain Res. 76, 64–72 (2000) , &
- Probing the activation-promoted structural rearrangements in preassembled receptor-G protein complexes. Nature Struct. Mol. Biol. 13, 778–786 (2006) et al.
- BU74, a complex oripavine derivative with potent kappa opioid receptor agonism and delayed opioid antagonism. Eur. J. Pharmacol. 509, 117–125 (2005) et al.
- The irreversible narcotic antagonistic and reversible agonistic properties of the fumaramate methyl ester derivative of naltrexone. Eur. J. Pharmacol. 70, 445–451 (1981) , &
- High-resolution crystal structure of human protease-activated receptor 1. Nature 492, 387–392 (2012) et al.
- Structural basis for allosteric regulation of GPCRs by sodium ions. Science 337, 232–236 (2012) et al.
- The 2.1 Å resolution structure of cyanopindolol-bound β1-adrenoceptor identifies an intramembrane Na+ ion that stabilises the ligand-free receptor. PLoS ONE 9, e92727 (2014) et al.
- Opiate agonists and antagonists discriminated by receptor binding in brain. Science 182, 1359–1361 (1973) , &
- Structural insights into the dynamic process of β2-adrenergic receptor signaling. Cell. 161, 1101–1111 (2015) et al.
- Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454, 183–187 (2008) , , , &
- Rhodopsin and 9-demethyl-retinal analog: effect of a partial agonist on displacement of transmembrane helix 6 in class A G protein-coupled receptors. J. Biol. Chem. 283, 4967–4974 (2008) , , , &
- A general protocol for the generation of nanobodies for structural biology. Nature Protocols 9, 674–693 (2014) et al.
- A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. Proc. Natl Acad. Sci. USA 104, 7682–7687 (2007) et al.
- Purification and functional reconstitution of monomeric mu-opioid receptors: allosteric modulation of agonist binding by Gi2. J. Biol. Chem. 284, 26732–26741 (2009) et al.
- The kinetics of competitive radioligand binding predicted by the law of mass action. Mol. Pharmacol. 25, 1–9 (1984) &
- Crystallizing membrane proteins using lipidic mesophases. Nature Protocols 4, 706–731 (2009) &
- XDS. Acta Crystallogr. D 66, 125–132 (2010)
- Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007) et al.
- Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004) &
- Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012) et al.
- MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010) et al.
- OPM: orientations of proteins in membranes database. Bioinformatics 22, 623–625 (2006) , , &
- CHARMM: The biomolecular simulation program. J. Comput. Chem. 30, 1545–1614 (2009) et al.
- Automated builder and database of protein/membrane complexes for molecular dynamics simulations. PLoS ONE 2, e880 (2007) , &
- CHARMM-GUI: a web-based graphical user interface for CHARMM. J. Comput. Chem. 29, 1859–1865 (2008) , , &
- CHARMM-GUI Membrane Builder toward realistic biological membrane simulations. J. Comput. Chem. 35, 1997–2004 (2014) et al.
- AMBER 14. (University of California, San Francisco, 2014) et al.
- SPFP: Speed without compromise—A mixed precision model for GPU accelerated molecular dynamics simulations. Comput. Phys. Commun. 184, 374–380 (2013) , &
- Routine microsecond molecular dynamics simulations with Amber on GPUs. 2. Explicit solvent particle mesh Ewald. J. Chem. Theory Comput. 9, 3878–3888 (2013) , , , &
- Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone ϕ, ψ and side-chain χ1 and χ2 dihedral angles. J. Chem. Theory Comput. 8, 3257–3273 (2012) et al.
- Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. J. Phys. Chem. B 114, 7830–7843 (2010) et al.
- All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 102, 3586–3616 (1998) et al.
- Extending the treatment of backbone energetics in protein force fields: limitations of gas‐phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. J. Comput. Chem. 25, 1400–1415 (2004) , &
- CHARMM general force field: a force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields. J. Comput. Chem. 31, 671–690 (2010) et al.
- VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996) , &
Extended data figures and tables
Extended Data Figures
- Extended Data Figure 1: Characterization of Nb39 and lattice interactions in μOR–Nb39 crystals. (756 KB)
a, 3H-diprenorphine (3H-DPN) competition binding shows increased affinity for μOR-selective agonists DAMGO and endomorphin-2 in the presence of Nb39. b, The dissociation half-life (t1/2) of BU72 was determined by measuring the association rate of the antagonist 3H-DPN in the presence of the indicated concentrations of BU72. The dissociation t1/2 of BU72 is 43 min and increases to 140 min in presence of Nb39. Panels a and b are representative of at least three experiments performed in triplicate, and the data and error bars represent the mean ± s.e.m. c, Crystal lattice packing of the μOR–Nb39 complex shows that most of the contacts are mediated by Nb39. The μOR extracellular domain is not involved in any contacts.
- Extended Data Figure 2: µOR–Nb39 interface. (950 KB)
a, Nb39 does not penetrate as deeply into the core of the µOR when compared with the β2AR–Nb80 complex and the M2R–Nb9-8 complex. In the β2AR–Nb80 and M2R–Nb9-8 complexes, nanobody CDR3 residues bind within the core of the receptor transmembrane bundle. In comparison, Nb39 binding involves more framework residues. Notably, seven residues of CDR3 remained unresolved in the final model of the µOR–Nb39 complex. b, Nb39 interacts primarily through hydrogen bonds with residues from ICL2, ICL3 and helix 8 of the µOR. c, Schematic representation of the interactions between µOR and Nb39 highlighting the numerous Nb39 framework interactions.
- Extended Data Figure 3: Cytoplasmic domain rearrangements in conserved regions. (644 KB)
a, The E/DRY sequence is a highly conserved motif within family A GPCRs responsible for constraining receptors in an inactive conformation. Comparisons of inactive- and active-state structures around the conserved E/DRY residues at the cytoplasmic surface of the μOR, the M2 muscarinic receptor (M2R), the β2 adrenergic receptor (β2AR) and rhodopsin (Rho) are shown here. Hydrogen bonds are shown as dotted lines. b, The NPxxY motif is a highly conserved sequence in TM7 among family A GPCRs. In the active state μOR, Y7.53 and N7.49 in TM7 interact with Y5.58 in TM5 and the backbone carbonyl of L3.43 in TM3 through a water-mediated polar network. A similar network is observed in the active state of rhodopsin. While waters are not observed in the lower-resolution structures of the β2AR and M2R, the positions of the side chains of Y7.53, N7.49 and Y5.58 suggest a similar water-mediated network with putative waters represented by red circles.
- Extended Data Figure 4: Conformation of the binding pocket and BU72. (957 KB)
The 2Fo − Fc electron density contoured at 2.0σ and within 1.8 A˚ of residues comprising the active μOR ligand-binding pocket is shown as grey mesh in a and b. The same views are shown in c and d with the omit Fo − Fc density for BU72 displayed as an orange mesh. Displayed Fo − Fc electron density is contoured at 3.0σ. e, Placement of an energetically minimized conformation of BU72 within the Fo − Fc electron density shows a poor fit for the pendant phenyl ring. The conformation of BU72 was minimized using quantum mechanical Hartree–Fock methods. f, An alternative possible ligand structure with sp2 geometry at the carbon adjacent to the phenyl (highlighted in red dashed circle) was initially considered due to a better fit within the electron density. This alternative ligand is predicted to be 2 Da smaller than BU72. g, In order to resolve potential ambiguity in the co-crystallized ligand, we performed mass spectrometry on the same protein sample used to generate crystals of the active μOR. The protein was precipitated in methanol and the supernatant was subjected to MALDI–MS which revealed a strong peak at m/z = 429.226, consistent with the expected mass of BU72. h, Shown is our final crystallographic model for BU72 within the Fo − Fc electron density. This model probably represents a high-energy conformation of BU72. Notably, the position of the morphinan scaffold is invariant between these alternative models for the crystallized ligand.
- Extended Data Figure 5: The N terminus of the μOR interacts with BU72. (708 KB)
a, Surface cut-away view showing that the N terminus forms a lid over the ligand-binding pocket. Shown in the lower panel is the ligand-binding pocket in the absence of the N terminus. b, Blue mesh shows the 2Fo − Fc omit map contoured at 1.0σ for the N terminus. c, Shown in green mesh is the Fo − Fc omit map contoured at 4.0σ of an unidentified density between BU72 and His54.
- Extended Data Figure 6: Molecular dynamics simulation of active μOR bound to antagonist BU74. (492 KB)
a, Structures of agonist BU72, and antagonists BU74 and β-funaltrexamine (β-FNA). The inactive-state structure of μOR was co-crystallized with β-FNA. b, BU74 was docked into the active-state structure of the μOR based on the crystallographic pose of BU72, but in a molecular dynamics simulation it rapidly moves away from this initial pose. The middle panel highlights the movements of BU74 after 560 ns of simulation and the rightmost panel shows the comparison of the BU74 pose as compared to the crystal structure of β-FNA bound to inactive μOR. c, Molecular dynamics trajectory measuring the distance between the phenolic hydroxyl of Y3267.43 and the tertiary amine of BU74. Dotted lines show the distance between Y3267.43 and the same amine of BU72 in the crystal structure of active μOR and β-FNA in the structure of inactive μOR.
- Extended Data Figure 7: Molecular dynamics simulation of inactive μOR bound to agonist β-FOA. (523 KB)
a, Structures of agonists BU72 and β-fuoxymorphamine (β-FOA) and antagonist β-funaltrexamine (β-FNA). b, Molecular dynamics simulation of inactive μOR with β-FOA docked into the same pose as β-FNA in the inactive-state crystal structure of μOR. β-FOA shifts towards TM3 with an accompanying rearrangement of TM3 residues D1473.32 and N1503.35 towards the active-state structure. The overall ligand-binding pocket resembles the active state after 455 ns of simulation. c, Trajectory of the W2936.48 χ2 dihedral angle (indicated in the middle panel in b) over 700 ns of simulation. In the presence of β-FOA, the preferred rotamer for W2936.48 rapidly approaches a conformation similar to the one observed in the structure of active μOR bound to BU72.
- Extended Data Figure 8: Comparison of polar networks involved in GPCR activation. (703 KB)
a, Residues involved in the polar network in the inactive state of the δOR (PDB ID: 4N6H) and conservation of those residues in β2AR, M2R, and rhodopsin. b, Residues involved in the polar network in active state μOR and conservation in β2AR, M2R, and rhodopsin. c, Water-mediated polar network in the inactive structure of the δOR involves residues from TM1, TM2, TM3, TM5, TM6 and TM7. d, An identical view as in c of the polar network in the active μOR. e, Residues involved in the polar network in inactive structures of δOR, β2AR and M2R are conserved both in sequence and conformation. f, In active μOR, β2AR and M2R, the residues within the polar network are again conserved in sequence and conformation.
- Extended Data Figure 9: Differences in TM6 polar network in opioid receptors and rhodopsin. (306 KB)
a, The entire set of contacts within the polar network that include a residue within TM6 is displayed for the inactive δOR, active μOR, and inactive and active rhodopsin (Rho). b, Helix wheel representation of TM6 showing polar contacts. Notably, the inactive δOR engages in many more polar contacts with neighbouring residues as compared to inactive rhodopsin. Additionally, the active states of both μOR and rhodopsin have fewer polar contacts than the inactive state.
Extended Data Tables
- Supplementary Information (117 KB)
This file contains an overview of molecular dynamics simulations and ligand parameterization.