Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Structure of the human κ-opioid receptor in complex with JDTic

Abstract

Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and—in the case of κ-opioid receptor (κ-OR)—dysphoria and psychotomimesis. Here we report the crystal structure of the human κ-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 Å resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human κ-OR. Modelling of other important κ-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5′-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure–activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for κ-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human κ-OR.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Crystal packing and overview of the human κ-OR structure in complex with JDTic, and comparison with the inactive CXCR4 and β 2 -AR structures.
Figure 2: Binding of the high-affinity selective antagonist JDTic in the human κ-OR crystal structure.
Figure 3: Putative interaction modes of morphine-based high-affinity κ-OR-selective antagonists nor-BNI and GNTI.
Figure 4: Model of covalently bound RB-64.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates and structure factors have been deposited in the Protein Data Bank under accession code 4DJH.

References

  1. Fredriksson, R., Lagerstrom, M. C., Lundin, L. G. & Schioth, H. B. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256–1272 (2003)

    CAS  Article  Google Scholar 

  2. Waldhoer, M., Bartlett, S. E. & Whistler, J. L. Opioid receptors. Annu. Rev. Biochem. 73, 953–990 (2004)

    CAS  Article  Google Scholar 

  3. Cherezov, V. et al. High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science 318, 1258–1265 (2007)

    ADS  CAS  Article  Google Scholar 

  4. Jaakola, V. P. et al. The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist. Science 322, 1211–1217 (2008)

    ADS  CAS  Article  Google Scholar 

  5. Chien, E. Y. et al. Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist. Science 330, 1091–1095 (2010)

    ADS  CAS  Article  Google Scholar 

  6. Warne, T. et al. Structure of a β1-adrenergic G-protein-coupled receptor. Nature 454, 486–491 (2008)

    ADS  CAS  Article  Google Scholar 

  7. Shimamura, T. et al. Structure of the human histamine H1 receptor complex with doxepin. Nature 475, 65–70 (2011)

    CAS  Article  Google Scholar 

  8. Wu, B. et al. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 330, 1066–1071 (2010)

    ADS  CAS  Article  Google Scholar 

  9. Rasmussen, S. G. et al. Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature 477, 549–555 (2011)

    ADS  CAS  Article  Google Scholar 

  10. Katritch, V., Cherezov, V. & Stevens, R. C. Diversity and modularity of G protein-coupled receptor structures. Trends Pharmacol. Sci. 33, 17–27 (2011)

    Article  Google Scholar 

  11. Congreve, M., Langmead, C. J., Mason, J. S. & Marshall, F. H. Progress in structure based drug design for G protein-coupled receptors. J. Med. Chem. 54, 4283–4311 (2011)

    CAS  Article  Google Scholar 

  12. Kufareva, I., Rueda, M., Katritch, V., Stevens, R. C. & Abagyan, R. Status of GPCR modeling and docking as reflected by community-wide GPCR Dock 2010 assessment. Structure 19, 1108–1126 (2011)

    CAS  Article  Google Scholar 

  13. Martin, W. R., Eades, C. G., Thompson, J. A., Huppler, R. E. & Gilbert, P. E. The effects of morphine- and nalorphine- like drugs in the nondependent and morphine-dependent chronic spinal dog. J. Pharmacol. Exp. Ther. 197, 517–532 (1976)

    CAS  PubMed  Google Scholar 

  14. Carlezon, W. A., Jr, Beguin, C., Knoll, A. T. & Cohen, B. M. Kappa-opioid ligands in the study and treatment of mood disorders. Pharmacol. Ther. 123, 334–343 (2009)

    CAS  Article  Google Scholar 

  15. Roth, B. L. et al. Salvinorin A: a potent naturally occurring nonnitrogenous κ opioid selective agonist. Proc. Natl Acad. Sci. USA 99, 11934–11939 (2002)

    ADS  CAS  Article  Google Scholar 

  16. Walsh, S. L., Strain, E. C., Abreu, M. E. & Bigelow, G. E. Enadoline, a selective kappa opioid agonist: comparison with butorphanol and hydromorphone in humans. Psychopharmacology (Berl.) 157, 151–162 (2001)

    CAS  Article  Google Scholar 

  17. Thomas, J. B. et al. Identification of the first trans-(3R,4R)- dimethyl-4-(3-hydroxyphenyl)piperidine derivative to possess highly potent and selective opioid κ receptor antagonist activity. J. Med. Chem. 44, 2687–2690 (2001)

    CAS  Article  Google Scholar 

  18. Carroll, F. I. et al. Pharmacological properties of JDTic: a novel κ-opioid receptor antagonist. Eur. J. Pharmacol. 501, 111–119 (2004)

    CAS  Article  Google Scholar 

  19. Jackson, K. J., Carroll, F. I., Negus, S. S. & Damaj, M. I. Effect of the selective kappa-opioid receptor antagonist JDTic on nicotine antinociception, reward, and withdrawal in the mouse. Psychopharmacology (Berl.) 210, 285–294 (2010)

    CAS  Article  Google Scholar 

  20. Salom, D. et al. Crystal structure of a photoactivated deprotonated intermediate of rhodopsin. Proc. Natl Acad. Sci. USA 103, 16123–16128 (2006)

    ADS  CAS  Article  Google Scholar 

  21. Mancia, F., Assur, Z., Herman, A. G., Siegel, R. & Hendrickson, W. A. Ligand sensitivity in dimeric associations of the serotonin 5HT2c receptor. EMBO Rep. 9, 363–369 (2008)

    CAS  Article  Google Scholar 

  22. Wang, J. B., Johnson, P. S., Wu, J. M., Wang, W. F. & Uhl, G. R. Human κ opiate receptor second extracellular loop elevates dynorphin’s affinity for human μ/κ chimeras. J. Biol. Chem. 269, 25966–25969 (1994)

    CAS  PubMed  Google Scholar 

  23. Ballesteros, J. A. & Weinstein, H. 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)

    CAS  Article  Google Scholar 

  24. Palczewski, K. et al. Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289, 739–745 (2000)

    ADS  CAS  Article  Google Scholar 

  25. Xu, F. et al. Structure of an agonist-bound human A2A adenosine receptor. Science 332, 322–327 (2011)

    ADS  CAS  Article  Google Scholar 

  26. Standfuss, J. et al. The structural basis of agonist-induced activation in constitutively active rhodopsin. Nature 471, 656–660 (2011)

    ADS  CAS  Article  Google Scholar 

  27. Subramanian, G., Paterlini, M. G., Larson, D. L., Portoghese, P. S. & Ferguson, D. M. Conformational analysis and automated receptor docking of selective arylacetamide-based κ-opioid agonists. J. Med. Chem. 41, 4777–4789 (1998)

    CAS  Article  Google Scholar 

  28. Cai, T. B. et al. Synthesis and in vitro opioid receptor functional antagonism of analogues of the selective kappa opioid receptor antagonist (3R)-7-hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-pipe ridinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (JDTic). J. Med. Chem. 51, 1849–1860 (2008)

    CAS  Article  Google Scholar 

  29. Thomas, J. B. et al. Importance of phenolic address groups in opioid kappa receptor selective antagonists. J. Med. Chem. 47, 1070–1073 (2004)

    CAS  Article  Google Scholar 

  30. Runyon, S. P. et al. Analogues of (3R)-7-hydroxy-N-[(1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-pipe ridinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (JDTic). Synthesis and in vitro and in vivo opioid receptor antagonist activity. J. Med. Chem. 53, 5290–5301 (2010)

    CAS  Article  Google Scholar 

  31. Zimmerman, D. M., Nickander, R., Horng, J. S. & Wong, D. T. New structural concepts for narcotic antagonists defined in a 4-phenylpiperidine series. Nature 275, 332–334 (1978)

    ADS  CAS  Article  Google Scholar 

  32. Vortherms, T. A., Mosier, P. D., Westkaemper, R. B. & Roth, B. L. Differential helical orientations among related G protein-coupled receptors provide a novel mechanism for selectivity. Studies with salvinorin A and the κ-opioid receptor. J. Biol. Chem. 282, 3146–3156 (2007)

    CAS  Article  Google Scholar 

  33. Surratt, C. K. et al. -mu opiate receptor. Charged transmembrane domain amino acids are critical for agonist recognition and intrinsic activity. J. Biol. Chem. 269, 20548–20553 (1994)

    CAS  PubMed  Google Scholar 

  34. Befort, K. et al. The conserved aspartate residue in the third putative transmembrane domain of the delta-opioid receptor is not the anionic counterpart for cationic opiate binding but is a constituent of the receptor binding site. Mol. Pharmacol. 49, 216–223 (1996)

    CAS  PubMed  Google Scholar 

  35. Totrov, M. & Abagyan, R. Flexible protein-ligand docking by global energy optimization in internal coordinates. Proteins 29, 215–220 (1997)

    Article  Google Scholar 

  36. Metzger, T. G., Paterlini, M. G., Portoghese, P. S. & Ferguson, D. M. Application of the message-address concept to the docking of naltrexone and selective naltrexone-derived opioid antagonists into opioid receptor models. Neurochem. Res. 21, 1287–1294 (1996)

    CAS  Article  Google Scholar 

  37. Chen, S. et al. Mutation of a single TMVI residue, Phe282, in the β2-adrenergic receptor results in structurally distinct activated receptor conformations. Biochemistry 41, 6045–6053 (2002)

    CAS  Article  Google Scholar 

  38. Chavkin, C. & Goldstein, A. Specific receptor for the opioid peptide dynorphin: structure–activity relationships. Proc. Natl Acad. Sci. USA 78, 6543–6547 (1981)

    ADS  CAS  Article  Google Scholar 

  39. Yan, F. et al. Structure-based design, synthesis, and biochemical and pharmacological characterization of novel salvinorin A analogues as active state probes of the κ-opioid receptor. Biochemistry 48, 6898–6908 (2009)

    CAS  Article  Google Scholar 

  40. Verdonk, M. L., Cole, J. C., Hartshorn, M., Murray, C. W. & Taylor, R. Improved protein-ligand docking using GOLD. Proteins 52, 609–623 (2003)

    CAS  Article  Google Scholar 

  41. Rosenbaum, D. M. et al. GPCR engineering yields high-resolution structural insights into β2-adrenergic receptor function. Science 318, 1266–1273 (2007)

    ADS  CAS  Article  Google Scholar 

  42. Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nature Protocols 4, 706–731 (2009)

    CAS  Article  Google Scholar 

  43. Cherezov, V., Peddi, A., Muthusubramaniam, L., Zheng, Y. F. & Caffrey, M. A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases. Acta Crystallogr. D 60, 1795–1807 (2004)

    Article  Google Scholar 

  44. Cherezov, V. et al. Rastering strategy for screening and centring of microcrystal samples of human membrane proteins with a sub-10 μm size X-ray synchrotron beam. J. R. Soc. Interface 6 (Suppl. 5). S587–S597 (2009)

    CAS  Article  Google Scholar 

  45. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    CAS  Article  Google Scholar 

  46. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007)

    CAS  Article  Google Scholar 

  47. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    CAS  Article  Google Scholar 

  48. Bricogne, G., et al. BUSTER v. 2.8.0 (Global Phasing, 2009)

  49. Adams, P. D., et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    CAS  Article  Google Scholar 

  50. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by PSI:Biology grant U54 GM094618 (V.K., V.C., R.C.S.) for biological studies and structure production, NIH Roadmap grant P50 GM073197 (V.C., R.C.S.) for technology development and R01 DA017624 (B.L.R., E.V., R.B.M., P.D.M.), R01 DA027170 (B.L.R.), the NIMH Psychoactive Drug Screening Program Contract (B.L.R., X.-P.H.), the Michael Hooker Distinguished Chair of Pharmacology (B.L.R.), and the NIH grant R01 DA009045 (F.I.C.). D.W. is supported by a Boehringer Ingelheim Fonds PhD Fellowship. The JDTic X-ray structure was determined by C. George at the Laboratory for the Structure of Matter, Naval Research Laboratory. We thank J. Velasquez for help on molecular biology; T. Trinh, K. Allin and M. Chu for help on baculovirus expression; V. Setola for help with functional activity assays; J. Evans for help acquiring compounds; the National Institute of Drug Abuse Drug Supply Program for supplying JDTic and other opioid ligands used in these studies; K. Kadyshevskaya for assistance with figure preparation; E. Abola for assistance with manuscript preparation; A. Walker for assistance with manuscript preparation; J. Smith, R. Fischetti and N. Sanishvili for assistance in the development and use of the minibeam and beamtime at GM/CA-CAT beamline 23-ID at the Advanced Photon Source, which is supported by National Cancer Institute grant Y1-CO-1020 and National Institute of General Medical Sciences grant Y1-GM-1104.

Author information

Authors and Affiliations

Authors

Contributions

H.W. assisted with protein expression, optimized the constructs, purified and crystallized the receptor in LCP, optimized crystallization conditions, grew crystals for data collection, collected the data and processed diffraction data, and prepared the manuscript. D.W. assisted with protein expression, purified the receptor, performed the thermal stability assay and assisted with preparing the manuscript. M.M. assisted with protein expression, purified the receptor, tested the JDTic compound, and performed the thermal stability assay. V.K. performed nor-BNI/GNTI-receptor docking and prepared the manuscript. G.W.H. processed diffraction data, solved and refined the structure and assisted with preparing the manuscript. E.V. created the initial tagged human κ-OR constructs and E.V. and X.-P.H. performed the ligand-binding and site-directed mutagenesis studies. W.L. assisted with construct optimization and crystallization in LCP. A.A.T. refined the structure and assisted with preparing the manuscript. F.I.C. and S.W.M. provided JDTic crystal structure, performed conformational studies of JDTic, and assisted with preparing the manuscript. R.B.W. and P.D.M. performed RB-64-receptor docking and prepared the manuscript. V.C. assisted with the crystallization in LCP, processed diffraction data, refined the structure and prepared the manuscript. B.L.R. suggested the JDTic compound for structural studies, supervised the pharmacology and mutagenesis studies and prepared the manuscript. R.C.S. was responsible for the overall project strategy and management and led the manuscript preparation and writing.

Corresponding author

Correspondence to Raymond C. Stevens.

Ethics declarations

Competing interests

R.C.S. is a founder and BOD member of Receptos, a GPCR drug discovery company.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-3, Supplementary Figures 1-12 and additional references. (PDF 2716 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Wu, H., Wacker, D., Mileni, M. et al. Structure of the human κ-opioid receptor in complex with JDTic. Nature 485, 327–332 (2012). https://doi.org/10.1038/nature10939

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature10939

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing