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.

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Accessions

Primary accessions

Protein Data Bank

Data deposits

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

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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

Affiliations

  1. Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA

    • Huixian Wu
    • , Daniel Wacker
    • , Mauro Mileni
    • , Vsevolod Katritch
    • , Gye Won Han
    • , Wei Liu
    • , Aaron A. Thompson
    • , Vadim Cherezov
    •  & Raymond C. Stevens
  2. National Institute of Mental Health Psychoactive Drug Screening Program, Department of Pharmacology and Division of Chemical Biology and Medicinal Chemistry, University of North Carolina Chapel Hill Medical School, 4072 Genetic Medicine Building, Chapel Hill, North Carolina 27599, USA

    • Eyal Vardy
    • , Xi-Ping Huang
    •  & Bryan L. Roth
  3. Center for Organic and Medicinal Chemistry, Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709, USA

    • F. Ivy Carroll
    •  & S. Wayne Mascarella
  4. Department of Medicinal Chemistry, Virginia Commonwealth University, 800 E. Leigh Street, Richmond, Virginia 23298, USA

    • Richard B. Westkaemper
    •  & Philip D. Mosier

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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.

Competing interests

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

Corresponding author

Correspondence to Raymond C. Stevens.

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