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Structural basis for bifunctional peptide recognition at human δ-opioid receptor

Nature Structural & Molecular Biology volume 22, pages 265–268 (2015)Cite this article

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Abstract

Bifunctional μ- and δ-opioid receptor (OR) ligands are potential therapeutic alternatives, with diminished side effects, to alkaloid opiate analgesics. We solved the structure of human δ-OR bound to the bifunctional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt and Tic. The observed receptor-peptide interactions are critical for understanding of the pharmacological profiles of opioid peptides and for development of improved analgesics.

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Figure 1: Structure of the BRILΔ36δ-OR–DIPP-NH2 complex.
Figure 2: Structural basis for the recognition of DIPP-NH2 by δ-OR.

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Acknowledgements

This work was supported by US National Institutes of Health (NIH), National Institute of General Medical Sciences grants U54 GM094618 (R.C.S., V.C. and V.K.), R01 GM108635 (V.C.), U54 GM094599 (P.F.), R01 GM095583 (P.F.) and P41 GM103393 (S. Boutet); US National Institute of Drug Abuse grants P01 DA035764 (V.C., V.K., B.L.R. and R.C.S.) and R01 DA017204 (B.L.R.); the US National Institute of Mental Health Psychoactive Drug Screening Program (P.G. and B.L.R.); the Michael Hooker Chair for Protein Therapeutics and Translational Proteomics to B.L.R.; and US National Science Foundation Science and Technology Center award 1231306 (J.C.H.S., P.F. and U.W.). Parts of this work were supported by the Helmholtz Association, the German Research Foundation (DFG) Cluster of Excellence ‘Center for Ultrafast Imaging’ and the German Federal Ministry of Education and Research (BMBF) projects FKZ 05K12CH1 (H.N.C., A.B., C.G., O.M.Y., T.A.W., D.O. and M. Metz) and 05K2012 (D.O. and H.N.C.). C.G. thanks the PIER Helmholtz Graduate School and the Helmholtz Association for financial support. M. Metz. acknowledges support from the Marie Curie Initial Training Network NanoMem (grant no. 317079). C.B., S. Ballet, D.T. and P.W.S. were supported by a collaboration convention between the Ministère du Développement Economique, de l'Innovation et de l'Exportation du Québec (PSR-SIIRI-417) and the Research Foundation–Flanders (FWO Vlaanderen, grant FWOAL570) and by grants to P.W.S. from the Canadian Institutes of Health Research (CIHR) (MOP-89716) and the NIH (DA-004443). We thank J. Velasquez, T. Trinh, M. Chu and A. Walker. Parts of this research were carried out at the Linac Coherent Light Source (LCLS), a US National User Facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences and at the GM/CA CAT, beamline 23ID-B, Advanced Photon Source, which is supported by the US National Cancer Institute grant Y1-CO-1020 and the US National Institute of General Medical Sciences grant Y1-GM-1104.

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Author notes

  1. Gustavo Fenalti, Gye Won Han, Andrii Ishchenko, Wei Liu, Haitao Zhang, Marc Messerschmidt, Vsevolod Katritch, Raymond C Stevens & Vadim Cherezov

    Present address: Present addresses: Celgene Corporation, San Diego, California, USA (G.F.), The Bridge Institute, University of Southern California, Los Angeles, California, USA (G.W.H., A.I., H.Z., V.K., R.C.S. and V.C.), Department of Chemistry, University of Southern California, Los Angeles, California, USA (G.W.H., A.I., R.C.S. and V.C.), Department of Biological Sciences, University of Southern California, Los Angeles, California, USA (H.Z., V.K. and R.C.S.), Center for Applied Structural Discovery at the Biodesign Institute, Arizona State University, Tempe, Arizona, USA (W.L.) and National Science Foundation BioXFEL Science and Technology Center, Buffalo, New York, USA (M. Messerschmidt).,

Authors and Affiliations

  1. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA

    Gustavo Fenalti, Gye Won Han, Andrii Ishchenko, Wei Liu, Haitao Zhang, Vsevolod Katritch, Raymond C Stevens & Vadim Cherezov

  2. Department of Physics, Arizona State University, Tempe, Arizona, USA

    Nadia A Zatsepin, Daniel James, Dingjie Wang, Uwe Weierstall & John C H Spence

  3. Department of Chemistry, Vrije Universiteit Brussel, Brussels, Belgium

    Cecilia Betti, Karel Guillemyn, Dirk Tourwé & Steven Ballet

  4. Department of Bioengineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium

    Cecilia Betti, Karel Guillemyn, Dirk Tourwé & Steven Ballet

  5. National Institute of Mental Health Psychoactive Drug Screening Program, University of North Carolina Chapel Hill Medical School, Chapel Hill, North Carolina, USA

    Patrick Giguere & Bryan L Roth

  6. Department of Pharmacology, University of North Carolina Chapel Hill Medical School, Chapel Hill, North Carolina, USA

    Patrick Giguere & Bryan L Roth

  7. Division of Chemical Biology and Medicinal Chemistry, University of North Carolina Chapel Hill Medical School, Chapel Hill, North Carolina, USA

    Patrick Giguere & Bryan L Roth

  8. Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, California, USA

    Sébastien Boutet, Marc Messerschmidt & Garth J Williams

  9. Center for Free Electron Laser Science, Deutsches Elektronen-Synchrotron (DESY), Hamburg, Germany

    Cornelius Gati, Oleksandr M Yefanov, Thomas A White, Dominik Oberthuer, Markus Metz, Chun Hong Yoon, Anton Barty & Henry N Chapman

  10. Institute of Biochemistry and Molecular Biology, University of Hamburg, Hamburg, Germany

    Dominik Oberthuer

  11. Department of Physics, University of Hamburg, Hamburg, Germany

    Markus Metz & Henry N Chapman

  12. European X-ray Free-Electron Laser Facility (XFEL GmbH), Hamburg, Germany

    Chun Hong Yoon

  13. Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona, USA

    Shibom Basu, Jesse Coe, Chelsie E Conrad, Raimund Fromme & Petra Fromme

  14. Center for Applied Structural Discovery at the Biodesign Institute, Arizona State University, Tempe, Arizona, USA

    Shibom Basu, Jesse Coe, Chelsie E Conrad, Raimund Fromme & Petra Fromme

  15. Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, Montreal, Quebec, Canada

    Peter W Schiller

Authors
  1. Gustavo Fenalti
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Contributions

G.F. designed, optimized and purified δ-OR receptor constructs for structural studies, crystallized the receptor in LCP, collected and processed synchrotron diffraction data, determined the synchrotron and XFEL structures, analyzed the data and wrote the paper; N.A.Z. collected and processed XFEL data; C.B. synthesized peptide ligands for structural and signaling studies; P.G. performed signaling studies, analyzed the data and wrote the paper; G.W.H. helped with structure refinement and analysis; A.I., H.Z. and W.L. collected XFEL data and helped with sample preparation; K.G. synthesized peptide ligands for structural and signaling studies; O.M.Y. refined the detector geometry and contributed to XFEL data processing; D.J., D.W., U.W. and J.C.H.S. designed the LCP injector and controlled it during XFEL data collection; S. Boutet, M. Messerschmidt and G.J.W. operated the CXI beamline at LCLS and contributed to XFEL data collection and processing; C.G., T.A.W., D.O., M. Metz, C.H.Y., A.B., H.N.C. and S. Basu participated in XFEL data collection and contributed to XFEL data processing; J.C., C.E.C., R.F. and P.F. collected and analyzed XFEL data and helped with biophysical characterization of crystals at LCLS; D.T. and P.W.S. helped with manuscript preparation; B.L.R. supervised the pharmacology studies, analyzed the data and wrote the paper; S. Ballet supervised the peptide synthesis and screening studies, synthesized peptide ligands for structural studies and wrote the paper; V.K. analyzed the data and wrote the paper; R.C.S. determined the overall project strategy, analyzed the data and wrote the paper; V.C. determined the overall project strategy and provided management, supervised XFEL data collection, analyzed the data and wrote the paper with contributions from all other coauthors.

Corresponding author

Correspondence to Vadim Cherezov.

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Integrated supplementary information

Supplementary Figure 1 Crystals of BRILΔ38δ-OR–DIPP-NH2 used for synchrotron and XFEL data collection.

a, Brightfield and b, cross-polarized images of BRILΔ38δ-OR–DIPP-NH2 crystals used to obtain the synchrotron structure. c, Brightfield and d, cross-polarized images of BRILΔ38δ-OR–DIPP-NH2 microcrystals after optimization of crystal growth conditions to yield crystals of ~5 μm, suitable for LCP crystallization in syringes. e,f, Cross-polarized images of BRILΔ38δ-OR–DIPP-NH2 LCP microcrystals grown in syringes and used to obtain the XFEL BRILΔ38δ-OR–DIPP-NH2 structure.

Supplementary Figure 2 Radioligand binding data for small-molecule and peptide ligands to μ-OR and δ-OR

Binding data of DIPP-NH2 and naltrindole to WT μ-OR, and of DIPP-NH2 and DADLE to both WT δ-OR and BRILΔ38δ-OR using the peptide [3H]DAMGO and small molecule [3H]naltrindole as competing radioligands. Results represent mean ± SEM of three independent experiments each in quadruplicate.

Supplementary Figure 3 XFEL single-crystal diffraction image.

Diffraction pattern from a BRILΔ38δ-OR–DIPP-NH2 microcrystal in LCP. Diffraction peaks identified by Cheetah program are circled. The edge of the detector corresponds to a resolution of 2.1 Å.

Supplementary Figure 4 Electron density around the peptide-binding site in the synchrotron and XFEL structures.

a and b, Two different binding conformations for Phe4-NH2 residue of the tetrapeptide DIPP-NH2 are found in molecule B of the Synchrotron (a) and XFEL (b) structures. The 2Fo-Fc electron density contoured at 1σ (blue mesh) is shown around the ligand.

c and d, DIPP-NH2 adopts the same binding mode in molecule A of the synchrotron (c) and XFEL structures (d). Synchrotron structure is shown as magenta cartoon with DIPP-NH2 as orange sticks, and XFEL structure is shown as light blue cartoon with DIPP-NH2 as blue sticks. A comparison between the quality of the electron density between Synchrotron and XFEL structures are shown in (c) and (d).

Supplementary Figure 5 Functional characterization of peptide ligands at the human μ-OR and δ-OR.

Efficacy and potency of the bi-functional ligand DIPP-NH2, the synthetic peptide ligand DAMGO as well as the endogenous opioid peptide endomorphin-1 and -2 were characterized at the μ-OR for arrestin recruitment using a BRET-based bioassay (a) and at the Gαi-protein pathway using a Glosensor-based bioassay (b). Results are reported as normalized concentration-responses induced by the selective μ-OR peptide ligand DAMGO. Similarly, the pharmacological profile of DIPP-NH2 toward the δ-OR was measured using a tango-based assay for arrestin recruitment (c,e) and Glosensor for Gαi activation (d,f). Normalized dose-response induced by the δ-OR peptide ligand DADLE is reported. e,f, Schild regression analysis at δ-OR arrestin recruitment and Gαi activity was performed with various concentration of DIPP-NH2 over a DADLE dose-response curve. Results represent mean ± SEM of three independent experiments each in quadruplicate.

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Fenalti, G., Zatsepin, N., Betti, C. et al. Structural basis for bifunctional peptide recognition at human δ-opioid receptor. Nat Struct Mol Biol 22, 265–268 (2015). https://doi.org/10.1038/nsmb.2965

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