Skip to main content

Thank you for visiting 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.

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


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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

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

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank


  1. Pasternak, G.W. Neuropharmacology 76 Pt B, 198–203 (2014).

    Article  CAS  Google Scholar 

  2. Abdelhamid, E.E., Sultana, M., Portoghese, P.S. & Takemori, A.E. J. Pharmacol. Exp. Ther. 258, 299–303 (1991).

    CAS  PubMed  Google Scholar 

  3. Schiller, P.W. Life Sci. 86, 598–603 (2010).

    Article  CAS  Google Scholar 

  4. Healy, J.R. et al. ACS Chem. Neurosci. 4, 1256–1266 (2013).

    Article  CAS  Google Scholar 

  5. Schiller, P.W. et al. J. Med. Chem. 36, 3182–3187 (1993).

    Article  CAS  Google Scholar 

  6. Bryant, S.D., Jinsmaa, Y., Salvadori, S., Okada, Y. & Lazarus, L.H. Biopolymers 71, 86–102 (2003).

    Article  CAS  Google Scholar 

  7. Schiller, P.W. et al. J. Med. Chem. 42, 3520–3526 (1999).

    Article  CAS  Google Scholar 

  8. Ballet, S. et al. Bioorg. Med. Chem. Lett. 19, 433–437 (2009).

    Article  CAS  Google Scholar 

  9. Schiller, P.W. et al. J. Recept. Signal Transduct. Res. 19, 573–588 (1999).

    Article  CAS  Google Scholar 

  10. Schiller, P.W. et al. Proc. Natl. Acad. Sci. USA 89, 11871–11875 (1992).

    Article  CAS  Google Scholar 

  11. Liu, W. et al. Science 342, 1521–1524 (2013).

    Article  CAS  Google Scholar 

  12. Weierstall, U. et al. Nat. Commun. 5, 3309 (2014).

    Article  Google Scholar 

  13. Fenalti, G. et al. Nature 506, 191–196 (2014).

    Article  CAS  Google Scholar 

  14. Granier, S. et al. Nature 485, 400–404 (2012).

    Article  CAS  Google Scholar 

  15. Manglik, A. et al. Nature 485, 321–326 (2012).

    Article  CAS  Google Scholar 

  16. Wu, H. et al. Nature 485, 327–332 (2012).

    Article  CAS  Google Scholar 

  17. Carpenter, K.A., Wilkes, B.C. & Schiller, P.W. Biopolymers 36, 735–749 (1995).

    Article  CAS  Google Scholar 

  18. Okada, Y. et al. Bioorg. Med. Chem. 11, 1983–1994 (2003).

    Article  CAS  Google Scholar 

  19. White, J.F. et al. Nature 490, 508–513 (2012).

    Article  CAS  Google Scholar 

  20. Chu, R. et al. J. Mol. Biol. 323, 253–262 (2002).

    Article  CAS  Google Scholar 

  21. Caffrey, M. & Cherezov, V. Nat. Protoc. 4, 706–731 (2009).

    Article  CAS  Google Scholar 

  22. Otwinowski, Z. & Minor, W. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  23. McCoy, A.J. et al. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  24. Liu, W. et al. Science 337, 232–236 (2012).

    Article  CAS  Google Scholar 

  25. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  26. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  27. Adams, P.D. et al. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  28. Liu, W., Ishchenko, A. & Cherezov, V. Nat. Protoc. 9, 2123–2134 (2014).

    Article  CAS  Google Scholar 

  29. Barty, A. et al. J. Appl. Crystallogr. 47, 1118–1131 (2014).

    Article  CAS  Google Scholar 

  30. White, T.A. et al. J. Appl. Crystallogr. 45, 335–341 (2012).

    Article  CAS  Google Scholar 

  31. Barnea, G. et al. Proc. Natl. Acad. Sci. USA 105, 64–69 (2008).

    Article  CAS  Google Scholar 

  32. Angers, S. et al. Proc. Natl. Acad. Sci. USA 97, 3684–3689 (2000).

    CAS  PubMed  Google Scholar 

Download references


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.

Author information

Authors and Affiliations



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.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

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.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Table 1 (PDF 2859 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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