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.

  • Article
  • Published:

Ligand binding to human prostaglandin E receptor EP4 at the lipid-bilayer interface

Nature Chemical Biology volume 15, pages 18–26 (2019)Cite this article

Subjects

Abstract

Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: Structure of the antagonist-bound human EP4 receptor in complex with the antibody Fab fragment.
Fig. 2: Structural comparison of EP4 structures with class A GPCRs.
Fig. 3: Orthosteric ligand-binding pocket of the EP4 receptor.
Fig. 4: Functional analyses of EP4 site-directed mutants.
Fig. 5: Docking of PGE2 to EP4.
Fig. 6: Allosteric inhibition of EP4 by antibody.

Similar content being viewed by others

Data availability

The atomic coordinates and structure factor files for the EP4–Fab001, EP4–Fab001_Br, and Fab001 have been deposited in the Protein Data Bank with accession codes 5YWY, 5YHL, and 5YFI, respectively. The raw diffraction images have been deposited in Zenodo data repository (https://doi.org/10.5281/zenodo.1173791).

References

  1. Hirata, T. & Narumiya, S. Prostanoid receptors. Chem. Rev. 111, 6209–6230 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. Fujino, H. & Regan, J. W. EP(4) prostanoid receptor coupling to a pertussis toxin-sensitive inhibitory G protein. Mol. Pharmacol. 69, 5–10 (2006).

    CAS  PubMed  Google Scholar 

  3. Buchanan, F. G. et al. Role of beta-arrestin 1 in the metastatic progression of colorectal cancer. Proc. Natl. Acad. Sci. USA 103, 1492–1497 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Yokoyama, U., Iwatsubo, K., Umemura, M., Fujita, T. & Ishikawa, Y. The prostanoid EP4 receptor and its signaling pathway. Pharmacol. Rev. 65, 1010–1052 (2013).

    Article  CAS  PubMed  Google Scholar 

  5. Yao, C. et al. Prostaglandin E2-EP4 signaling promotes immune inflammation through Th1 cell differentiation and Th17 cell expansion. Nat. Med. 15, 633–640 (2009).

    Article  CAS  PubMed  Google Scholar 

  6. Libioulle, C. et al. Novel Crohn disease locus identified by genome-wide association maps to a gene desert on 5p13.1 and modulates expression of PTGER4. PLoS. Genet. 3, e58 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Shi, Y. et al. A genome-wide association study identifies new susceptibility loci for non-cardia gastric cancer at 3q13.31 and 5p13.1. Nat. Genet. 43, 1215–1218 (2011).

    Article  CAS  PubMed  Google Scholar 

  8. Hinds, D. A. et al. A genome-wide association meta-analysis of self-reported allergy identifies shared and allergy-specific susceptibility loci. Nat. Genet. 45, 907–911 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Markovič, T., Jakopin, Ž., Dolenc, M. S. & Mlinarič-Raščan, I. Structural features of subtype-selective EP receptor modulators. Drug Discov. Today 22, 57–71 (2017).

    Article  CAS  PubMed  Google Scholar 

  10. Ward, C. L. et al. First clinical experience with ONO-4232: a randomized, double-blind, placebo-controlled healthy volunteer study of a novel lusitropic agent for acutely decompensated heart failure. Clin. Ther. 38, 1109–1121 (2016).

    Article  CAS  PubMed  Google Scholar 

  11. Watanabe, Y. et al. KAG-308, a newly-identified EP4-selective agonist shows efficacy for treating ulcerative colitis and can bring about lower risk of colorectal carcinogenesis by oral administration. Eur. J. Pharmacol. 754, 179–189 (2015).

    Article  CAS  PubMed  Google Scholar 

  12. Rausch-Derra, L., Huebner, M., Wofford, J. & Rhodes, L. A prospective, randomized, masked, placebo-controlled multisite clinical study of grapiprant, an EP4 prostaglandin receptor antagonist (PRA), in dogs with osteoarthritis. J. Vet. Intern. Med. 30, 756–763 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Bao, X. et al. Combination of EP4 antagonist and checkpoint inhibitors promotes anti-tumor effector T cells in preclinical tumor models. J. Immunother. Cancer 3, 350 (2015).

    Article  Google Scholar 

  14. Kobayashi, T. et al. Identification of domains conferring ligand binding specificity to the prostanoid receptor. Studies on chimeric prostacyclin/prostaglandin D receptors. J. Biol. Chem. 272, 15154–15160 (1997).

    Article  CAS  PubMed  Google Scholar 

  15. Stillman, B. A., Audoly, L. & Breyer, R. M. A conserved threonine in the second extracellular loop of the human EP2 and EP4 receptors is required for ligand binding. Eur. J. Pharmacol. 357, 73–82 (1998).

    Article  CAS  PubMed  Google Scholar 

  16. Narumiya, S., Sugimoto, Y. & Ushikubi, F. Prostanoid receptors: structures, properties, and functions. Physiol. Rev. 79, 1193–1226 (1999).

    Article  CAS  PubMed  Google Scholar 

  17. Shiroishi, M. et al. Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae. Microb. Cell. Fact. 11, 78 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Vaidehi, N., Grisshammer, R. & Tate, C. G. How can mutations thermostabilize G-protein-coupled receptors? Trends Pharmacol. Sci. 37, 37–46 (2016).

    Article  CAS  PubMed  Google Scholar 

  19. Yasuda, S. et al. Hot-spot residues to be mutated common in G protein-coupled receptors of class A: identification of thermostabilizing mutations followed by determination of three-dimensional structures for two example receptors. J. Phys. Chem. B 121, 6341–6350 (2017).

    Article  CAS  PubMed  Google Scholar 

  20. 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).

    Article  CAS  Google Scholar 

  21. Katritch, V. et al. Allosteric sodium in class A GPCR signaling. Trends. Biochem. Sci. 39, 233–244 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Takayama, K., Shimizu, T., Urushibata, Y. & Sugimoto, Y. Antibody against human prostaglandin E2 receptor EP4. US patent 20130197199 (2013).

  23. Kabashima, K. et al. The prostaglandin receptor EP4 suppresses colitis, mucosal damage and CD4 cell activation in the gut. J. Clin. Invest. 109, 883–893 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Venkatakrishnan, A. J. et al. Molecular signatures of G-protein-coupled receptors. Nature 494, 185–194 (2013).

    Article  CAS  PubMed  Google Scholar 

  25. Hanson, M. A. et al. Crystal structure of a lipid G protein-coupled receptor. Science 335, 851–855 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Chrencik, J. E. et al. Crystal structure of antagonist bound human lysophosphatidic acid receptor 1. Cell 161, 1633–1643 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Shao, Z. et al. High-resolution crystal structure of the human CB1 cannabinoid receptor. Nature 540, 602–606 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Srivastava, A. et al. High-resolution structure of the human GPR40 receptor bound to allosteric agonist TAK-875. Nature 513, 124–127 (2014).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  30. Kappel, K., Miao, Y. & McCammon, J. A. Accelerated molecular dynamics simulations of ligand binding to a muscarinic G-protein-coupled receptor. Q. Rev. Biophys. 48, 479–487 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Stanley, N., Pardo, L. & Fabritiis, G. D. The pathway of ligand entry from the membrane bilayer to a lipid G protein-coupled receptor. Sci. Rep. 6, 22639 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Mutoh, M. et al. Involvement of prostaglandin E receptor subtype EP4 in colon carcinogenesis. Cancer Res. 62, 28–32 (2002).

    CAS  PubMed  Google Scholar 

  33. Kedzie, K. M., Donello, J. E., Krauss, H. A., Regan, J. W. & Gil, D. W. A single amino-acid substitution in the EP2 prostaglandin receptor confers responsiveness to prostacyclin analogs. Mol. Pharmacol. 54, 584–590 (1998).

    Article  CAS  PubMed  Google Scholar 

  34. Audoly, L. & Breyer, R. M. Substitution of charged amino acid residues in transmembrane regions 6 and 7 affect ligand binding and signal transduction of the prostaglandin EP3 receptor. Mol. Pharmacol. 51, 61–68 (1997).

    Article  CAS  PubMed  Google Scholar 

  35. Stitham, J., Stojanovic, A., Merenick, B. L., O’Hara, K. A. & Hwa, J. The unique ligand-binding pocket for the human prostacyclin receptor. Site-directed mutagenesis and molecular modeling. J. Biol. Chem. 278, 4250–4257 (2003).

    Article  CAS  PubMed  Google Scholar 

  36. Neuschäfer-Rube, F., Engemaier, E., Koch, S., Böer, U. & Püschel, G. P. Identification by site-directed mutagenesis of amino acids contributing to ligand-binding specificity or signal transduction properties of the human FP prostanoid receptor. Biochem. J. 371, 443–449 (2003).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Funk, C. D., Furci, L., Moran, N. & Fitzgerald, G. A. Point mutation in the seventh hydrophobic domain of the human thromboxane A2 receptor allows discrimination between agonist and antagonist binding sites. Mol. Pharmacol. 44, 934–939 (1993).

    CAS  PubMed  Google Scholar 

  38. Natarajan, C., Hata, A. N., Hamm, H. E., Zent, R. & Breyer, R. M. Extracellular loop II modulates GTP sensitivity of the prostaglandin EP3 receptor. Mol. Pharmacol. 83, 206–216 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Margan, D., Borota, A., Mracec, M. & Mracec, M. 3D homology model of the human prostaglandin E2 receptor EP4 subtype. Rev. Roum. Chim. 57, 39–44 (2012).

    CAS  Google Scholar 

  40. Zare, B., Madadkar-Sobhani, A., Dastmalchi, S. & Mahmoudian, M. Prediction of the human EP1 receptor binding site by homology modeling and molecular dynamics simulation. Sci. Pharm. 79, 793–816 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Hutchings, C. J., Koglin, M., Olson, W. C. & Marshall, F. H. Opportunities for therapeutic antibodies directed at G-protein-coupled receptors. Nat. Rev. Drug. Discov. 16, 787–810 (2017).

    Article  CAS  PubMed  Google Scholar 

  42. Cheng, R. K. Y. et al. Structural insight into allosteric modulation of protease-activated receptor 2. Nature 545, 112–115 (2017).

    Article  CAS  PubMed  Google Scholar 

  43. Zhang, H. et al. Structure of the full-length glucagon class B G-protein-coupled receptor. Nature 546, 259–264 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Leduc, M. et al. Functional selectivity of natural and synthetic prostaglandin EP4 receptor ligands. J. Pharmacol. Exp. Ther. 331, 297–307 (2009).

    Article  CAS  PubMed  Google Scholar 

  45. Inoue, A. et al. TGFα shedding assay: an accurate and versatile method for detecting GPCR activation. Nat. Methods 9, 1021–1029 (2012).

    Article  CAS  PubMed  Google Scholar 

  46. Nomura, Y. et al. The intervening removable affinity tag (iRAT) production system facilitates Fv antibody fragment-mediated crystallography. Protein Sci. 25, 2268–2276 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nat. Protoc. 4, 706–731 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Ueno, G. et al. Remote access and automation of SPring-8 MX beamlines. AIP Conf. Proc. 1741, 050021 (2016).

    Article  Google Scholar 

  49. Yamashita, K., Hirata, K. & Yamamoto, M. KAMO: towards automated data processing for microcrystals. Acta Crystallogr. D Struct. Biol. 74, 441–449 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Foadi, J. et al. Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography. Acta Crystallogr. D. Biol. Crystallogr. 69, 1617–1632 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A. 64, 112–122 (2008).

    Article  CAS  PubMed  Google Scholar 

  56. Thorn, A. & Sheldrick, G. M. ANODE: anomalous and heavy-atom density calculation. J. Appl. Crystallogr. 44, 1285–1287 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    Article  CAS  PubMed  Google Scholar 

  58. Halgren, T. A. Identifying and characterizing binding sites and assessing druggability. J. Chem. Inf. Model. 49, 377–389 (2009).

    Article  CAS  PubMed  Google Scholar 

  59. Sherman, W., Day, T., Jacobson, M. P., Friesner, R. A. & Farid, R. Novel procedure for modeling ligand/receptor induced fit effects. J. Med. Chem. 49, 534–553 (2006).

    Article  CAS  PubMed  Google Scholar 

  60. Harder, E. et al. OPLS3: a force field providing broad coverage of drug-like small molecules and proteins. J. Chem. Theory. Comput. 12, 281–296 (2016).

    Article  CAS  PubMed  Google Scholar 

  61. Case, D. A. et al. AMBER 11. (University of California, San Francisco, 2010).

    Google Scholar 

Download references

Acknowledgements

We are grateful to Ono Pharmaceutical Company for supplying EP4 antagonists; to the beamline scientists at BL32XU and BL41XU of SPring-8 (Hyogo, Japan) for their technical assistance during data collection; to A. Inoue at Tohoku University for the TGF-α shedding assay; to B.K. Kobilka (Tsinghua University and Stanford University), W. Shihoya and R. Taniguchi (The University of Tokyo) for their useful comments; and to H. Tsujimoto, M. Sasanuma and members of the Iwata lab at Kyoto University for technical assistance. DNA sequencing analysis was performed at the Medical Research Support Center, Graduate School of Medicine, Kyoto University. This work was supported by the Strategic Basic Research Program, JST (S.I.); the Toray Science Foundation (T.K.); the Takeda Science Foundation (T.K. and R.S.); the Naito Foundation (T.K.); Koyanagi Foundation (T.K.); the Platform for Drug Discovery, Informatics, and Structural Life Science (PDIS) funded by the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) and the Japan Agency for Medical Research and Development (AMED) (T.K., T.M., M. Shiroishi, T.H. and M.Y.); Core Research for Evolutional Science and Technology (CREST) funded by AMED (Y. Su., S.N. and T.K.); the ImPACT Program of the Council for Science, Technology and Innovation (Cabinet Office, Government of Japan; T.M. and M.K.); MEXT as a “Priority Issue on Post-K computer” (Building Innovative Drug Discovery Infrastructure Through Functional Control of Biomolecular Systems) (hp160213) (T. Hi.); and the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant Nos. 15K08268 to R.S., 15J00102 to K.M., 15J04343 to S.H., 15H06862 to K.Y., and 15H05905 to Y. Su.). K.M. and S.H. are recipients of JSPS postdoctoral fellowships. X-ray crystallographic data were collected at SPring-8 (Proposal Nos. 2013A1379, 2013B1184, 2013B1092, 2014A1301, 2014B1355, 2014B1273, 2015A1080, 2015A1044, 2015B1092, 2015B2044, and 2015B2080).

Author information

Author notes

  1. Yosuke Toyoda

    Present address: School of Medicine, Tsinghua University, Beijing, China

  2. These authors contributed equally: Yosuke Toyoda, Kazushi Morimoto, Ryoji Suno.

Authors and Affiliations

  1. Department of Medical Chemistry and Cell Biology, Kyoto University Graduate School of Medicine, Konoe-cho, Yoshida, Sakyo-ku, Kyoto, Japan

    Yosuke Toyoda, Kazushi Morimoto, Ryoji Suno, Shoichiro Horita, Yusuke Sekiguchi, Yunhon Hotta, Hidetsugu Asada, Takanori Nakane, Yuki Shiimura, Tomoya Nakagita, Norimichi Nomura, So Iwata & Takuya Kobayashi

  2. RIKEN SPring-8 Center, Sayo-cho, Sayo-gun, Hyogo, Japan

    Keitaro Yamashita, Kunio Hirata, Masaki Yamamoto & So Iwata

  3. Department of Chemistry, Graduate School of Science, Chiba University, Inage, Chiba, Japan

    Satoshi Yasuda & Takeshi Murata

  4. Institute of Advanced Energy, Kyoto University, Uji, Kyoto, Japan

    Satoshi Yasuda & Masahiro Kinoshita

  5. Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan

    Mitsunori Shiroishi

  6. Japan Science and Technology Agency (JST), Platform for Drug Discovery, Informatics, and Structural Life Science, Konoe-cho, Yoshida, Sakyo-ku, Kyoto, Japan

    Mitsunori Shiroishi, Takamitsu Hosoya, Takatsugu Hirokawa, Takeshi Murata, Masaki Yamamoto & Takuya Kobayashi

  7. NB Health Laboratory Co. Ltd., Kita Ward Sapporo City, Hokkaido, Japan

    Tomoko Shimizu, Yuji Urushibata & Kiyoshi Takayama

  8. Graduate School of Energy Science, Kyoto University, Uji, Kyoto, Japan

    Yuta Kajiwara

  9. Molecular Profiling Research Center for Drug Discovery (MOLPROF), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan

    Yuta Kajiwara & Takatsugu Hirokawa

  10. Department of Pharmaceutical Biochemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, Oe-Honmachi, Kumamoto, Japan

    Tomoaki Inazumi, Kyoshiro Tsuge & Yukihiko Sugimoto

  11. JST, the Strategic Basic Research Program, Membrane Protein Crystallography Project, Kyoto, Japan

    Hidetsugu Asada, Yuki Shiimura, Norimichi Nomura, So Iwata & Takuya Kobayashi

  12. Laboratory of Chemical Bioscience, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo, Japan

    Suguru Yoshida, Tomoko Kuribara & Takamitsu Hosoya

  13. Japan Agency for Medical Research and Development (AMED), Core Research for Evolutional Science and Technology (CREST), Chiyoda-ku, Tokyo, Japan

    Yukihiko Sugimoto, Shuh Narumiya & Takuya Kobayashi

  14. Biomedical Department, Mitsui Knowledge Industry Co., Minato-ku, Tokyo, Japan

    Miwa Sato

  15. Division of Biomedical Science, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan

    Takatsugu Hirokawa

  16. Medical Innovation Center, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto, Japan

    Shuh Narumiya

Authors
  1. Yosuke Toyoda
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kazushi Morimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ryoji Suno
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shoichiro Horita
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Keitaro Yamashita
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kunio Hirata
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yusuke Sekiguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Satoshi Yasuda
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Mitsunori Shiroishi
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Tomoko Shimizu
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Yuji Urushibata
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Yuta Kajiwara
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Tomoaki Inazumi
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Yunhon Hotta
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Hidetsugu Asada
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Takanori Nakane
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Yuki Shiimura
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Tomoya Nakagita
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Kyoshiro Tsuge
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Suguru Yoshida
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Tomoko Kuribara
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Takamitsu Hosoya
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Yukihiko Sugimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Norimichi Nomura
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Miwa Sato
    View author publications

    You can also search for this author in PubMed Google Scholar

  26. Takatsugu Hirokawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  27. Masahiro Kinoshita
    View author publications

    You can also search for this author in PubMed Google Scholar

  28. Takeshi Murata
    View author publications

    You can also search for this author in PubMed Google Scholar

  29. Kiyoshi Takayama
    View author publications

    You can also search for this author in PubMed Google Scholar

  30. Masaki Yamamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  31. Shuh Narumiya
    View author publications

    You can also search for this author in PubMed Google Scholar

  32. So Iwata
    View author publications

    You can also search for this author in PubMed Google Scholar

  33. Takuya Kobayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.K., Y.T., S.I., and S.N. designed the project. Initial trials of EP4 were conducted by Y.T., H.A., and T. Nakane. Purification and crystallization of EP4 and EP4–Fab001 were performed by Y.T. and Y. Sekiguchi. The thermostabilizing mutation Gly1063.39Arg was discovered by S. Yasuda, Y.K., T.M., and M.K. using a theoretical strategy developed by M.K., S. Yasuda, and T.M. The alanine-scanning mutations were designed by T.K., Y.T., K.M., and T. Nakane. The construction and binding assays of EP4 mutants were performed by K.M. and Y.T. FSEC-TS was performed by K.M., Y.T. and Y.H. TGF-α shedding assays were performed by K.M. ITC experiments were performed by M. Shiroishi. The generation, expression, purification, and evaluation of the antibody were performed by K. Ta., Y. Su., T.S., Y.U., T.I., and K. Tsu. Fab and Fv fragments were prepared by Y.T., N.N., Y. Sekiguchi, Y.H., and Y. Shiimura. The synthesis of CHEMBL1644016 was performed by S. Yoshida, T. Ku., and T.H. The data collection was performed by Y.T., R.S., K.Y., and K.H., and supervised by S.I. and M.Y. Structure determination and refinement were performed by S.H., R.S., K.Y., K.H., Y.T., T. Nakane, and T. Nakagita. The molecular dynamics simulations and computational modeling were performed by T. Hi and M. Sato. The manuscript was prepared by Y.T., T.K., S.I., K.M., S.H., and R.S., and all authors discussed the results and commented on the manuscript. The research was supervised by T.K., R.S., S.I., and S.N.

Corresponding authors

Correspondence to Shuh Narumiya, So Iwata or Takuya Kobayashi.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11, Supplementary Tables 1–2

Reporting Summary

Supplementary Video 1

Molecular dynamics simulations of ligand access of EP4

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Toyoda, Y., Morimoto, K., Suno, R. et al. Ligand binding to human prostaglandin E receptor EP4 at the lipid-bilayer interface. Nat Chem Biol 15, 18–26 (2019). https://doi.org/10.1038/s41589-018-0131-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-018-0131-3

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research