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.

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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).

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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

    • Yosuke Toyoda

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

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

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

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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.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Shuh Narumiya or So Iwata or Takuya Kobayashi.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–11, Supplementary Tables 1–2

  2. Reporting Summary

  3. Supplementary Video 1

    Molecular dynamics simulations of ligand access of EP4

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DOI

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

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