Abstract

Endothelin receptors (ETRs) have crucial roles in vascular control and are targets for drugs designed to treat circulatory-system diseases and cancer progression. The nonpeptide dual-ETR antagonist bosentan is the first oral drug approved to treat pulmonary arterial hypertension. Here we report crystal structures of human endothelin ETB receptor bound to bosentan and to the ETB-selective analog K-8794, at 3.6-Å and 2.2-Å resolution, respectively. The K-8794-bound structure reveals the detailed water-mediated hydrogen-bonding network at the transmembrane core, which could account for the weak negative allosteric modulation of ETB by Na+ ions. The bosentan-bound structure reveals detailed interactions with ETB, which are probably conserved in the ETA receptor. A comparison of the two structures shows unexpected similarity between antagonist and agonist binding. Despite this similarity, bosentan sterically prevents the inward movement of transmembrane helix 6 (TM6), and thus exerts its antagonistic activity. These structural insights will facilitate the rational design of new ETR-targeting drugs.

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Acknowledgements

We thank the members of the Nureki lab and the beamline staff at BL32XU of SPring-8 (Sayo, Japan) for technical assistance during data collection. We also thank Kowa Co., Ltd., for providing K-8794. pCAGGS expression plasmid vector was a kind gift from J. Miyazaki (Osaka University, Osaka, Japan). The diffraction experiments were performed at SPring-8 BL32XU (proposals 2015A1024, 2015A1057, 2015B2024, and 2015B2057). This work was supported by JSPS KAKENHI grants 16K07172 (T.D.), 26640102 (T.D.), 16H06294 (O.N.), 15H05775 (F.Y.), 15J09780 (S.W.), 17J30010 (S.W.), 17H05000 (T.N.) and 15H06862 (K.Y.), the Core Research for Evolutional Science, PRESTO from the Japan Science and Technology (JST) Technology Program; the Platform for Drug Discovery, Information, and Structural Life Science from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; the Japan Agency for Medical Research and Development (AMED); and the National Institute of Biomedical Innovation. A.I. was funded by JST, PRESTO (grant JPMJPR1331), and the PRIME from AMED. J.A. received funding from AMED-CREST and AMED, and a MEXT Grant-in-Aid for Scientific Research on Innovative Areas (grant 15H05897).

Author information

Author notes

    • Akiko Okuta

    Deceased.

Affiliations

  1. Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Nagoya, Japan.

    • Wataru Shihoya
    •  & Yoshinori Fujiyoshi
  2. Cellular and Structural Physiology Institute, Nagoya University, Nagoya, Japan.

    • Wataru Shihoya
    • , Akiko Okuta
    • , Kazutoshi Tani
    •  & Yoshinori Fujiyoshi
  3. Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan.

    • Wataru Shihoya
    • , Tomohiro Nishizawa
    •  & Osamu Nureki
  4. Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Kawaguchi, Japan.

    • Tomohiro Nishizawa
    • , Asuka Inoue
    •  & Kunio Hirata
  5. RIKEN SPring-8 Center, Sayo, Japan.

    • Keitaro Yamashita
    •  & Kunio Hirata
  6. Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.

    • Asuka Inoue
    • , Francois Marie Ngako Kadji
    •  & Junken Aoki
  7. Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology (AMED-CREST), Tokyo, Japan.

    • Junken Aoki
  8. Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan.

    • Tomoko Doi

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Contributions

W.S. designed experiments; expressed, purified, and crystallized the antagonist-bound ETB receptor; collected data; and refined the structures. T.N. initially crystallized the K-8794-bound ETB receptor, assisted with the structural determination, and designed the construct ETB-Y4-mT4L. K.Y. and K.H. developed a pipeline for data collection and processing, and assisted with the structural determination. A.I., F.M.N.K., and J.A. performed and oversaw the cell-based assays. A.O. introduced K-8794 in the experimental design and characterized its pharmacology. K.T. initially designed the T4L-fused construct. T.D. performed the radiobinding assays. The manuscript was prepared by W.S., T.N., K.Y., A.I., K.H., K.T., Y.F., T.D., and O.N. Y.F., T.D., and O.N. supervised the research.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Tomoko Doi or Osamu Nureki.

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https://doi.org/10.1038/nsmb.3450