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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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).
The authors declare no competing financial interests.
Integrated supplementary information
a, Crystallization constructs of the ETB receptor are shown, with all of the modifications to the human wild-type the ETB receptor indicated. Residues interacting with both bosentan and K-8794 are colored green, the residue only interacting with bosentan is blue, and those only interacting with K-8794 are red, as shown in the figure. b, c, Effects of ET-1 on the release of AP-TGFα and antagonists on the ET-1-induced release of AP-TGFα in HEK293 cells expressing the endothelin receptors. In the competitive assays, the concentration of the agonist ET-1 was 0.2 nM, and the AP-TGFα release response in the ET-1 treatment alone was normalized to 100%. Symbols and error bars are means and s.e.m. (standard error of the mean), respectively. For most data points, the error bars are smaller than the symbols. d, e, Crystallographic data of the ETB-Y5-mT4L protein bound to K-8794 (d) and the ETB-Y4-mT4L protein bound to bosentan (e). The left panels show the crystals of the antagonist-bound ETB receptors. The middle and right panels show their crystal packings. T4L is shown as a grey cartoon, and the K-8794- and bosentan-bound ETB receptors are shown as orange and turquoise cartoons, respectively. Crystal lattices are indicated by black lines.
a, b, Fo − Fc omit maps for K-8794 (a) and bosentan (b), contoured at 3.0 σ and 4.0 σ, respectively. TM6 and TM7 are omitted. c, The bosentan binding site, in which the colors represent the temperature factors ranging from 20 Å2 (blue) to 120 Å2 (red). d, Stereo view of the 2Fo−Fc map, contoured at 1.0 σ, for the residues within 4 Å contact distances of the ligand in the K-8794-bound ETB structure. e, Stereo views of the 2Fo−Fc maps, contoured at 1.0 σ, for the residues within 4 Å contact distances of the ligand in the bosentan-bound ETB structure. f, Stereo view of the composite omit map, contoured at 1.0 σ, for the residues within 4 Å contact distances of the ligand in the bosentan-bound ETB structure.
a–c, Comparison of the antagonist binding sites of the peptide-activated GPCRs. Ribbon representations of the ETB receptor in complex with bosentan (a), Orexin receptor OX2 in complex with suvorexant (PDB accession number 4RNB) (b), and NOP receptor in complex with the peptidomimetic antagonist C-24 (PDB accession number 4EA3) (c) are aligned according to the position of Trp6.48, which is indicated by the stick model in each figure. The black dashed line indicates the position of the Cα atoms of Trp6.48. The small-molecule antagonists are represented by stick models. Like the ETB receptor, OX2 belongs to the β subfamily of the class A GPCRs, while NOP belongs to the γ subfamily. d, e, Electrostatic surfaces of the ETB structures bound to bosentan (d) and ET-1 (e), viewed from the extracellular side (left) and within the membrane plane (right). Bosentan and ET-1 are shown as sticks and transparent surfaces, colored blue and pink, respectively.
Interaction diagrams of K-8794 (a) and bosentan (b) with the ETB receptor. Interactions within 4 Å are shown. Polar and hydrophobic contacts are represented as red dashed and green lines, respectively.
Amino acid sequence alignment of the human ETB (UniProt ID: P24530) and ETA (P25101) receptors. Secondary structure elements for α-helices and β-strands are indicated by cylinders and arrows, respectively. Conservation of the residues between ETA and ETB is indicated as follows: red panels for completely conserved, red letters for partially conserved, and black letters for not conserved. The residues involved in bosentan and K-8794 binding are shown as blue squares and orange diamonds, respectively.
Chemical structures of major small-molecule endothelin receptor antagonists. Endothelin receptor antagonists commonly have negatively-charged moieties (sulfonamide or carboxylate).
a, b, Comparison of the ET-1 and bosentan binding modes, coloured as in Fig. 6. Receptors and ET-1 are represented by ribbons, and the side chains of ET-118-21 and bosentan are shown as sticks with transparent surfaces. c, The residues that interact with both ET-1 and bosentan are superimposed. d–f, Comparison of the structural changes upon ET-1 (d), bosentan (e), and K-8794 (f) binding, coloured as in Figs. 1 and 4.
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Shihoya, W., Nishizawa, T., Yamashita, K. et al. X-ray structures of endothelin ETB receptor bound to clinical antagonist bosentan and its analog. Nat Struct Mol Biol 24, 758–764 (2017). https://doi.org/10.1038/nsmb.3450
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