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Activation mechanism of endothelin ETB receptor by endothelin-1

Abstract

Endothelin, a 21-amino-acid peptide, participates in various physiological processes, such as regulation of vascular tone, humoral homeostasis, neural crest cell development and neurotransmission. Endothelin and its G-protein-coupled receptor are involved in the development of various diseases, such as pulmonary arterial hypertension, and thus are important therapeutic targets. Here we report crystal structures of human endothelin type B receptor in the ligand-free form and in complex with the endogenous agonist endothelin-1. The structures and mutation analysis reveal the mechanism for the isopeptide selectivity between endothelin-1 and -3. Transmembrane helices 1, 2, 6 and 7 move and envelop the entire endothelin peptide, in a virtually irreversible manner. The agonist-induced conformational changes are propagated to the receptor core and the cytoplasmic G-protein coupling interface, and probably induce conformational flexibility in TM6. A comparison with the M2 muscarinic receptor suggests a shared mechanism for signal transduction in class A G-protein-coupled receptors.

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Figure 1: ET-1 bound and ligand-free structures of ETB receptor.
Figure 2: Orthosteric pocket of ETB receptor.
Figure 3: Structural comparison between ET-1-bound and ligand-free ETB receptors.
Figure 4: Allosteric coupling through TM6.
Figure 5: Polar interaction network reorganization.
Figure 6: Schematic representation of ETB receptor activation by ET-1.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in the Protein Data Bank under accession number 5GLH for the ET-1-bound and 5GLI for the ligand-free ETB receptors.

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Acknowledgements

We thank S. Nishimura and K. Kumazaki for discussions, Y. Oomae and K. Yamaguchi for ETB mutant preparations, the beamline staff at BL32XU of SPring-8 (Hyogo, Japan) for technical help during data collection, and T. Suzuki for the mass spectrometry. This work was supported by Japan Society for the Promotion of Science KAKENHI grant numbers 15J09780, 22227004, 24227004, 25650019, 26440024, and 26640102; the Core Research for Evolutional Science and Technology Program; Platform for Drug Discovery, Information, and Structural Life Science from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; the Japan New Energy and Industrial Technology Development Organization (NEDO), the Japan Agency for Medical Research and Development (AMED) and the National Institute of Biomedical Innovation.

Author information

Authors and Affiliations

Authors

Contributions

W.S. optimized the construct for crystallization, performed the thermostability assay, developed the purification procedure, expressed and purified the ET-1-bound and ligand-free receptors, and crystallized and collected data for the ligand-free structure. T.N. crystallized the ET-1-bound receptor and collected data, and solved and refined the structures. A.O. performed the thermostabilization screening and established the ETBR-Y5 construct, and made the initial T4L-fused construct for crystallization. K.T. designed the T4L-fused construct and assisted with the structural determination. N.D. analysed the purified protein by mass spectrometry. T.D. performed the pharmacological analyses. The manuscript was prepared by W.S., T.N., K.T., Y.F., O.N. and T.D.; Y.F., O.N. and T.D. supervised the research.

Corresponding authors

Correspondence to Osamu Nureki or Tomoko Doi.

Additional information

Reviewer Information

Nature thanks M. Barton, A. Davenport and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Thermostabilized construct of ETB receptor.

a, Crystallization construct of ETB receptor, shown with all of the modifications to the human wild-type ETB receptor. The thermostabilizing mutations R124Y1.55, D154A2.57, K270A5.35, S342A6.54 and I381A7.48, and the three cysteine mutations, C396A, C400A and C405A, are coloured red and cyan, respectively. The C-terminal residues after S407 were truncated, and the T4L or mT4L was inserted between Lys303 and Leu311. The most conserved residues in each TM helix are coloured gold. Dashed lines indicate disulfide bonds. A Flag epitope tag was added after the N-terminal signal sequence, and a TEV protease site was introduced between Gly57 and Leu66. b, Thermostability profiles of the GFP-fused wild-type ETB and ETBR-Y5, measured by the FSEC-TS method50. Each fluorescent signal intensity at the monomeric peak was normalized to that of the unheated sample as 100%. Data are given as means ± s.e.m. of three independent experiments. The wild-type ETB–GFP (closed circles) has a melting temperature (Tm) of 36.7 °C and ETBR-Y5–GFP (open circles) has a Tm of 50.1 °C, as calculated from the fitting curves. c, d, Apparent 125I-labelled ET-1 equilibrium dissociation constants (Kd). Values of the apparent dissociation constants for the wild-type (WT), thermostabilized (ETBR-Y5), T4-fused (ETB-Y4-T4L) and mT4L-fused (ETBR-Y5-mT4L) constructs are shown. Each experiment was performed three or four times. d, The apparent inhibition constants (Ki) for 125I-labelled ET-1 binding and half-maximal effective concentrations (EC50) for Gi activation by ET-1 and ET-3. Values for wild-type (WT) and thermostabilized (ETBR-Y5) constructs are shown. e, Time courses for GTP-γS binding to the G protein Gi mediated by wild-type (circles) and thermostabilized (squares) ETB receptors reconstituted into phospholipid vesicles, in the presence (open symbols) or absence (filled symbols) of ET-1. The assays were repeated four or five times. f, g, ET-1-dependent (f) and ET-3-dependent (g) Gi activation mediated by wild-type (closed circles) and thermostabilized (open circles) ETB receptors.

Extended Data Figure 2 Ligand-free and ET-1-bound crystal structures of the ETB receptor.

a, b, Ligand-free (a) and ET-1-bound (b) structures of ETB, and the crystallized constructs. Two crystal structures were obtained, using the different constructs indicated in each panel. The thermostabilizing mutations and the Cys-to-Ala mutations to avoid lipid modification are indicated with red and blue circles, respectively. ce, Crystal packing of the ligand-free structure of ETBR-Y5-mT4L (c) and the architectures of the cytoplasmic (d) and extracellular (e) sides. The 2FoFc electron density map contoured at 0.8σ (blue mesh) revealed a sulfate ion bound to the cytoplasmic surface, which stabilizes the cytoplasmic architecture. The extracellular view shows a strong positive FoFc density (green and red meshes contoured at 2.5 and −2.5σ, respectively) within the orthosteric pocket, which was assigned as the C-terminal tag residues from the adjacent molecule in the crystal lattice. f, Close-up extracellular view and the C-terminal tag sequence modelled in the density. These residues are not included in the deposited coordinate files, because we could not exclude the possibility that contaminant peptides are bound to the receptor. g, h, Crystal packing of the ET-1-bound structure of ETBR-Y5-T4L (g) and close-up view of the crystal packing contacts between the adjacent molecules (h). TM6 is partly involved in the crystal packing with the adjacent molecule. TM5 forms a continuous helix, together with the first helix of T4L.

Extended Data Figure 3 Structural comparison of the peptide-activated GPCRs.

ad, Comparison of the orthosteric pockets of the peptide-activated GPCRs bound to agonists (a, b, d) or an antagonist (c). Ribbon representations (top) and cutaway surfaces (bottom) for ETB in complex with ET-1 (a), NTSR1 in complex with the NTS8–13 peptide (PDB accession number 4GRV) (b), chemokine receptor CXCR4 in complex with the virus chemokine vMIP-II (PDB accession number 4RWS) (c) and the μ-opioid receptor in complex with the small-drug agonist BU72 and the nanobody Nb39 (PDB accession number 5C1M) (d) are aligned, according to the position of W6.48. The peptidic and small-drug agonist/antagonist are represented by ribbons and sticks. Interaction ranges are indicated by black brackets (top), and the approximate interacting surface areas for their ligands are indicated (bottom). The extent of the penetration of the C-terminal tail of ET-1 is similar to the small drug-agonist (BU72) bound to the μ-opioid receptor, and is much deeper than the peptide agonist bound to NTSR1. The internal electric charges of the orthosteric pockets are complementary to the terminal charges of their peptide ligands: ETB and NTSR1 are positively charged, while CXCR4 and the μ-opioid receptor are negatively charged.

Extended Data Figure 4 Comparison of ET-1 structures.

a, The FoFc omit map for ET-1, contoured at 2.0σ, is shown. ET-1 is depicted by sticks and ribbons. The N-terminal end of the α-helical region is capped by the D8 and E10 side chains. The distances between the nitrogen at the N terminus and the carboxyl oxygens of D18 and W21 are indicated with red dotted lines (Å). The N-terminal and α-helical regions of ET-1 are stabilized by intra-peptide interactions; the negatively charged D8 and E10 side chains coordinate the backbone amides of the K9, E10 and C11 residues, supporting the α-helical folding, and the short hairpin at the M7 residue is stabilized by a hydrogen bond between the S6 carbonyl and the D8 amide. be, Reported structures of ET-1 and related peptides. NMR structures of ET-1: full-length model from 20 conformers28 (b) (PDB accession number 1V6R) and a partial model that includes an unmodelled region (from L17 to W21)29 (c) (PDB accession number 1EDP). X-ray crystal structure of the N-terminal-extended ET-1-like peptide63 (PDB accession number 1T7H) (d). X-ray crystal structure of ET-1 (ref. 27) (PDB accession number 1EDN) (e). All structures are represented by sticks and ribbons, and the colour code is the same as in Fig. 1. The X-ray crystal structure of ET-1 (e) probably represents a rather deformed conformation affected by crystal packing interactions. Close-up views in ac highlight the intra-peptide interactions that stabilize the common architecture of these peptides. Hydrogen bonds are indicated by yellow dotted lines with their respective distance values (Å).

Extended Data Figure 5 Stereo views of the orthosteric pocket.

a, b, Stereo views showing the detailed interactions between ET-1 and ETB receptor in the orthosteric pocket, viewed from different viewpoints. Residues involved in the major interactions between ET-1 and ETB receptor are shown, at the N-terminal and C-terminal regions of ET-1 (a) and at the α-helical and C-terminal regions (b). Hydrogen bonds are indicated with yellow dotted lines.

Extended Data Figure 6 Ligand–receptor interactions.

a, Schematic drawing of the orthosteric pocket. The residues shown here are within a radius of 4 Å around the ligand in the crystal structure. Amino-acid residues of ET-1 are represented by capital letters enclosed within circles. Blue and red ovals indicate main chain amide, and carbonyl and carboxyl groups of ET-1, respectively. All residues of the ETB receptor involved in the interactions are indicated by large boxes and amino-acid letters, and the types of interaction are indicated with dotted lines. b, c, ET-1 interactions at the C-terminal region (b) and at the α-helical and N-terminal regions (c) analysed by LIGPLOT64. The labels and stick drawings of ET-1 residues are coloured cyan (N-terminal region), orange (α-helical region) and pink (C-terminal region), according to the same colour code used in Fig. 1. The ETB receptor residues involved in the hydrophobic and hydrogen bond interactions are indicated by black and green letters, respectively. Intermolecular hydrogen bonds are indicated as green dashed lines, and disulfide bonds are indicated as yellow dashed lines.

Extended Data Figure 7 Competitive binding assays of mutant ETB receptors.

a, Competitive binding of ET-1 to wild-type and mutant ETB receptors. The IC50 values, representing the apparent half-maximal inhibitory concentration of ET-1 or ET-3 on 125I-labelled ET-1 binding to mutant ETB receptors, are indicated. The corresponding residues in ETA are also indicated in the table, with the non-conserved residues represented in cyan. The letters a and b in the table indicate the host cells for expression: a, expressed in SF+ cell membranes; b, expressed in HEK293 cell membranes. b, c, Mutations that significantly affect ET-1 (b) and ET-3 (c) binding are indicated in the ET-1-bound structure. Mutated residues of ETB are coloured according to the degree of decreased affinity, as shown in Extended Data Fig. 7a. ETs are indicated in ribbon representation with the colours as in d. d, Amino-acid sequences and selectivities of the ET isopeptides. The residues different from ET-1 are highlighted in red. Intra-peptide disulfide bonds are indicated by yellow lines. e, Sequence conservation between human ETA and ETB receptors, mapped on the ET-1-bound ETB structure. The residues of the receptor core are highly conserved between ETA and ETB receptors, and the amino-acid sequences of the C-terminal regions of the three endothelin isopeptides are identical, as shown in Fig. 2f, suggesting that the interactions between the C-terminal region of ET and the receptor core are conserved in any combination of ET isopeptides and receptor subtypes. However, the sequence conservation between ETA and ETB receptors suggests slightly divergent interactions through the extracellular potions of the receptors, including TM5, ECL1 and ECL2, where the N-terminal and α-helical regions of the ET-1 interact with the receptor.

Extended Data Figure 8 Sequence conservation between ETA and ETB receptors.

Amino-acid sequences of the thermostabilized crystallized construct (hETBR-Y5), human ETB (UniProt ID: P24530), rat ETB (P26684), human ETA (P25101) and rat ETA (P26684) are aligned65. 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 partly conserved; and black letters for not conserved. The thermostabilizing and the Cys-to-Ala mutations in the crystallized constructs are indicated with red and cyan letters, respectively. The residues with the Ballesteros–Weinstein number of X.50 in each TM helix are highlighted with yellow panels. The residues involved in the ET-1 binding are indicated by triangles, coloured according to the interacting regions of ET-1 (cyan, N-terminal region; orange, α-helical region; pink, C-terminal region).

Extended Data Figure 9 Structural comparison of ETB receptor and class A GPCRs.

a, b, Cytoplasmic views of ligand-free and ET-1 bound ETB receptors (a) and active and inactive M2R (b). The cytoplasmic architecture is similar between ligand-free and ET-1-bound ETB receptors, while M2R shows the outward displacement of TM6 upon activation. Panels show close-up views of the E/DRY motif, with the important residues represented by sticks. The intra-helical salt bridge interaction is disrupted upon activation in M2R, and R3.50 points towards the centre of the receptor in the active conformation. Although the similar salt bridge formation is prevented by the sulfate ion in the ligand-free ETB receptor, the rotamer orientations of R3.50 in both the ligand-free and ET-1 bound ETB receptors represent the features of the inactive conformation. cf, The structural comparisons of the β2-adrenergic receptor bound to an antagonist (PDB accession number 2RH1) and bound to an agonist and Gs (PDB accession number 3SN6) (c), the μ-opioid receptor bound to an antagonist (PDB accession number 4DKL) and bound to an agonist and nanobody (PDB accession number 5C1M) (d), rhodopsin in the ground state (PDB accession number 3PXO) and in the active state (PDB accession number 2X72) (e), and the A2A receptor bound to an antagonist (PDB accession number 4EIY) and bound to an agonist (PDB accession number 3QAK) (f). Cytoplasmic view (top), E/DRY motif on TM3 (middle upper), CWXP motif on TM6 (middle lower) and NPXXY motif on TM7 (bottom) of each receptor are shown. Red arrows in the upper panels indicate the outward displacement of TM6 that occurs upon receptor activation. The putative water molecule at the NPXXY motif in the β2-adrenergic receptor is represented by red circle. Residues involved in the structural rearrangement during receptor activation are represented by sticks, and hydrogen bonding interactions are indicated with yellow dotted lines. The agonist-bound A2A receptor retains the structural features of the inactive conformation. The intra-helical salt bridge is formed in the E/DRY motif, and Tyr7.53 in the NPXXY motif is too far away to form a water-mediated hydrogen bonding interaction with Tyr5.58, although TM7 is shifted inwards (middle upper and lower panels in f).

Extended Data Figure 10 ET-1-induced conformational changes in ETB receptor.

ad, Cutaway representation and hydrophobic packing interaction in the receptor core for the ligand-free (a, c) and ET-1-bound (b, d) ETB receptor. ET-1 binding induces the tightly packed hydrophobic core in the receptor. e, f, Collapse of the putative Na+ binding pocket in the ETB receptor. Asp1472.50 and its surrounding residues are shown for the ligand-free (e) and ET-1-bound (f) ETB receptor. Cross-sectional representations of the orthosteric pocket are overlaid. The putative Na+ binding site is indicated with a purple-shaded circle. The electron density for the Na+ ion was not observed, probably because of the low resolution of the structure. g, h, TM6–7 interactions in the ligand-free (g) and ET-1-bound (f) ETB receptors. TM1, TM2, TM3 and TM7 are shown as surface representations. The residues of TM6 directed towards TM7 are represented by CPK models.

Extended Data Table 1 Crystallographic statistics of the ET-1-bound and ligand-free structures of the ETB receptor

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Shihoya, W., Nishizawa, T., Okuta, A. et al. Activation mechanism of endothelin ETB receptor by endothelin-1. Nature 537, 363–368 (2016). https://doi.org/10.1038/nature19319

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