TITLE Crystal Structure of Human Endothelin ETB Receptor in Complex

bioRxiv preprint doi: https://doi.org/10.1101/460410; this version posted November 2, 2018. The copyright holder has placed this preprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, remix, or adapt this material for any purpose without crediting the original authors.

1 TITLE

2 Crystal structure of human endothelin ETB receptor in complex

3 with peptide inverse agonist IRL2500.

4 Chisae Nagiri1*, Wataru Shihoya1*‡, Asuka Inoue2, Francois Marie Ngako Kadji2, Junken

5 Aoki2, Osamu Nureki1‡.

7 Affiliations:

8 1 Department of Biological Sciences, Graduate School of Science, The University of Tokyo,

9 Bunkyo, Tokyo 113-0033, Japan.

10 2 Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3, Aoba, Aramaki,

11 Aoba-ku, Sendai, Miyagi 980-8578, Japan.

13 *These authors contributed equally to this work.

14 ‡To whom correspondence should be addressed. E-mail: [email protected] (W.S.),

15 [email protected] (O.N.)

– 1 –

16 Abstract

17 Endothelin receptors (ETA and ETB) are G-protein coupled receptors activated

18 by endothelin-1 and are involved in blood pressure regulation. IRL2500 is a peptide-

19 mimetic of the C-terminal tripeptide of endothelin-1, and has been characterized as a

20 potent ETB-selective antagonist, which has preventive effects against brain edema. Here,

21 we report the crystal structure of the human ETB receptor in complex with IRL2500 at

22 2.7 Å-resolution. The structure revealed the different binding modes between IRL2500

23 and ET-1, and provides structural insights into its ETB-selectivity. Notably, the biphenyl

24 group of IRL2500 penetrates into the transmembrane core proximal to D2.50, stabilizing

25 the inactive conformation. Using the newly-established constitutively active mutant, we

26 clearly demonstrate that IRL2500 functions as an inverse agonist for the ETB receptor.

27 The current findings will expand the chemical space of ETR antagonists and facilitate the

28 design of inverse agonists for other class A GPCRs.

– 2 –

29 Main text

30 Introduction

31 Endothelin receptors (ETR) are G-protein coupled receptors activated by vaso

1 32 active peptide, endothelins . Two endothelin receptor subtypes (ETA and ETB) are widely

33 expressed in the vascular endothelium, brain, and other circulatory organs2,3. Endothelin-

34 1 (ET-1) activates the endothelin receptors (ETRs) with sub-nanomolar affinities. The

35 activation of the ETA receptor leads to potent and long-lasting vasoconstriction, whereas

36 that of the ETB receptor induces nitric oxide-mediated vasorelaxation. Therefore, the up-

37 regulation of ET-1 is significantly related to circulatory-system diseases, including

38 pulmonary arterial hypertension (PAH)4–7. Moreover, the autocrine and paracrine

39 signaling functions of ET-1 through the ETA receptor play a critical role in tumor growth

40 and survival8. Therefore, ETR antagonists have been developed for the treatment of

41 circulatory-system diseases and cancers6,7. Bosentan is the first orally-active ETR

9,10 42 antagonist , and is used to treat PAH. The ETB receptor is the prominent ET receptor

11 43 subtype in the brain, with high expression levels in astrocytes . Stimulation of the ETB

44 receptor modulates astrocytic responses, indicating its important roles in regulating

12 45 astrocytic functions . The up-regulation of the astrocytic ETB receptor by ET-1 increases

46 the vascular permeability and reduces the AQP4 levels, thereby aggravating vasogenic

11 47 brain edema . The application of ETB-selective antagonists may provide preventive

48 effects against brain edema in the acute phase of brain insults13–16.

49 To date, most endothelin receptor antagonists have been developed based on

50 bosentan17,18. The ETR antagonists that have been developed till now are mostly N- – 3 –

51 heterocyclic sulfonamides with similar structures and molecular weights, and non-

52 sulfonamide antagonists (atrasentan, ambrisentan, darusentan, and enrasentan) still retain

53 high similarities with each other and with the sulfonamides7. Therefore, the ETR agents

54 are chemically very similar, and expanded chemical space should be exploited. IRL2500

55 is a peptide ETR antagonist developed based on the partial structure of ET-119, rather than

56 bosentan. IRL2500 has been characterized as an ETB-selective antagonist with an IC50

57 value of 1.2 nM20, which shows higher affinity than that of bosentan. In an animal model,

58 the intracerebroventricular administration of IRL2500 attenuated cold injury-mediated

59 brain edema and disruption of the blood-brain barrier, indicating the neuroprotective

60 effect of IRL250014,15. An understanding of the IRL2500 binding mode would facilitate

61 the expansion of the chemical space of ET agents.

21 62 We previously reported the crystal structures of the ETB receptor bound to ET-1

22 63 and bosentan ; however, both the binding mode and ETB-selectivity of IRL2500

64 remained to be elucidated. Here, we present the crystal structure of the ETB receptor in

65 complex with IRL2500. This structure revealed the unique binding mode of IRL2500,

66 which differs from those of ET-1 and bosentan. Structure-guided functional analyses

67 clearly demonstrate that IRL2500 functions as an inverse agonist for the ETB receptor,

68 and thus will provide the basis for design of inverse agonists for other class A GPCRs.

– 4 –

71 Results

72 Overall structure

73 For crystallization, we used the previously established, thermostabilized ETB

22,23 74 receptor (ETB-Y4) . The IC50 value of IRL2500 for ETB-Y4 was similar to that for the

75 wild type receptor in the TGFα shedding assay24 (Fig. 1), suggesting that the

76 themostabilizing mutations minimally affect the IRL2500 binding. In contrast, the IC50

77 value of IRL2500 for the ETA receptor is over 3 M (Fig. 1), indicating that IRL2500 has

20 78 over 100-fold ETB-selectivity, consistent with the previous pharmacological analysis .

79 To facilitate crystallization, we replaced the third intracellular loop (ICL3) of the receptor

25 26 80 with minimal T4 Lysozyme (ETB-Y4-mT4L). Using in meso crystallization , we

81 obtained crystals of ETB-Y4-mT4L in complex with IRL2500 (Supplementary Fig. 1a, b).

82 In total, 58 datasets were collected and merged by the data processing system KAMO27.

83 Eventually, we determined the ETB structure in complex with IRL2500 at 2.7 Å resolution,

84 by molecular replacement using the antagonist-bound ETB structure (PDB code: 5X93)

85 (Table 1).

86 The overall structure consists of the canonical 7 transmembrane helices (TM), the

87 amphipathic helix 8 at the C-terminus (H8), and two antiparallel β-strands in the

88 extracellular loop 2 (ECL2), as in the previously determined ETB structures (Fig. 2a). The

89 IRL2500-bound structure is similar to the bosentan-bound structure, rather than the ET-

90 1-bound structure (R.M.S.D. values for Cα atoms=1.34 and 1.95 Ǻ, respectively),

91 reflecting the inactive conformation. We observed a remarkable difference in the

92 conformation of ECL2. The β strands are opened up by 9 Å, as compared with those in – 5 –

93 the ligand-free structure (Fig. 2b and Supplementary Fig. 2a), and are the widest among

94 the peptide-activated class A GPCRs (Supplementary Fig. 2b). This structural feature

95 indicates the innate flexibility of ECL2, to capture the large peptide ligand endothelin, in

96 the inactive conformation of the ETB receptor.

98 IRL2500 binding site

99 We first describe the IRL2500 binding mode. IRL2500 consists of a tryptophan,

100 a 3,5-dimethylbenzoyl group, and a biphenyl group19, which are connected by two peptide

101 bonds (Fig. 2c). IRL2500 binds to the transmembrane binding cleft exposed to the

102 extracellular side, with a clear electron density in the Fo − Fc omit map (Supplementary

103 Fig. 3a, b). The carboxylate group of the tryptophan moiety in IRL2500 forms salt bridges

104 with K1823.33 and R3436.55 (superscripts indicate Ballesteros–Weinstein numbers28) (Fig.

105 2c, d). The tryptophan side chain of IRL2500 hydrogen bonds with the carbonyl group of

106 the N1582.61 side chain, and forms extensive van der Waals interactions with N1582.61,

107 K1612.64, V1773.28, P1783.29, and F2404.64 (Fig. 2d). The dimethyl phenyl group of

108 IRL2500 forms van der Waals interactions with the hydrophobic pocket, and is

109 surrounded by V1853.36, L2775.42, Y2815.46, W3366.48, L3396.51, and H3406.52. The

110 biphenyl group penetrates deeply into the receptor core proximal to D1472.50, and forms

111 van der Waals interactions with D1472.50, H1502.53, W3366.48, and S3767.43. Overall, the

112 carboxylate of IRL2500 is specifically recognized by the positively charged residues of

113 the ETB receptor, and the other moieties fill the space within the transmembrane binding

114 pocket.

115 To elucidate the structural basis for the ETB-selectivity of IRL2500, we compared – 6 –

116 the residues constituting the IRL2500 binding site between the ETB and ETA receptors

117 (Fig. 3a and Supplementary Fig. 4). Although most of the residues are conserved, three

118 residues are replaced with bulkier residues in the ETA receptor (H150Y, V177F, and

119 S376T). These replacements may cause steric clashes with the aromatic groups of

120 IRL2500 and reduce its affinity. To investigate this hypothesis, we measured the IC50

121 values of IRL2500 for the H150Y, V177F, and S346T ETB receptor mutants. These

122 mutants showed similar responses for ET-1 in the TGFα shedding assay (Supplementary

123 Fig. 5), and only V177F showed a 4-fold higher IC50 value with IRL2500 (Fig. 3b). These

124 data suggest that the V177F mutation in the ETA receptor sterically clashes with the

125 tryptophan moiety of IRL2500 and reduces its affinity, thus partially accounting for the

126 ETB-selectivity of IRL2500. This is consistent with the previous study, in which the

127 replacement of the tryptophan moiety with the smaller valine residue in IRL2500

29 128 weakened its ETB-selectivity .

129

130 Comparison of the binding modes of IRL2500, ET-1, and bosentan

131 IRL2500 is designed to mimic the Y13, F14, I19, I20, and W21 residues in ET-

19 132 1, which play critical roles in ligand binding to the ETB receptor . The tryptophan and

133 dimethyl phenyl group of IRL2500 seem to be equivalent to W21 and I20 in ET-1,

134 respectively, while the biphenyl group of IRL2500 seems to be equivalent to F14 and I19

135 of ET-1. However, a comparison between IRL2500 and ET-1 binding revealed an

136 unexpected difference in their binding interactions (Fig. 4a). The carboxylate of the

137 tryptophan in IRL2500 superimposes well with that of W21 in ET-1, and is coordinated

138 by similar positively charged residues. The tryptophan moiety and dimethyl phenyl group – 7 –

139 of IRL2500 superimpose well with I20 and W21 of ET-1, respectively. In contrast, the

140 biphenyl group of IRL2500 penetrates into the receptor core, in an opposite manner to

141 the F14 and I19 of ET-1. Overall, the electrostatic interactions between the carboxylates

142 and the positively charged residues are conserved in IRL2500 and ET-1 binding, but the

143 other moieties form totally distinct interactions with the receptor. The volume of the

144 ligand binding pocket in the ligand-free structure is large, thereby allowing the aromatic

145 moieties of IRL2500 to flip.

146 IRL2500 has distinct chemical moieties as compared with bosentan, because

147 IRL2500 was not developed based on bosentan. To reveal the similarities and differences

148 in their binding modes, we compared the binding modes of IRL2500 and bosentan in

149 detail (Fig. 4b, c). The carboxylate of IRL2500 and the sulfonamide of bosentan are

150 similarly coordinated by the positively charged residue R3436.55, suggesting that this

151 electrostatic interaction is a common feature of the antagonist binding to the ETB receptor.

152 In addition, like bosentan, the aromatic moieties of IRL2500 fit within the local

153 hydrophobic pockets in the ETB receptor. Overall, IRL2500 has moieties that form similar

154 binding interactions to those of bosentan. However, bosentan lacks the moiety

155 corresponding to the biphenyl group of IRL2500, which deeply penetrates into the

156 receptor core. Thus, IRL2500 fits into the pocket more tightly as compared with bosentan,

157 contributing to its higher affinity.

158

159 IRL2500 function an inverse agonist for ETB

160 To obtain mechanistic insights into the receptor inactivation by IRL2500, we

161 compared the ETB structures bound to ET-1, bosentan, and IRL2500. Previous structural – 8 –

162 studies showed that ET-1 binding induces the inward moment of the extracellular portion

163 of TM6 including W3366.48, leading to receptor activation on the intracellular side21 (Fig.

164 5a). Bosentan binding sterically prevents the inward motion of W3366.48 with its 2-

165 methoxyphenoxy group, and thus functions as an antagonist22 (Fig. 5b). The

166 dimethylphenyl group of IRL2500 superimposes well with the 2-methoxyphenoxy group

167 of bosentan and similarly prevents the inward motion. Moreover, the dimethyl phenyl and

168 biphenyl groups of IRL2500 sandwich the W3366.48 side chain, tightly preventing its

169 inward rotation. These observations suggest that IRL2500 strongly prevents the transition

170 to the active state, as compared with bosentan, thereby possibly working as an inverse

171 agonist that reduces the basal activity.

172 To investigate the inverse agonist activity of IRL2500 for the ETB receptor, we

173 first measured ligand-induced AP-TGF release responses. The EC50 value of the agonist

174 ET-1 was 0.11 nM, while IRL2500 and the antagonist bosentan did not change the

175 receptor activation level (Fig. 5c). These data suggested that IRL2500 does not have the

176 inverse agonist activity or that the assay is not sensitive enough to detect inverse agonist

177 activity. Indeed, we observed that the basal activity of the ETB receptor was very low in

178 the assay (Fig. 5d) and thus we could not distinguish whether IRL2500 functions as an

179 antagonist or an inverse agonist by this assay.

180 Therefore, we tried the same assay using a constitutively active mutant of the ETB

181 receptor. Constitutively active mutant GPCRs have been employed in pharmacological

182 characterizations of inverse agonists30, because such mutant GPCRs allow the assay to

183 measure signals in a larger detection window. The substitution of the highly conserved

184 L3.43 to glutamine has been identified as a causative activating mutation in the TSHR31

– 9 –

185 and CYSLTR232 genes, which are related to hyperthyroidism and uveal melanoma,

3.43 186 respectively. Therefore, we transferred the L3.43Q mutation into ETB (ETB-L192 Q)

187 and examined its constitutive activity. We found that ETB-L3.43Q induced spontaneous

188 AP-TGFα release in a plasmid volume-dependent manner (Fig. 5d), indicating that

189 L3.43Q works as a constitutive active mutation in the ETB receptor. We evaluated the

190 dose response effects of bosentan and IRL2500, using the constitutive active mutant ETB-

191 L3.43Q (Fig. 5e). Again, the antagonist bosentan did not change the receptor activation

192 from the baseline level, whereas IRL2500 reduced the basal activity (EC50=1.2 nM).

193 These data indicate that IRL2500 works as a potent inverse agonist for the ETB receptor,

194 consistent with the structural observations. The biphenyl group of IRL2500 prevents the

195 inward motion of W3366.48 and stabilizes the inactive conformation, and thus IRL2500

196 functions as an inverse agonist.

197

198

199

200

201

202

203

204

205

– 10 –

206 Discussion

207 We have determined the crystal structure of the ETB receptor in complex with the

208 peptide compound IRL2500, and thus elucidated the detailed receptor interactions and

209 the structural basis for its ETB selectivity. Although IRL2500 is designed to mimic the

210 partial structure of ET-1, the binding mode is quite different. Moreover, using the

211 constitutively active mutant that we established in the current study, we first revealed that

212 IRL2500 functions as a potent inverse agonist for the ETB receptor, and provided the

213 structural basis for the inverse agonistic mechanism.

214 Small-molecule ETR antagonists have been developed over the years; however,

215 most ETR antagonists have been designed based on bosentan. Thus, the presently

216 available ET agents are chemically very similar. IRL2500 was developed based on ET-1

217 and has totally distinct chemical moieties, as compared with bosentan. However, the

218 comparison of the IRL2500 and bosentan binding modes revealed the unexpected

219 similarity in their binding interactions. This observation suggests that the charge-

220 complementary interactions in the center of the pocket form the core of the receptor-

221 antagonist interactions, and the other aromatic moieties fit the local hydrophobic pocket.

222 The ligand binding pocket in the inactive ETB structures is larger than those in other

223 GPCR structures, and thus aromatic moieties may be necessary to fit well within the

224 pocket.

225 We revealed that the biphenyl group of IRL2500 penetrates deeply into the

226 receptor core proximal to D1472.50, preventing the inward motion of W3366.48 in TM6,

227 and thus IRL2500 functions as an inverse agonist (Fig. 6a). This D2.50 constitutes a

– 11 –

228 sodium binding site adjacent to the orthosteric site, which is highly conserved among the

229 class A GPCRs33. Sodium has negative allosteric effects on ligand binding in most class

230 A GPCRs, by stabilizing the inactive conformations. Therefore, this sodium binding site

231 is the hot spot for the design of allosteric modulators and inverse agonists to fix receptors

232 in the inactive conformations. In the BLT1 structure bound to the inverse agonist BIIL260,

233 the benzamidine group of BIIL260 directly hydrogen bonds with D2.50, stabilizing the

234 inactive conformation instead of the sodium34 (Fig. 6b). The biphenyl group of IRL2500

235 superimposes well with the benzamidine group of BIIL260 (Fig. 6c). Although the

236 biphenyl group of IRL2500 does not form any hydrogen-bonding interactions with the

237 receptor, it prevents the conformational change around the D2.50 in a similar manner to

238 the benzamidine moiety of BIIL260. For the design of effective inverse agonists, the

239 biphenyl moiety would be also useful as a modulation part along with another moiety that

240 exerts specific and tight binding to the orthosteric site, as well as a benzamidine group.

241

242

243

244

245

246

247

– 12 –

248 Materials and methods

249 Expression and purification

250 The haemagglutinin signal peptide, followed by the Flag epitope tag

251 (DYKDDDDK) and a nine-amino-acid linker, was added to the N-terminus of the

252 receptor, and a tobacco etch virus (TEV) protease recognition sequence was introduced

253 between G57 and L66, to remove the disordered N-terminus during the purification

254 process. The C-terminus was truncated after S407, and three cysteine residues were

255 mutated to alanine (C396A, C400A, and C405A) to avoid heterogeneous palmitoylation.

256 To improve crystallogenesis, we introduced four thermostabilizing mutations () and

257 inserted minimal T4 lysozyme25 into intracellular loop 3, between L3035.68 and L3116.23

22 258 (ETB-Y4-mT4L ).

259 The thermostabilized construct ETB-Y4-mT4L was subcloned into a modified

260 pFastBac vector, with the resulting construct encoding a TEV cleavage site followed by

261 a GFP-His10 tag at the C-terminus. The recombinant baculovirus was prepared using the

262 Bac-to-Bac baculovirus expression system (Invitrogen). Sf9 insect cells were infected

263 with the virus at a cell density of 4.0 × 106 cells per millilitre in Sf900 II medium, and

264 grown for 48 h at 27 °C. The harvested cells were disrupted by sonication, in buffer

265 containing 20 mM Tris-HCl, pH 7.5, and 20% glycerol. The crude membrane fraction

266 was collected by ultracentrifugation at 180,000g for 1 h. The membrane fraction was

267 solubilized in buffer, containing 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1% DDM,

268 0.2% cholesterol hemisuccinate, 10 μM IRL2500, and 2 mg ml−1 iodoacetamide, for

269 1 h at 4 °C. The supernatant was separated from the insoluble material by

– 13 –

270 ultracentrifugation at 180,000g for 20 min, and incubated with TALON resin

271 (Clontech) for 30 min. The resin was washed with ten column volumes of buffer,

272 containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% LMNG, 0.01% CHS, 10 μM

273 IRL2500, and 15 mM imidazole. The receptor was eluted in buffer, containing 20 mM

274 Tris-HCl, pH 7.5, 500 mM NaCl, 0.01% LMNG, 0.001% CHS, 10 μM IRL2500, and

275 200 mM imidazole. The eluate was treated with TEV protease and dialysed against

276 buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 10 μM IRL2500). The cleaved

2+ 277 GFP–His10 tag and the TEV protease were removed with Co -NTA resin. The receptor

278 was concentrated and loaded onto a Superdex200 10/300 Increase size-exclusion

279 column, equilibrated in buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl,

280 0.01% LMNG, 0.001% CHS, and 10 μM IRL2500. Peak fractions were pooled,

281 concentrated to 40 mg ml−1 using a centrifugal filter device (Millipore 50 kDa MW

282 cutoff), and frozen until crystallization. During the concentration, IRL2500 was added

283 to a final concentration of 100 μM.

284

285 Crystallization

286 The purified receptor was reconstituted into molten lipid (monoolein and

287 cholesterol 10:1 by mass) at a weight ratio of 1:1.5 (protein:lipid). The protein-laden

288 mesophase was dispensed into 96-well glass plates in 30 nl drops and overlaid with 800

289 nl precipitant solution by a Gryphon LCP robot (Art Robbins Instruments)26. Crystals of

290 ETB-Y4-mT4L bound to IRL2500 were grown at 20ºC in precipitant conditions

291 containing 30% PEG300, 100 mM Bis-tris, pH 7.5, 150 mM sodium phosphate

292 monobasic, and 10 mM TCEP hydrochloride. The crystals were harvested directly from – 14 –

293 the LCP using micromounts (MiTeGen) or LithoLoops (Protein Wave) and frozen in

294 liquid nitrogen, without adding any extra cryoprotectant.

295

296 Data collection and structure determination

297 X-ray diffraction data were collected at the SPring-8 beamline BL32XU, with 10

298 × 15 μm2 (width × height) micro-focused beams and an EIGER X 9M detector (Dectris).

299 Various wedge data sets (10°) per crystal were mainly collected with the ZOO system, an

300 automatic data-collection system developed at SPring-8 (K.Y., G.U., K.H., M.Y., and

301 K.H., submitted). The loop-harvested microcrystals were identified by raster scanning

302 and subsequently analyzed by SHIKA35. Each data set was indexed and integrated with

303 XDS36, and the datasets were hierarchically clustered by using the correlation coefficients

304 of the intensities between datasets. After the rejection of outliers, 58 data sets were finally

305 merged with XSCALE36. The IRL2500-bound structure was determined by molecular

37 306 replacement with PHASER , using the K8794-bound ETB structure (PDB code: 5X93).

307 Subsequently, the model was rebuilt and refined using COOT38 and PHENIX39,

308 respectively. The final model of IRL2500-bound ETB-Y4-T4L contained residues 91-207,

309 214-303, and 311-403 of ETB, 1-14 and 22-117 of mT4L, IRL2500, 6 monoolein

310 molecules, two phosphoric acids, and 41 water molecules. The model quality was

311 assessed by MolProbity40. Figures were prepared using CueMol

312 (http://www.cuemol.org/ja/)

313

– 15 –

314 TGFα shedding assay

315 The TGFα shedding assay, which measures the activation of Gq and G12

24 22 316 signaling , was performed as described previously . Briefly, a plasmid encoding an ETB

317 construct with an internal FLAG epitope tag or an ETA construct was transfected, together

318 with a plasmid encoding alkaline phosphatase (AP)-tagged TGFα (AP-TGFα), into

319 HEK293A cells by using a polyethylenimine (PEI) transfection reagent (1 µg ETR

320 plasmid, 2.5 µg AP-TGFα plasmid, and 25 µl of 1 mg/ml PEI solution per 10-cm culture

321 dish). After a one day culture, the transfected cells were harvested by trypsinization,

322 washed, and resuspended in 30 ml of Hank's Balanced Salt Solution (HBSS) containing

323 5 mM HEPES (pH 7.4). The cell suspension was seeded in a 96 well plate (cell plate) at

324 a volume of 80 μl per well and incubated for 30 min in a CO2 incubator. For the

325 measurement of antagonist activity, IRL2500 was diluted in 0.01% bovine serum albumin

326 (BSA) and HEPES-containing HBSS (assay buffer) and added to the cell plate at a volume

327 of 10 µl per well. After 5 min, ET-1, at a final concentration of 0.2 nM, was added to the

328 cell plate at a volume of 10 µl per well. For the measurement of agonistic activity, after

329 adding 10 µl of the assay buffer, serially diluted ET-1 was mixed with the cells at a volume

330 of 10 µl per well. After a 1 h incubation in the CO2 incubator, aliquots of the conditioned

331 media (80 μl) were transferred to an empty 96-well plate (conditioned media (CM) plate).

332 Similarly, for the measurement of inverse agonist activity, the cells were mixed with 10

333 µl of the assay buffer, followed by the addition of serially diluted IRL2500, and incubated

334 for 4 h before the transfer of the conditioned media. The AP reaction solution (10 mM p-

335 nitrophenylphosphate (p-NPP), 120 mM Tris–HCl (pH 9.5), 40 mM NaCl, and 10 mM

– 16 –

336 MgCl2) was dispensed into the cell plates and the CM plates (80 µl per well). The

337 absorbance at 405 nm (Abs405) of the plates was measured, using a microplate reader

338 (SpectraMax 340 PC384, Molecular Devices), before and after a 1 h incubation at room

339 temperature. AP-TGFα release was calculated as described previously22. The AP-TGFα

340 release signals were fitted to a four-parameter sigmoidal concentration-response curve,

341 using the Prism 7 software (GraphPad Prism), and the pEC50 (equal to -Log10 EC50) and

342 Emax values were obtained.

343 To measure the constitutive activity in a plasmid volume-dependent manner,

344 HEK293 cells were seeded in a 96-well plate at a concentration of 4 x 105 cells per ml in

345 Opti-MEM I Reduced Serum Media (Thermo Fisher Scientific), in a volume of 80 μl per

346 well. A transfection mixture was prepared by mixing the PEI transfection reagent (0.2 µl

347 per well) and plasmids (20 ng AP-TGFα plasmid, titrated ETR plasmid, and an empty

348 vector to balance the total plasmid volume) in Opti-MEM I Reduced Serum Media (20

349 µl). The mixture was added to the cells, which were then incubated for 24 h before the

350 transfer of the conditioned media. After adding the AP reaction solution, the absorbances

351 of the cells and the CM plates were measured at 20 min intervals. The AP-TGFα release

352 signals were calculated as described above, and the signal in the mock-transfected

353 conditions was set at the baseline.

354

355

356

– 17 –

357 Acknowledgements

358 The diffraction experiments were performed at SPring-8 BL32XU (proposal

359 2017A2527). We thank the beamline staff at BL32XU of SPring-8 (Sayo, Japan) for

360 technical assistance during data collection. We also thank Kouki Kawakami, Takeaki

361 Shibata and Ayumi Inoue (Tohoku University, Japan) for technical assistance in the

362 characterization of the L3.43Q-mutant ETB receptor. This work was supported by grants

363 from the Platform for Drug Discovery, Informatics and Structural Life Science by the

364 Ministry of Education, Culture, Sports, Science and Technology (MEXT), JSPS

365 KAKENHI grants 16H06294 (O.N.), 17J30010 (W.S.), 30809421 (W.S.), 17K08264

366 (A.I.), and the Japan Agency for Medical Research and Development (AMED) grants:

367 the PRIME JP17gm5910013 (A.I.) and the LEAP JP17gm0010004 (A.I. and J.A.), and

368 the National Institute of Biomedical Innovation.

369

370 Author contributions

371 C.N. expressed, purified, and crystallized the IRL2500-bound ETB receptor,

372 collected data, and refined the structures. W.S. designed all of the experiments, initially

373 crystallized the receptor, and refined the structure. A.I., F.M.N.K., and J.A. performed

374 and oversaw the cell-based assays. The manuscript was prepared by C.N., W.S., A.I., and

375 O.N. W.S. and O.N. supervised the research. Coordinates and structure factors have been

376 deposited in the Protein Data Bank, under the accession number XXXX for the IRL2500-

377 bound structure. The X-ray diffraction images are also available at SBGrid Data Bank

378 (https://data.sbgrid.org/), under the ID YYYY. – 18 –

379

380 Competing interests

381 The authors declare no competing interests

– 19 –

382 References

383 384 1. Yanagisawa, M. et al. A novel potent vasoconstrictor peptide produced by 385 vascular endothelial cells. Nature 332, 411–415 (1988). 386 2. Arai, H. et al. Cloning and expression of a cDNA encoding an endothelin 387 receptor. Nature 348, 730–732 (1990). 388 3. Sakurai, T. et al. Cloning of a cDNA encoding a non-isopeptide-selective 389 subtype of the endothelin receptor. Nature 348, 732–735 (1990). 390 4. Channick, R. N. et al. Effects of the dual endothelin-receptor antagonist bosentan 391 in patients with pulmonary hypertension: a randomised placebo-controlled study. 392 Lancet 358, 1119–1123 (2001). 393 5. Rubin, L. J. et al. Bosentan therapy for pulmonary arterial hypertension. N. Engl. 394 J. Med. 346, 896–903 (2002). 395 6. Maguire, J. J. & Davenport, A. P. Endothelin@25 - new agonists, antagonists, 396 inhibitors and emerging research frontiers: IUPHAR Review 12. Br. J. 397 Pharmacol. 171, 5555–5572 (2014). 398 7. Davenport, A. P. et al. Endothelin. Pharmacol. Rev. 68, 357–418 (2016). 399 8. Rosanò, L., Spinella, F. & Bagnato, A. Endothelin 1 in cancer: biological 400 implications and therapeutic opportunities. Nat. Rev. Cancer 13, 637–651 (2013). 401 9. Clozel, M. et al. Pathophysiological role of endothelin revealed by the first orally 402 active endothelin receptor antagonist. Nature 365, 759–761 (1993). 403 10. Clozel, M. et al. Pharmacological characterization of bosentan, a new potent 404 orally active nonpeptide endothelin receptor antagonist. J. Pharmacol. Exp. Ther. 405 270, 228–235 (1994). 406 11. Koyama, Y. & Michinaga, S. Regulations of Astrocytic Functions by 407 Endothelins: Roles in the Pathophysiological Responses of Damaged Brains. J. 408 Pharmacol. Sci. 118, 401–407 (2012). 409 12. Hammond, T. R. et al. Endothelin-B Receptor Activation in Astrocytes Regulates 410 the Rate of Oligodendrocyte Regeneration during Remyelination. Cell Rep. 13, 411 2090–2097 (2015). 412 13. Moldes, O. et al. Neuroprotection afforded by antagonists of endothelin-1 413 receptors in experimental stroke. Neuropharmacology 63, 1279–1285 (2012). 414 14. Michinaga, S. et al. Amelioration of Cold Injury-Induced Cortical Brain Edema 415 Formation by Selective Endothelin ETB Receptor Antagonists in Mice. PLoS

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416 One 9, e102009 (2014). 417 15. Michinaga, S. et al. Improvement of cold injury-induced mouse brain edema by

418 endothelin ETB antagonists is accompanied by decreases in 419 matrixmetalloproteinase 9 and vascular endothelial growth factor-A. Eur. J. 420 Neurosci. 42, 2356–2370 (2015).

421 16. Michinaga, S. et al. Delayed Administration of BQ788, an ETB Antagonist, after 422 Experimental Traumatic Brain Injury Promotes Recovery of Blood–Brain Barrier 423 Function and a Reduction of Cerebral Edema in Mice. J. Neurotrauma 35, 1481– 424 1494 (2018). 425 17. Palmer, M. J. Endothelin Receptor Antagonists: Status and Learning 20 Years 426 On. Prog. Med. Chem. 47, 203–237 (2009). 427 18. Mucke, H. A. M. Small-molecule endothelin receptor antagonists: a review of 428 patenting activity across therapeutic areas. IDrugs 12, 366–375 (2009).

429 19. Friih, T. et al. IRL 2500: A potent ETB selective endothelin antagonist. 430 Bioorganic Med. Chem. Lett. 6, 2323–2328 (1996). 431 20. Balwierczak, J. L. et al. Characterization of a potent and selective endothelin-B 432 receptor antagonist, IRL 2500. J. Cardiovasc. Pharmacol. 26 Suppl 3, S393-6 433 (1995).

434 21. Shihoya, W. et al. Activation mechanism of endothelin ETB receptor by 435 endothelin-1. Nature 537, 363–368 (2016).

436 22. Shihoya, W. et al. X-ray structures of endothelin ETB receptor bound to clinical 437 antagonist bosentan and its analog. Nat. Struct. Mol. Biol. 24, 758–764 (2017). 438 23. Okuta, A. et al.Thermostabilization of the Human Endothelin Type B Receptor. 439 J. Mol. Biol. 428, 2265–2274 (2016). 440 24. Inoue, A. et al. TGFα shedding assay: an accurate and versatile method for 441 detecting GPCR activation. Nat. Methods 9, 1021–1029 (2012). 442 25. Thorsen, T. S. et al. Modified T4 Lysozyme Fusion Proteins Facilitate G Protein- 443 Coupled Receptor Crystallogenesis. Structure 22, 1657–1664 (2014). 444 26. Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic 445 mesophases. Nat. Protoc. 4, 706–731 (2009). 446 27. Yamashita, K., Hirata, K. & Yamamoto, M. KAMO: towards automated data 447 processing for microcrystals. Acta Crystallogr. Sect. D Struct. Biol. 74, 441–449 448 (2018). 449 28. Ballesteros, J. A. & Weinstein, H. Integrated methods for the construction of 450 three-dimensional models and computational probing of structure-function

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451 relations in G protein-coupled receptors. Methods Neurosci. 25, 366–428 (1995). 452 29. Sakaki, J. et al. Discovery of IRL 3461: a novel and potent endothelin antagonist

453 with balanced ETA/ETB affinity. Bioorg. Med. Chem. Lett. 8, 2241–2246 (1998). 454 30. Soudijn, W., van Wijngaarden, I. & Ijzerman, A. P. Structure-activity 455 relationships of inverse agonists for G-protein-coupled receptors. Med. Res. Rev. 456 25, 398–426 (2005). 457 31. Trülzsch, B. et al. Detection of thyroid-stimulating hormone receptor and 458 Gsalpha mutations: in 75 toxic thyroid nodules by denaturing gradient gel 459 electrophoresis. J. Mol. Med. 78, 684–691 (2001). 460 32. Moore, A. R. et al. Recurrent activating mutations of G-protein-coupled receptor 461 CYSLTR2 in uveal melanoma. Nat. Genet. 48, 675–680 (2016). 462 33. Katritch, V. et al. Allosteric sodium in class A GPCR signaling. Trends Biochem. 463 Sci. 39, 233–244 (2014). 464 34. Hori, T. et al. Na+-mimicking ligands stabilize the inactive state of leukotriene 465 B4 receptor BLT1. Nat. Chem. Biol. 14, 262–269 (2018). 466 35. Ueno, G. et al. Remote access and automation of SPring-8 MX beamlines. in AIP 467 Conf. Proc. 1741, 050021 (2016). 468 36. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010). 469 37. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 470 658–674 (2007). 471 38. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development 472 of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010). 473 39. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for 474 macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 475 213–221 (2010). 476 40. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular 477 crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010). 478

479

480

481

482 – 22 –

483 Figure Legends

484 Fig. 1. Inhibition of ET-1 binding by IRL2500.

485 Effect of IRL2500 on the ET-1 (0.2 nM)-induced release of AP-TGFα in HEK293 cells

486 expressing the endothelin receptors. For each experiment, the AP-TGFα release response

487 in the absence of IRL2500 is set at 100%. Data are displayed as means ± s.e.m. (standard

488 error of the mean) from seven to nine independent experiments, with each performed in

489 triplicate.

490

491 Fig. 2. ETB structure in complex with IRL2500.

492 a, The overall structure of the IRL2500-bound ETB receptor. The receptor is shown as a

493 sky blue ribbon model. IRL2500 is shown as a deep sky blue stick with a transparent

494 surface model. b, Superimposition of the IRL2500-bound and ligand-free ETB structures

495 (PDB code: 5GLI), colored sky blue and light green, respectively. c, Schematic

496 representation of the interactions between ETB and IRL2500 within 4.5 Å. The dashed

497 lines show hydrogen bonds. d, Binding pocket for IRL2500, viewed from the

498 extracellular side (over) and the membrane plane (lower). The receptor is shown in a sky

499 blue ribbon representation. IRL2500 and receptor residues involved in ligand binding are

500 shown as sticks, coloured deep sky blue and sky blue, respectively. The dashed lines show

501 hydrogen bonds.

502

– 23 –

503 Fig. 3. Conservation of the IRL2500 binding site.

504 a, Sequence conservation of the IRL2500 binding site between ETA and ETB, mapped

505 onto the IRL2500-bound structure. Conserved and non-conserved residues are coloured

506 sky blue and gray, respectively. The receptor residues involved in IRL2500 binding are

507 shown as sticks. The dashed lines show hydrogen bonds. b, Effect of IRL2500 on the ET-

508 1 (0.2 nM)-induced release of AP-TGFα in HEK293 cells expressing the mutant ETB

509 receptors. For each experiment, the AP-TGFα release response in the absence of IRL2500

510 is set at 100%. Data are displayed as means ± s.e.m. (standard error of the mean) from

511 four to six independent experiments, with each performed in triplicate.

512

513 Fig. 4. Comparison of binding modes of IRL2500, ET-1, and bosentan.

514 a, Superimposition of the IRL2500- and ET-1-bound ETB receptors (PDB code: 5GLH).

515 The ET-1- and IRL2500-bound receptors are shown as pink and sky blue ribbons,

516 respectively. IRL2500 is shown as a stick model. ET-1 is shown as a magenta ribbon with

517 stick models of the peptide residues (Y13, F14, I19, I20, and W21). b, c, Binding pockets

518 for bosentan (b) and IRL2500 (c). The bosentan-bound receptor (PDB code: 5XPR) is

519 shown as a thin orange ribbon model. The residues involved in bosentan binding (D1542.57,

520 Q1813.32, K1823.33, K2735.38, W3366.48, and R3436.55) and D1472.50 are shown as sticks.

521 Bosentan is shown as an orange stick model. IRL2500 and the IRL2500-bound receptor

522 are coloured as in panel (a). The residues involved in IRL2500 binding (N1582.61,

523 K1823.33, R3436.55, D1472.50, and W3366.48) are shown as sticks.

524

– 24 –

525 Fig. 5. Inverse agonistic activity of IRL2500.

526 a, b, Structural changes upon ET-1 and IRL2500 binding, as compared with the bosentan-

527 bound structure, coloured as in Fig. 4. Black arrows indicate the inward movements of

528 TM6 and W3366.48. c, Effects of IRL2500 and bosentan on the ET-1-induced AP-TGFα

529 release for the ETB receptor. Data are displayed as means ± s.e.m. (standard error of the

530 mean) from three to four independent experiments. d, Constitutive activity of ETB.

531 HEK293 cells were transfected with titrated volume of a plasmid encoding the wild-type

532 ETB (ETB-WT) or the L3.43Q-mutant ETB (ETB-L3.43Q) and accumulated AP-TGF

533 release during 24 h after transfection was measured. AP-TGF release signal in 0 ng

534 receptor plasmid was set as a baseline. Data are displayed as means ± s.e.m. from three

535 independent experiments.. e, Effects of IRL2500 and bosentan on the ET-1-induced AP-

536 TGFα release for the constitutive active ETB receptor (ETB-L3.43Q). Data are displayed

537 as means ± s.e.m. from three to four independent experiments.

538

539 Fig. 6. Structural comparison of ETB and BLT1 in complex with inverse

540 agonists.

541 a, b, The overall structures of the IRL2500-bound ETB receptor (a) and the BIIL260-

542 bound BLT1 receptor (PDB code: 5X33) (b). The ETB and BLT1 receptors are shown as

543 sky blue and light green ribbons, respectively. The inverse agonists IRL2500 and BIIL260

544 are shown as blue and dark green sticks, respectively. The lower panels show the binding

545 interactions around the sodium binding site. The residues involved in ligand binding are

546 represented with sticks. Hydrogen bonds are indicated by black dashed lines. In the – 25 –

547 IRL2500-bound structure, the biphenyl group of IRL2500 forms van der Waals

548 interactions with the receptor, and does not form any hydrogen-binding interactions. In

549 the BIIL260 structure, BIIL260 forms hydrogen bonds with D662.50, S1063.39, and

7.45 550 S276 . c, Superimposition of the ETB and BLT1 structures in complex with the inverse

551 agonists.

552

553

554

555

556

557

558

559

560

561

562

– 26 –

563 Supplementary Figure Legends

564 Supplementary Fig. 1. Crystallization.

565 a, Gel filtration chromatogram and SDS-PAGE of the purified IRL2500-bound ETB

566 receptor. b, Crystals of the IRL2500-bound ETB receptor.

567

568 Supplementary Fig. 2. Comparison of the ECL2 structure with those of

569 other peptide-activated GPCRs.

570 a, Superimposition of the ETB structures determined to date. The IRL2500-bound and

571 other structures are coloured sky blue and gray, respectively. b, Superimposition of the

572 IRL2500-bound ETB structure with other peptide-activated class A GPCRs.

573

574 Supplementary Fig. 3. Electron density.

575 a, Fo − Fc omit maps for IRL2500, contoured at 3.0σ. b, 2Fo − Fc map around the IRL2500

576 binding site, contoured at 2.5σ.

577

578 Supplementary Fig. 4. Alignment of the human ETA and ETB receptors.

579 Alignment of the amino acid sequences of the human ETB receptor (UniProt ID: P24530)

580 and the human ETA receptor (P25101). Secondary structure elements for α-helices and β-

581 strands are indicated by cylinders and arrows, respectively. Conservation of the residues

582 between ETA and ETB is indicated as follows: red panels for completely conserved; red – 27 –

583 letters for partially conserved; and black letters for not conserved. The residues involved

584 in IRL2500 binding are indicated with squares.

585

586 Supplementary Fig. 5. ET-1 responses of mutant receptors.

587 Concentration response-curves of AP-TGFα release in the ET-1 treatment of HEK293

588 cells expressing the endothelin receptors. Symbols and error bars are means and s.e.m.

589 (standard error of the mean) of four or six independent experiments, each performed in

590 triplicate.

– 28 –

ETB (WT) ETB-Y4 ETA

100 100 100 relea se) 80 80 80 60 60 60 TG F 40 40 40 AP - 20 20 20 0 0 0 GPCR activatio n (%max

0 -9 -8 -7 -6 -5 0 -9 -8 -7 -6 -5 0 -9 -8 -7 -6 -5 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 IRL-2500 (M)

ETB (n=7) ETB-Y4 (n=8) ETA (n=9)

IC50 20 nM 44 nM > 3 M (pIC50 ± SEM) (7.70 ± 0.05) (7.35 ± 0.07) (>5.5)

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a b

ECL2 ECL2

TM7 TM2 TM2 TM1

TM6 TM1 TM5 TM4 TM3 TM5 TM4 TM7

TM3 H8 TM6

c d 4.64 4.64 F240 F240 K1612.64 2.64 2.64 TM5 TM4 K161 4.64 TM3 TM2 6.55 H2N K161 F240 3.28 R343 V177 N1582.61 V1773.28 NH TM3 N H3C CH3 R3436.55 H N1582.61 CH3 NH NH TM2 3.29 5.42 P178 2.53 L277 3.29 P178 3.29 H150 2 O H N P178 3.28 3 5.42 V177 CH 2.61 L277 TM1 N158 3.32 CH3 2 Q181 H N 3.33 2.50 L3396.51 K182 D147 6.51 CH3 H2N 3.32 L339 Q181 O N TM6 3.33 TM4 3.35 OH 3.33 K182 S184 K182 7.43 HN S376 2.53 H150 3.36 7.46 3.32 2.53 V185 S379 Q181 5.46 H150 5.46 H3C CH3 Y281 Y281 6.48 TM1 7.41 3.36 HO 3.35 W336 3.36 3.35 A375 V185 HO S184 V185 S184 W3366.48 NH TM7 7.43 5.42 H3C S376 2.50 L277 D147 6.51 2.50 6.55 L339 7.41 OH O D147 TM5 R343 A375 7.46 S379 Y2815.46 W3366.48 OH

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a b K1612.64 ETB (WT) ETB (H150Y) TM3 100 100 F2404.64 TM2 V1773.28 80 80 60 60 N1582.61 40 40 20 20 0 0 GPCR activatio n (%max )

3.29 -9 -8 -7 -6 -9 -8 -7 -6 P178 0 -10 0 -10 2.53 H150 10 10 10 10 10 10 10 10 10 10 IRL-2500 (M) TM1 3.32 Q181 ETB (V177F) ETB (S376T) 3.33 2.50 K182 D147 100 100 80 80 TM4 S1843.35 60 60 7.43 40 40 S376 20 20 7.46 3.36 S379 0 0

V185 GPCR activatio n (%max )

-9 -8 -7 -6 -9 -8 -7 -6 A3757.41 0 -10 0 -10 10 10 10 10 10 10 10 10 10 10 TM7 IRL-2500 (M) 5.42 L277 ETB (WT) ETB (H150Y) ETB (V177F) ETB (S376T) L3396.51 TM5 6.55 IC50 21 nM 18 nM 130 nM 6.1 nM R343 6.48 5.46 (pIC50 ± SEM) (7.67 ± 0.09) (7.74 ± 0.15) (6.87 ± 0.13) (8.22 ± 0.15) Y281 W336

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a ET-1 IRL2500 b Bosentan c IRL2500

TM4 TM4 Y13 K2735.38 TM3 N1582.61 F14 K1823.33 3.32 3.33 I20 6.55 Q181 2.57 K182 R343 D154 R3436.55 TM4 WIRL2500 I19 TM1 TM2 TM2 TM2 W21 6.48 2.50 W336 6.48 D147 D1472.50 W336

TM6 TM5 TM5 TM5 TM6

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a Bosentan ET-1 b Bosentan IRL2500

TM5 TM5 TM4 TM4

TM6 TM2 TM6 TM2

TM3 TM3

W3366.48 W3366.48

c Bosentan IRL-2500 ET-1 d 50 Plasmid DNA (ng per well) ETB-WT 40 60 50 30 0 Ligand pEC50 ± SEM release ) release ) 40 0.5 30 Bosentan NA 20 20 2 NA 10 IRL-2500 10 8 0 ET-1 -9.75 ± 0.05 0 (%AP-TG F GPCR activatio n (%AP-TG F -10 Constitutive activit y 32

-9 -8 -7 -6 -5 0 -11 -10 ETB-WT ETB-L3.43Q 10 10 10 10 10 10 10 Ligand (M)

e ETB-L3.43Q 30 20 pEC50 ± SEM 10 Ligand release ) 0 Bosentan NA -10 IRL-2500 -8.92 ± 0.06 -20 ET-1 -9.97 ± 0.07 -30 GPCR activatio n (%AP-TG F

-9 -8 -7 -6 -5 0 -11 -10 10 10 10 10 10 10 10 Ligand (M)

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a b c

K1823.33

TM7 TM7 2.53 H150 6.48 2.53 W336 V69 W2366.48

3.36 CO (D66) 7.45 S1843.35 V185 S276 7.46 TM3 TM3 S277 2.50 D1472.50 TM6 D66 TM6

S1063.39 TM2 TM2

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IRL2500-ETB Data collection Space group I422 Cell dimensions a, b, c (Å) 110.0, 110.0, 291.7 α, β, γ (°) 90, 90, 90 Resolution (Å)* 43.88-2.70 (2.797-2.70)

* Rmeas 0.295 (3.946) * 10.74 (1.08)

* CC1/2 0.995 (0.798) Completeness (%)* 99.66 (99.35) Redundancy* 19.3 (19.6)

Refinement Resolution (Å) 43.88-2.70 No. reflections 25,069

Rwork / Rfree 0.2244 / 0.2628 No. atoms Protein 3256 Ligand 43 Water/Ion/Lipid 181 Averaged B-factors (Å2) Protein 82.9 Ligand/ion 52.3 Water 87.7 R.m.s. deviations from ideal Bond lengths (Å) 0.004 Bond angles (°) 0.92 Ramachandran plot Favored (%) 97.77 Allowed (%) 2.23 Outlier (%) 0

*Values in parentheses are for highest-resolution shell.

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a b

kDa 2500 75

2000 50 ETBR 37 1500

25 1000 UV280 absorbance (mAU)

500

0 10 µm 0 5 10 15 20 25 mL

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a b

ECL2 ECL2

TM2

TM1

TM5 TM7

TM3 TM6 TM4

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a b K1612.64

TM5 TM4 4.64 TM3 TM2 F240 3.28 V177 2.61 TM3 N158

TM2 P1783.29 H1502.53

TM1 Q1813.32 K1823.33 D1472.50

TM6 TM4 S1843.35 S3767.43

7.46 V1853.36 S379

A3757.41

TM7

L2775.42 6.51 TM1 6.55 L339 TM5 R343 5.46 Y281 W3366.48

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1 1 0 2 0 30 4 0 50 6 0 70

ETB_Human MQPPP SLC GRA LVA L VL A CGLSR I WG E ERGF P PDRA T P L L Q T AE IM T PPTK T LWPKGSNAS L A R S LA P AE ETA_Human ...ME T LC L RA SFW L A L VG...C V IS DN... P ERYS T N L S NH VD DF T TFRG T ELS.....F L V T T HQ P TN

TM1

8 0 9 0 1 00 1 1 0 1 20 130 140

ETB_Human V PK G DR T AGS PPRTISPPP C QGPIE I KET FKYINTV V SC L VF VL G II GN STLLRIIY KNKCMRNGPN ILI ETA_Human L VL P .. S NGS .....MHNY C PQQTK I TSA FKYINTV I SC T IF IV G MV GN ATLLRIIY QNKCMRNGPN ALI

TM2 TM3 TM3 150 1 6 0 1 7 0 180 1 9 0 200

ETB_Human ASLALGDL LH I VID IPINV Y KLLA ED WP ..... FG A E M CKL V PF I QK A SVGITVL SLCALS I DRYRAVAS ETA_Human ASLALGDL I Y VVID LPINV FKLLA GR WP FDHND FG V F LCKL FPF LQK SSVGITVL NLCALS VDRYRAVAS

TM4 β1 β2

210 2 2 0 2 3 0 2 4 0 250 2 6 0 270

ETB_Human WSR IK GIG V PKW TA V EIV L IW VV SV V LA V PEAIGF D II TM D Y K GSYL RI C L LHPV Q K T A FM Q FY KT A KDW ETA_Human WSR V Q GIG I P LV TA I EIV S IW IL S F ILA I PEAIGF VMV PF E Y RG EQH K T C ML N..AT S K FM EFY QD V KDW

TM5 TM6

280 2 9 0 3 0 0 3 1 0 3 20 3 3 0 340

ETB_Human WLF SFYFC LPL A ITA FFYTLMTCEML R KKS G . M QIAL ND HLKQRREVAKTVFCLV LV FALCW LPLHLSRI ETA_Human WLF G FYFC MPL VC TA I FYTLMTCEML N RRN G S L RIAL SE HLKQRREVAKTVFCLV VI FALCW F PLHLSRI

TM7 H8

3 50 3 60 3 70 3 8 0 3 90 4 0 0 410

ETB_Human LK LT LYN QN D PNRCELLSFLL VL DYIGIN MA SL NSCINPIALY LVSK RFKNCF KSSCLCC WC .QS FEEK Q S ETA_Human LK K TV YN EM D K NRCELLSFLL LM DYIGIN L A TM NSCINPIALY F VSK KFKNCF QS SCLCC C C Y QS KSLM T S

420 4 3 0 440 Interaction with IRL2500 ETB_Human LEEKQ SC LK F KAN DHG.YD NF RSS N KY SS S ETA_Human V PMNG T S I Q WK NH D QNNHN T DRSS HK DS M N

Nagiri et al. Fig. S4 bioRxiv preprint doi: https://doi.org/10.1101/460410; this version posted November 2, 2018. The copyright holder has placed this preprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, remix, or adapt this material for any purpose without crediting the original authors.

ETB (WT) ETB (H150Y) ETB (V177F) ETB (S376T) 60 60 60 60 50 50 50 50

release ) 40 40 40 40 30 30 30 30 20 20 20 20 10 10 10 10 0 0 0 0 GPCR activatio n (%AP-TG F -9 -8 -7 -9 -8 -7 -9 -8 -7 -9 -8 -7 0 -12 -11 -10 0 -12 -11 -10 0 -12 -11 -10 0 -12 -11 -10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 ET-1 (M)

ETB (WT) ETB (H150Y) ETB (V177F) ETB (S376T)

EC50 0.14 nM 0.090 nM 0.093 nM 0.16 nM (pEC50 ± SEM) (9.85 ± 0.07) (10.05 ± 0.10) (10.03 ± 0.11) (9.79 ± 0.04)

Nagiri et al. Fig. S5