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 The copyright holder has placed this preprint (which was not certified by peer review) in the Public Domain.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‡. 6 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. 12 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 – 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 The copyright holder has placed this preprint (which was not certified by peer review) in the Public Domain.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. 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 – 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. 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 – 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. 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. 69 70 – 4 – 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. 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 – 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. 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. 97 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).