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Crystal structure of human endothelin ETB receptor in complex with sarafotoxin S6b
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Crystal structure of human endothelin ETB receptor in complex with sarafotoxin S6b Tamaki Izume, Hirotake Miyauchi, Wataru Shihoya**, Osamu Nureki* Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo, Tokyo, 113-0033, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 December 2019 Accepted 27 December 2019 Available online xxx
Sarafotoxins (SRTXs) are endothelin-like peptides extracted from snake venom. SRTXs stimulate the endothelin ETA and ETB receptors and enhance vasoconstriction, followed by left ventricular dysfunction and bronchoconstriction. SRTXs include four major isopeptides, S6a-d, with different subtype selectivities. Here, we report the crystal structure of the human ETB receptor in complex with the non-selective sarafotoxin S6b at 3.0 Å resolution. This structure reveals the similarities and differences between the binding modes of the endothelins and S6b. Moreover, molecular dynamics simulations based on the S6bbound receptor provides structural insight into the subtype selectivity of the sarafotoxins. Our study clarifies the recognition mechanism of the endothelin-like peptide families. © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: Crystal structure GPCR Toxin
1. Introduction Endothelin-1 (ET-1) is a 21 amino-acid peptide hormone with potent vasoconstrictor activity [1]. ET-1 and its related isopeptides, ET-2 and ET-3, perform several physiological functions in neural crest development, cell proliferation, sodium excretion, salt homeostasis, and regulation of vascular tone and cell growth [2]. Endothelins stimulate the two endothelin receptor subtypes (ETA and ETB) [3,4], which share approximately 60% sequence similarity and belong to the class A G-protein-coupled receptors (GPCRs). The endothelin receptors are widely expressed in the vascular endothelium, brain, and other circulatory organs. ETB is expressed in the vascular endothelium and its stimulation induces nitric oxidemediated vasorelaxation, while ETA and ETB are both expressed in the vascular smooth muscle and their stimulation leads to potent and long-lasting vasoconstriction. Therefore, systemic administration of ET-1 causes transient vasodilation (initial endothelial ETB activation) and hypotension, followed by prolonged vasoconstriction and hypertension (smooth muscle ETA and ETB activation). ET1 is one of the most potent vasoconstrictors and is highly toxic (LD50 for mice is about 0.015 mg/kg body weight). The upregulation of ET-1 is significantly related to circulatory-system
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (W. Shihoya), [email protected] (O. Nureki).
diseases [5], including pulmonary arterial hypertension (PAH) [6]. Thus, endothelin receptor antagonists have been developed for the treatment of circulatory system diseases. Recently, the structures of the human ETB receptor have been determined in complex with agonists ET-1 and ET-3, [7,8], a partial agonist IRL1620, an antagonist bosentan [9], and inverse agonists K-8794 and IRL2500 [10]. Notably, the 2.0 Å resolution structure of the ET-3-bound receptor elucidated the detailed activation process of the receptor. Sarafotoxins (SRTXs) are a group of toxins present in a venom of Atractaspididae family of snakes [11e13], and share high structural and functional homology with the endothelins. SRTXs include the four major isopeptides S6a-d, composed of 21 amino acids. SRTXs stimulate the endothelin receptors and increase vasoconstriction, which is followed by left ventricular dysfunction, bronchoconstriction, and increase of the airway resistance. S6b binds to the ETA and ETB receptors with similar affinities to ET-1, and thus are highly lethal by causing cardiac arrest and death in mice within minutes of intravenous administration (LD50 for mice is about 0.015 mg/kg body weight) [13]. S6b is the most potent sarafotoxin. By contrast, S6c shows 100- to 10,000-fold reduced affinity for the ETA receptor and thus functions as an ETB-selective agonist. Therefore, S6c shows the modest toxicity as compared with S6b (LD50 for mice is about 0.30 mg/kg body weight). Moreover, S6b possesses modest matrix metalloproteinase inhibitory activity, because S6b shares a common fold with the core region of the tissue inhibitors of metalloproteinases (TIMPs) [14]. A number of ETB structures have been determined; however, the binding modes and selectivities of the sarafotoxins remained to be elucidated. Here we report the crystal
https://doi.org/10.1016/j.bbrc.2019.12.091 0006-291X/© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Please cite this article as: T. Izume et al., Crystal structure of human endothelin ETB receptor in complex with sarafotoxin S6b, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.091
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structure of the sarafotoxin S6b-bound human ETB receptor at 3.0 Å resolution. This structure revealed the binding mode of sarafotoxins and the structural basis for their subtype selectivities.
micromounts (MiTeGen) or LithoLoops (Protein Wave) and frozen in liquid nitrogen, without adding any extra cryoprotectant. 2.3. Data collection and structure determination
2. Material and method 2.1. Expression and purification The haemagglutinin signal peptide, followed by the Flag epitope tag (DYKDDDDK) and a nine-amino-acid linker, was added to the Nterminus of the receptor, and a tobacco etch virus (TEV) protease recognition sequence was introduced between G57 and L66, to remove the disordered N-terminus during the purification process. The C-terminus was truncated after S407, and three cysteine residues were mutated to alanine (C396A, C400A, and C405A) to avoid heterogeneous palmitoylation. To improve crystallogenesis, we introduced four thermostabilizing mutations R124Y1.55, D154A2.57, K270A5.35, DS342A6.54 and I381A7.48 (superscripts indicate BallesteroseWeinstein numbers) and inserted T4 lysozyme into intracellular loop 3, between L3035.68 and L3116.23 (ETB-Y5-T4L) [15]. The thermostabilized construct ETB-Y5-T4L was subcloned into a modified pFastBac vector, with the resulting construct encoding a TEV cleavage site followed by a GFP-His10 tag at the C-terminus. The recombinant baculovirus was prepared using the Bac-to-Bac baculovirus expression system (Invitrogen). Sf9 insect cells were infected with the virus at a cell density of 4.0 106 cells per millilitre in Sf900 II medium, and grown for 48 h at 27 C. The harvested cells were disrupted by sonication, in buffer containing 20 mM Tris-HCl, pH 7.5, and 20% glycerol. The crude membrane fraction was collected by ultracentrifugation at 180,000g for 1 h. The membrane fraction was solubilized in buffer, containing 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1% DDM, 0.2% cholesterol hemisuccinate (CHS), and 2 mg ml1 iodoacetamide, for 1 h at 4 C. The supernatant was separated from the insoluble material by ultracentrifugation at 180,000g for 20 min, and incubated with TALON resin (Clontech) for 30 min. The resin was washed with ten column volumes of buffer, containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% LMNG, 0.01% CHS, and 15 mM imidazole. The receptor was eluted in buffer, containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.01% LMNG, 0.001% CHS, and 200 mM imidazole. The eluate was treated with TEV protease and dialysed against buffer (20 mM Tris-HCl, pH 7.5 and 500 mM NaCl). The cleaved GFPeHis10 tag and the TEV protease were removed with the TALON resin. The receptor was concentrated and loaded onto a Superdex200 10/300 Increase size-exclusion column, equilibrated in buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.01% LMNG, and 0.001% CHS. Peak fractions were pooled, concentrated to 40 mg ml1 using a centrifugal filter device (Millipore 50 kDa MW cutoff), and frozen until crystallization. During the concentration, S6b was added to a final concentration of 100 mM.
X-ray diffraction data were collected at the SPring-8 beamline BL32XU, with 10 15 mm2 (width height) micro-focused beams and an EIGER X 9 M detector (Dectris). We manually collected 32 data sets (10 per crystal), and the collected images were automatically processed with KAMO [17] (https://github.com/ keitaroyam/yamtbx). Each data set was indexed and integrated with XDS [18] and then subjected to a hierarchical clustering analysis based on the unit cell parameters using BLEND [19]. After the rejection of outliers, 16 data sets were finally merged with XSCALE [18]. The S6b-bound structure was determined by molecular replacement with PHASER [20], using the ET-3-bound ETB structure (PDB code: 6IGK). Subsequently, the model was rebuilt and refined using COOT [21] and PHENIX [22], respectively. The final model of the S6b-bound ETB-Y5-T4L contained residues 86e210, 214e303, and 311e403 of ETB, 1e160 of T4L, and s6b. The model quality was assessed by MolProbity [23]. Figures were prepared using CueMol (http://www.cuemol.org/ja/). 2.4. Simulation system setup The initial structures of S6b and S6c-bound receptors were prepared by using the program MODELLER [24] and the N-and Cterminus were truncated as observed in the crystal structure of the ETB-S6b complex. All MD simulations were performed with NAMD2.12 [25]. The simulation system was set at the condition of 96 96 96 Å3 with 1-Palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) membrane bilayer, 150 mM NaCl, and TIP3 water molecules. The missing hydrogen atoms were modified by the psfgen plugin of VMD [26]. The disulfide bonds and protonation states were properly set as the physiological condition. The net charge was neutralized by adding Cl-ions. The molecular topology and force field parameter from Charmm36 [27,28] were used for all simulations. The simulation systems were energy minimized for 1,000 steps with fixed positions of the non-hydrogen atoms. After minimization, another 1,000 steps of energy minimization with 10 kcal mol1 restraints for the non-hydrogen atoms, except for the lipid molecules within 5.0 Å from the proteins. Next, equilibrations were performed for 0.1 ns under NVT conditions, with 10 kcal mol1 Å2 restraints for the heavy atoms of the proteins. Finally, equilibration was performed for 1.0 ns under NPT conditions with the 1.0 kcal mol1 Å2 restraints for all Ca atoms of the proteins. The production runs of the equilibrium simulations were performed for 50 ns without restraints, maintaining the constant temperature at 310 K using Langevin dynamics and the -Hoover Langevin piston [29]. constant pressure at 1 atm using Nose The long-range electrostatic interactions were calculated by the particle mesh Ewald method [30].
2.2. Crystallization 3. Results The purified receptor was reconstituted into molten lipid (monoolein and cholesterol 10:1 by mass) at a weight ratio of 1:1.5 (protein:lipid). The protein-laden mesophase was dispensed into 96-well glass plates in 30 nl drops and overlaid with 800 nl precipitant solution by a Gryphon LCP robot (Art Robbins Instruments) [16]. Crystals of ETB-Y5-T4L bound to S6b were grown at 20 C in precipitant conditions containing 30% PEG600, 100 mM Citrate, pH 5.0, 100 mM ammonium sulfate. Crystals of ETB-Y5-T4L bound to S6c were grown at 20 C in precipitant conditions containing 30% PEG500dme, 100 mM Tris, pH 7.8, 100 mM potassium thiocyanate. The crystals were harvested directly from the LCP using
3.1. Overall structure For crystallization, we used the thermostabilized ETB receptor (ETB-Y5), and inserted T4L into ICL3 as described previously (ETBY5-T4L) [7]. Using in meso crystallization, we obtained crystals of ETB-Y5-T4L in complex with S6b or S6c. The crystals of the S6cbound receptor were tiny and diffracted X-rays to a maximum of only 7.0 Å resolution (Supplementary Fig. 1a). The crystals of the S6b-bound receptor grew to their full size (20 20 20 mm3) and diffracted X-rays to a maximum of 3.0 Å resolution (Supplementary
Please cite this article as: T. Izume et al., Crystal structure of human endothelin ETB receptor in complex with sarafotoxin S6b, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.091
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Fig. 1b). Therefore, 32 datasets were collected for the S6b-bound receptor and 16 datasets were merged by the data processing system KAMO. Eventually, we determined the ETB structures in complex with S6b at 3.0 Å resolution, by molecular replacement using the ET-3-bound receptor (PDB 6IGK) [8] (Supplementary Table 1). The ETB receptor consists of the canonical 7 transmembrane helices (TM), the amphipathic helix 8 at the C-terminus (H8), and two antiparallel b-strands in the extracellular loop 2 (ECL2), as in the previously determined ETB structures (Fig. 1a). The electron density of S6b was clearly observed in the ligand binding pocket (Supplementary Fig. 1c). The overall structure is essentially similar to those of the ET-1- and ET-3-bound receptors (Fig. 1b). However, there is a remarkable difference on the intracellular side. In the S6b-bound receptor, the intracellular end of TM5 is opened up by 4 Å, as compared with those in the ET-1- and ET-3-bound receptors (Fig. 1c). A number of previous studies have shown that the intracellular portion of TM6 moves outwardly by 10e14 Å upon Gprotein coupling [31]. Moreover, in NTSR1 (neurotensin receptor type 1), agonist and G-protein binding induce the outward displacement of TM5 by 2e4 Å [32] (Fig. 1d). Although T4L insertion affects the conformations of TM5 and TM6, agonist binding may cause the outward displacement of the TM5 in the ETB receptor, as in NTSR1. The previous ETB structures revealed that the agonist binding disrupts the hydrogen-bonding network between W3366.48 and D1472.50, which stabilizes the inactive conformation by connecting TMs 2, 3, 6, and 7 at the receptor core [8] (Fig. 2a and b). In the S6bbound receptor, W3366.48 rotates inwardly as compared with that in the K-8794-bound inactive receptor, and forms a hydrogen bond with N3787.45 (Fig. 2c). D1472.50 forms hydrogen bonding interactions with N1191.50 and T1883.39. Therefore, S6b binding also induces the rearrangement of the hydrogen-bonding interaction in the receptor core, as similarly to the endothelin ligands (Fig. 2c and d).
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3.2. Sarafotoxin S6b binding site We next describe the detailed interactions between S6b and ETB. 8 residues in S6b are different from those in ET-1 and ET-3; however, 4 cysteines are completely conserved (Fig. 3a). Thus, S6b adopts a similar bicyclic architecture, comprising the N-terminal region (residues 1e7), a-helical region (residues 8e17), and C-terminal region (residues 18e21). The N-terminal region is attached to the central a-helical region by the intrachain disulfide bond pairs (C1eC15 and C3eC11) (Fig. 3b). A previous NMR study of S6b revealed that the N-terminal and a-helical regions form a stable conformation in solution, whereas the C-terminal region has a conformational flexibility (Fig. 3c). Therefore, the C-terminal region adopts its fixed conformation by the interaction with the receptor, as in the other endothelin ligands. S6b superimposes well with ET-1 and ET-3 (R.M.S.D. values for Ca ¼ 0.39 and 0.40 Å, respectively) (Fig. 3d). Despite the sequence differences, S6b highly mimics the endothelins and activates the receptor. Next we describe the differences in the binding modes of S6b, ET-1, and ET-3. In the N-terminal region, 5 residues of S6b are different from those of ET-1 and ET-3. As in the endothelin-bound structures, the N-terminal region of S6b is exposed to the solvent and thus the receptor can tolerate the sequence difference (Fig. 3eeh). Notably, K4, D5, and T7 form an intramolecular hydrogen-bonding network in S6b, and the Q352ECL3 side chain participates in this network and form a hydrogen bond with D5 (Fig. 3e). V12 in ET-1 and ET-3 is replaced with L12 in S6b, forming more extensive hydrophobic interactions with ECL2 (Fig. 3i). L17 in ET-1 and ET-3 is replaced with the polar residue Q17 in S6b. Instead of the hydrophobic interaction, Q17 forms a hydrogen bond with E165ECL1 (Fig. 3j). I19 in ET-1 and ET-3 is replaced with V19, forming similar hydrophobic interactions (Fig. 3k). These differences are subtle, and thus the conserved residues among S6b, ET-1, and ET-3 form essentially similar interactions with the receptor
Fig. 1. Overall structure of the S6b-bound ETB receptor. a, Overall structure of the S6b-bound ETB receptor, viewed from the membrane plane (left) and extracellular side (right). The receptor and T4L are colored purple and grey, respectively. S6b is shown as a transparent surface representation and a ribbon model, with its N-terminal region colored cyan, a-helical region orange, and C-terminal region deep pink. b, c, Superimposed ETB structures in complex with S6b (purple), ET-1 (pink), and ET-3 (orange), viewed from the membrane plane (b) and intracellular side (c). d, Intracellular views of the superimposed structures of the C-state hNTSR1 in complex with Gi1 (blue), active-state rNTSR1 (green), and inactive-state rNTSR1 (grey).
Please cite this article as: T. Izume et al., Crystal structure of human endothelin ETB receptor in complex with sarafotoxin S6b, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.091
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Fig. 2. Comparisons of the receptor core regions. a-c, Receptor core interactions in the K-8794- (a, blue), ET-3- (b, orange), and S6b- (c, purple) bound ETB receptors. The receptors are shown as ribbon representations, and the side chains of N1191.50, D1472.50, T1883.39, F3326.44, W3366.48, and N3787.45 are shown as stick models. Waters are shown as red spheres. The dashed lines show hydrogen bonds colored according to the respective structures. d, Superimposition of the receptor core interactions.
(Supplementary Figs. 2aec). 3.3. Insight into the subtype selectivity of sarafotoxins S6b and S6c have similar affinities towards the ETB receptor, whereas S6c has reduced affinity for the ETA receptor by about 100-
to 10,000-fold [11]. Between S6b and S6c, three residues are diverged (S2T, K9E, and Y13N) (Fig. 3a). Moreover, a previous study showed that [E9]S6b functions as a ETB-selective agonist [33] and is approximately equipotent with S6c, suggesting that the charge of the 9th residues in the sarafotoxins determines the subtype selectivity of the sarafotoxins. To obtain structural insight into the
Fig. 3. Interactions between S6b and ETB. a, Comparison of the amino acid sequences of ET-1, ET-3, S6b, and S6c. b, c, Comparison of the S6b structures in the complex (b) and solution (c). d, Superimposition of S6b (purple), ET-1 (pink), and ET-3 (orange). e-g, Interactions between the N-terminal region and the receptor in the S6b- (e), ET-1- (f), and ET-3- (g) bound structures. h, Superimposed structures of the S6b-, ET-1-, and ET-3-bound receptors. i-k, Superimposition of the S6b-, ET-1-, and ET-3-bound structures, focused on residues 12 (i), 17 (j), and 19 (k) of the peptide ligands.
Please cite this article as: T. Izume et al., Crystal structure of human endothelin ETB receptor in complex with sarafotoxin S6b, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.091
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TM6 and ECL3 in the S6b-bound receptor. The intracellular side of the S6b-bound receptor moves outwardly as compared with those of the endothelin-bound receptors. This observation suggests that TM5 adopt more active conformation for G-protein coupling, as in NTSR1. Moreover, the MD simulations based on the S6bbound ETB structure provides structural clues for the ETB-selectivity of S6c. Author contributions T.I. expressed, purified, and crystallized the S6b-bound ETB receptor, collected data, and refined the structures. H.M. performed the molecular dynamics simulation. W.S. designed the study and mainly wrote the manuscript with T.I. W.S. and O.N. supervised the research. Data availability
Fig. 4. Models of the sarafotoxin-bound endothelin receptor. a-d, Models of the human ETA and ETB receptors in complex with S6b or S6c, obtained by molecular dynamics simulations. Interactions between sarafottoxins and ECL2 and N-termini of the receptors are shows as sticks.
subtype selectivity of the sarafotoxins, we performed molecular dynamics simulations of the human ETB and ETA receptors in complex with S6b or S6c. We focused on the receptor interactions with the 9th residues of the sarafotoxins, which are located at the N-terminal end of the a-helical regions. In the S6b-bound ETB receptor, K9 forms a salt bridge with D246ECL2 in ECL2, anchoring the a-helical region to ECL2 (Fig. 4a). In the S6c-bound ETB receptor, D246ECL2 repels E9, displacing the a-helical region toward TM6-7 (Fig. 4b), thereby E9 forms a hydrogen bond with Y247ECL2. Nevertheless, S6b and S6c have similar affinities for the ETB receptor, suggesting that the polar interaction between the 9th residue and receptor is not critical for sarafotoxin binding. Instead, the extensive hydrophobic residues in ECL2 play an important role in sarafotoxin binding (Fig. 4a and b). However, the hydrophobic residues L252ECL2 and I254ECL2 in the ETB receptor are replaced with the polar residues H236ECL2 and T238ECL2 in the ETA receptor, respectively. These replacements would reduce the hydrophobic interactions with the sarafotoxin ahelical region. Instead, in the S6b-bound ETA receptor, R232ECL2 forms a salt bridge with E10 (Fig. 4c), and the N-terminal residues of the ETA receptor cover the a-helical region. These interactions compensate for the weaker hydrophobic interactions with the ahelical region and allow the high-affinity binding of S6b. In the S6cbound ETA receptor, E9 forms an ionic interaction with R232ECL2 together with E10 (Fig. 4d), but still repels E230ECL2. Due to the interaction, the N-terminal residues of the ETA receptor cannot cover the a-helical region of S6c, thus opening up the ligand binding pocket. Overall, E9 in S6c may rearrange the interaction over the a-helical region, consequently destabilize the interaction with the ETA receptor, and reduce the affinity.
4. Discussion Our study determined the crystal structure of the S6b-bound ETB receptor and elucidated that S6b structurally mimics the endothelins and activates the receptor. The different residues between S6b and the endothelins form the additional contacts with
Coordinates and structure factors have been deposited in the Protein Data Bank, under the accession number 6LRY. The raw X-ray diffraction images are also available at Zenodo (https://doi.org/10. 5281/zenodo.3603541). All other data are available from the authors upon reasonable request. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The diffraction experiments were performed at SPring-8 BL32XU (proposal 2016A2527). We thank the beamline staff at BL32XU of SPring-8 (Sayo, Japan) for technical assistance during data collection, and K. Yamashita for the assistance of the PDB deposition and uploading the raw data. This research was partially supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under grant number JP19am0101070 (support number 1628). This work was also supported by grants from the Platform for Drug Discovery, Informatics and Structural Life Science by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), JSPS KAKENHI grants 16H06294 (O.N.), 17J30010, 30809421 (W.S.), 19J13421 (T.I.), and 17J02425 (H.M.). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.091. References [1] M. Yanagisawa, H. Kurihara, S. Kimura, A novel potent vasoconstrictor peptide produced by vascular endothelial cells, Nature 332 (1988) 411e415. [2] J.J. Maguire, A.P. Davenport, Endothelin@25 - new agonists, antagonists, inhibitors and emerging research frontiers: IUPHAR Review 12, Br. J. Pharmacol. 171 (2014) 5555e5572. [3] H. Arai, S. Hori, I. Aramori, H. Ohkubo, S. Nakanishi, Cloning and expression of a cDNA encoding an endothelin receptor, Nature 348 (1990) 730e732. [4] T. Sakurai, et al., Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor, Nature 348 (1990) 732e735. [5] G. Remuzzi, N. Perico, A. Benigni, New therapeutics that antagonize endothelin: promises and frustrations, Nat. Rev. Drug Discov. 1 (2002) 986e1001. [6] L.J. Rubin, et al., Bosentan therapy for pulmonary arterial hypertension, N. Engl. J. Med. 346 (2002) 896e903. [7] W. Shihoya, et al., Activation mechanism of endothelin ET B receptor by
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Please cite this article as: T. Izume et al., Crystal structure of human endothelin ETB receptor in complex with sarafotoxin S6b, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.091
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Crystal structure of human endothelin ETB receptor in complex with sarafotoxin S6b Tamaki Izume, Hirotake Miyauchi, Wataru Shihoya**, Osamu Nureki* Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo, Tokyo, 113-0033, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 December 2019 Accepted 27 December 2019 Available online xxx
Sarafotoxins (SRTXs) are endothelin-like peptides extracted from snake venom. SRTXs stimulate the endothelin ETA and ETB receptors and enhance vasoconstriction, followed by left ventricular dysfunction and bronchoconstriction. SRTXs include four major isopeptides, S6a-d, with different subtype selectivities. Here, we report the crystal structure of the human ETB receptor in complex with the non-selective sarafotoxin S6b at 3.0 Å resolution. This structure reveals the similarities and differences between the binding modes of the endothelins and S6b. Moreover, molecular dynamics simulations based on the S6bbound receptor provides structural insight into the subtype selectivity of the sarafotoxins. Our study clarifies the recognition mechanism of the endothelin-like peptide families. © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: Crystal structure GPCR Toxin
1. Introduction Endothelin-1 (ET-1) is a 21 amino-acid peptide hormone with potent vasoconstrictor activity [1]. ET-1 and its related isopeptides, ET-2 and ET-3, perform several physiological functions in neural crest development, cell proliferation, sodium excretion, salt homeostasis, and regulation of vascular tone and cell growth [2]. Endothelins stimulate the two endothelin receptor subtypes (ETA and ETB) [3,4], which share approximately 60% sequence similarity and belong to the class A G-protein-coupled receptors (GPCRs). The endothelin receptors are widely expressed in the vascular endothelium, brain, and other circulatory organs. ETB is expressed in the vascular endothelium and its stimulation induces nitric oxidemediated vasorelaxation, while ETA and ETB are both expressed in the vascular smooth muscle and their stimulation leads to potent and long-lasting vasoconstriction. Therefore, systemic administration of ET-1 causes transient vasodilation (initial endothelial ETB activation) and hypotension, followed by prolonged vasoconstriction and hypertension (smooth muscle ETA and ETB activation). ET1 is one of the most potent vasoconstrictors and is highly toxic (LD50 for mice is about 0.015 mg/kg body weight). The upregulation of ET-1 is significantly related to circulatory-system
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (W. Shihoya), [email protected] (O. Nureki).
diseases [5], including pulmonary arterial hypertension (PAH) [6]. Thus, endothelin receptor antagonists have been developed for the treatment of circulatory system diseases. Recently, the structures of the human ETB receptor have been determined in complex with agonists ET-1 and ET-3, [7,8], a partial agonist IRL1620, an antagonist bosentan [9], and inverse agonists K-8794 and IRL2500 [10]. Notably, the 2.0 Å resolution structure of the ET-3-bound receptor elucidated the detailed activation process of the receptor. Sarafotoxins (SRTXs) are a group of toxins present in a venom of Atractaspididae family of snakes [11e13], and share high structural and functional homology with the endothelins. SRTXs include the four major isopeptides S6a-d, composed of 21 amino acids. SRTXs stimulate the endothelin receptors and increase vasoconstriction, which is followed by left ventricular dysfunction, bronchoconstriction, and increase of the airway resistance. S6b binds to the ETA and ETB receptors with similar affinities to ET-1, and thus are highly lethal by causing cardiac arrest and death in mice within minutes of intravenous administration (LD50 for mice is about 0.015 mg/kg body weight) [13]. S6b is the most potent sarafotoxin. By contrast, S6c shows 100- to 10,000-fold reduced affinity for the ETA receptor and thus functions as an ETB-selective agonist. Therefore, S6c shows the modest toxicity as compared with S6b (LD50 for mice is about 0.30 mg/kg body weight). Moreover, S6b possesses modest matrix metalloproteinase inhibitory activity, because S6b shares a common fold with the core region of the tissue inhibitors of metalloproteinases (TIMPs) [14]. A number of ETB structures have been determined; however, the binding modes and selectivities of the sarafotoxins remained to be elucidated. Here we report the crystal
https://doi.org/10.1016/j.bbrc.2019.12.091 0006-291X/© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Please cite this article as: T. Izume et al., Crystal structure of human endothelin ETB receptor in complex with sarafotoxin S6b, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.091
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structure of the sarafotoxin S6b-bound human ETB receptor at 3.0 Å resolution. This structure revealed the binding mode of sarafotoxins and the structural basis for their subtype selectivities.
micromounts (MiTeGen) or LithoLoops (Protein Wave) and frozen in liquid nitrogen, without adding any extra cryoprotectant. 2.3. Data collection and structure determination
2. Material and method 2.1. Expression and purification The haemagglutinin signal peptide, followed by the Flag epitope tag (DYKDDDDK) and a nine-amino-acid linker, was added to the Nterminus of the receptor, and a tobacco etch virus (TEV) protease recognition sequence was introduced between G57 and L66, to remove the disordered N-terminus during the purification process. The C-terminus was truncated after S407, and three cysteine residues were mutated to alanine (C396A, C400A, and C405A) to avoid heterogeneous palmitoylation. To improve crystallogenesis, we introduced four thermostabilizing mutations R124Y1.55, D154A2.57, K270A5.35, DS342A6.54 and I381A7.48 (superscripts indicate BallesteroseWeinstein numbers) and inserted T4 lysozyme into intracellular loop 3, between L3035.68 and L3116.23 (ETB-Y5-T4L) [15]. The thermostabilized construct ETB-Y5-T4L was subcloned into a modified pFastBac vector, with the resulting construct encoding a TEV cleavage site followed by a GFP-His10 tag at the C-terminus. The recombinant baculovirus was prepared using the Bac-to-Bac baculovirus expression system (Invitrogen). Sf9 insect cells were infected with the virus at a cell density of 4.0 106 cells per millilitre in Sf900 II medium, and grown for 48 h at 27 C. The harvested cells were disrupted by sonication, in buffer containing 20 mM Tris-HCl, pH 7.5, and 20% glycerol. The crude membrane fraction was collected by ultracentrifugation at 180,000g for 1 h. The membrane fraction was solubilized in buffer, containing 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1% DDM, 0.2% cholesterol hemisuccinate (CHS), and 2 mg ml1 iodoacetamide, for 1 h at 4 C. The supernatant was separated from the insoluble material by ultracentrifugation at 180,000g for 20 min, and incubated with TALON resin (Clontech) for 30 min. The resin was washed with ten column volumes of buffer, containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% LMNG, 0.01% CHS, and 15 mM imidazole. The receptor was eluted in buffer, containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.01% LMNG, 0.001% CHS, and 200 mM imidazole. The eluate was treated with TEV protease and dialysed against buffer (20 mM Tris-HCl, pH 7.5 and 500 mM NaCl). The cleaved GFPeHis10 tag and the TEV protease were removed with the TALON resin. The receptor was concentrated and loaded onto a Superdex200 10/300 Increase size-exclusion column, equilibrated in buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.01% LMNG, and 0.001% CHS. Peak fractions were pooled, concentrated to 40 mg ml1 using a centrifugal filter device (Millipore 50 kDa MW cutoff), and frozen until crystallization. During the concentration, S6b was added to a final concentration of 100 mM.
X-ray diffraction data were collected at the SPring-8 beamline BL32XU, with 10 15 mm2 (width height) micro-focused beams and an EIGER X 9 M detector (Dectris). We manually collected 32 data sets (10 per crystal), and the collected images were automatically processed with KAMO [17] (https://github.com/ keitaroyam/yamtbx). Each data set was indexed and integrated with XDS [18] and then subjected to a hierarchical clustering analysis based on the unit cell parameters using BLEND [19]. After the rejection of outliers, 16 data sets were finally merged with XSCALE [18]. The S6b-bound structure was determined by molecular replacement with PHASER [20], using the ET-3-bound ETB structure (PDB code: 6IGK). Subsequently, the model was rebuilt and refined using COOT [21] and PHENIX [22], respectively. The final model of the S6b-bound ETB-Y5-T4L contained residues 86e210, 214e303, and 311e403 of ETB, 1e160 of T4L, and s6b. The model quality was assessed by MolProbity [23]. Figures were prepared using CueMol (http://www.cuemol.org/ja/). 2.4. Simulation system setup The initial structures of S6b and S6c-bound receptors were prepared by using the program MODELLER [24] and the N-and Cterminus were truncated as observed in the crystal structure of the ETB-S6b complex. All MD simulations were performed with NAMD2.12 [25]. The simulation system was set at the condition of 96 96 96 Å3 with 1-Palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) membrane bilayer, 150 mM NaCl, and TIP3 water molecules. The missing hydrogen atoms were modified by the psfgen plugin of VMD [26]. The disulfide bonds and protonation states were properly set as the physiological condition. The net charge was neutralized by adding Cl-ions. The molecular topology and force field parameter from Charmm36 [27,28] were used for all simulations. The simulation systems were energy minimized for 1,000 steps with fixed positions of the non-hydrogen atoms. After minimization, another 1,000 steps of energy minimization with 10 kcal mol1 restraints for the non-hydrogen atoms, except for the lipid molecules within 5.0 Å from the proteins. Next, equilibrations were performed for 0.1 ns under NVT conditions, with 10 kcal mol1 Å2 restraints for the heavy atoms of the proteins. Finally, equilibration was performed for 1.0 ns under NPT conditions with the 1.0 kcal mol1 Å2 restraints for all Ca atoms of the proteins. The production runs of the equilibrium simulations were performed for 50 ns without restraints, maintaining the constant temperature at 310 K using Langevin dynamics and the -Hoover Langevin piston [29]. constant pressure at 1 atm using Nose The long-range electrostatic interactions were calculated by the particle mesh Ewald method [30].
2.2. Crystallization 3. Results The purified receptor was reconstituted into molten lipid (monoolein and cholesterol 10:1 by mass) at a weight ratio of 1:1.5 (protein:lipid). The protein-laden mesophase was dispensed into 96-well glass plates in 30 nl drops and overlaid with 800 nl precipitant solution by a Gryphon LCP robot (Art Robbins Instruments) [16]. Crystals of ETB-Y5-T4L bound to S6b were grown at 20 C in precipitant conditions containing 30% PEG600, 100 mM Citrate, pH 5.0, 100 mM ammonium sulfate. Crystals of ETB-Y5-T4L bound to S6c were grown at 20 C in precipitant conditions containing 30% PEG500dme, 100 mM Tris, pH 7.8, 100 mM potassium thiocyanate. The crystals were harvested directly from the LCP using
3.1. Overall structure For crystallization, we used the thermostabilized ETB receptor (ETB-Y5), and inserted T4L into ICL3 as described previously (ETBY5-T4L) [7]. Using in meso crystallization, we obtained crystals of ETB-Y5-T4L in complex with S6b or S6c. The crystals of the S6cbound receptor were tiny and diffracted X-rays to a maximum of only 7.0 Å resolution (Supplementary Fig. 1a). The crystals of the S6b-bound receptor grew to their full size (20 20 20 mm3) and diffracted X-rays to a maximum of 3.0 Å resolution (Supplementary
Please cite this article as: T. Izume et al., Crystal structure of human endothelin ETB receptor in complex with sarafotoxin S6b, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.091
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Fig. 1b). Therefore, 32 datasets were collected for the S6b-bound receptor and 16 datasets were merged by the data processing system KAMO. Eventually, we determined the ETB structures in complex with S6b at 3.0 Å resolution, by molecular replacement using the ET-3-bound receptor (PDB 6IGK) [8] (Supplementary Table 1). The ETB receptor consists of the canonical 7 transmembrane helices (TM), the amphipathic helix 8 at the C-terminus (H8), and two antiparallel b-strands in the extracellular loop 2 (ECL2), as in the previously determined ETB structures (Fig. 1a). The electron density of S6b was clearly observed in the ligand binding pocket (Supplementary Fig. 1c). The overall structure is essentially similar to those of the ET-1- and ET-3-bound receptors (Fig. 1b). However, there is a remarkable difference on the intracellular side. In the S6b-bound receptor, the intracellular end of TM5 is opened up by 4 Å, as compared with those in the ET-1- and ET-3-bound receptors (Fig. 1c). A number of previous studies have shown that the intracellular portion of TM6 moves outwardly by 10e14 Å upon Gprotein coupling [31]. Moreover, in NTSR1 (neurotensin receptor type 1), agonist and G-protein binding induce the outward displacement of TM5 by 2e4 Å [32] (Fig. 1d). Although T4L insertion affects the conformations of TM5 and TM6, agonist binding may cause the outward displacement of the TM5 in the ETB receptor, as in NTSR1. The previous ETB structures revealed that the agonist binding disrupts the hydrogen-bonding network between W3366.48 and D1472.50, which stabilizes the inactive conformation by connecting TMs 2, 3, 6, and 7 at the receptor core [8] (Fig. 2a and b). In the S6bbound receptor, W3366.48 rotates inwardly as compared with that in the K-8794-bound inactive receptor, and forms a hydrogen bond with N3787.45 (Fig. 2c). D1472.50 forms hydrogen bonding interactions with N1191.50 and T1883.39. Therefore, S6b binding also induces the rearrangement of the hydrogen-bonding interaction in the receptor core, as similarly to the endothelin ligands (Fig. 2c and d).
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3.2. Sarafotoxin S6b binding site We next describe the detailed interactions between S6b and ETB. 8 residues in S6b are different from those in ET-1 and ET-3; however, 4 cysteines are completely conserved (Fig. 3a). Thus, S6b adopts a similar bicyclic architecture, comprising the N-terminal region (residues 1e7), a-helical region (residues 8e17), and C-terminal region (residues 18e21). The N-terminal region is attached to the central a-helical region by the intrachain disulfide bond pairs (C1eC15 and C3eC11) (Fig. 3b). A previous NMR study of S6b revealed that the N-terminal and a-helical regions form a stable conformation in solution, whereas the C-terminal region has a conformational flexibility (Fig. 3c). Therefore, the C-terminal region adopts its fixed conformation by the interaction with the receptor, as in the other endothelin ligands. S6b superimposes well with ET-1 and ET-3 (R.M.S.D. values for Ca ¼ 0.39 and 0.40 Å, respectively) (Fig. 3d). Despite the sequence differences, S6b highly mimics the endothelins and activates the receptor. Next we describe the differences in the binding modes of S6b, ET-1, and ET-3. In the N-terminal region, 5 residues of S6b are different from those of ET-1 and ET-3. As in the endothelin-bound structures, the N-terminal region of S6b is exposed to the solvent and thus the receptor can tolerate the sequence difference (Fig. 3eeh). Notably, K4, D5, and T7 form an intramolecular hydrogen-bonding network in S6b, and the Q352ECL3 side chain participates in this network and form a hydrogen bond with D5 (Fig. 3e). V12 in ET-1 and ET-3 is replaced with L12 in S6b, forming more extensive hydrophobic interactions with ECL2 (Fig. 3i). L17 in ET-1 and ET-3 is replaced with the polar residue Q17 in S6b. Instead of the hydrophobic interaction, Q17 forms a hydrogen bond with E165ECL1 (Fig. 3j). I19 in ET-1 and ET-3 is replaced with V19, forming similar hydrophobic interactions (Fig. 3k). These differences are subtle, and thus the conserved residues among S6b, ET-1, and ET-3 form essentially similar interactions with the receptor
Fig. 1. Overall structure of the S6b-bound ETB receptor. a, Overall structure of the S6b-bound ETB receptor, viewed from the membrane plane (left) and extracellular side (right). The receptor and T4L are colored purple and grey, respectively. S6b is shown as a transparent surface representation and a ribbon model, with its N-terminal region colored cyan, a-helical region orange, and C-terminal region deep pink. b, c, Superimposed ETB structures in complex with S6b (purple), ET-1 (pink), and ET-3 (orange), viewed from the membrane plane (b) and intracellular side (c). d, Intracellular views of the superimposed structures of the C-state hNTSR1 in complex with Gi1 (blue), active-state rNTSR1 (green), and inactive-state rNTSR1 (grey).
Please cite this article as: T. Izume et al., Crystal structure of human endothelin ETB receptor in complex with sarafotoxin S6b, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.091
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Fig. 2. Comparisons of the receptor core regions. a-c, Receptor core interactions in the K-8794- (a, blue), ET-3- (b, orange), and S6b- (c, purple) bound ETB receptors. The receptors are shown as ribbon representations, and the side chains of N1191.50, D1472.50, T1883.39, F3326.44, W3366.48, and N3787.45 are shown as stick models. Waters are shown as red spheres. The dashed lines show hydrogen bonds colored according to the respective structures. d, Superimposition of the receptor core interactions.
(Supplementary Figs. 2aec). 3.3. Insight into the subtype selectivity of sarafotoxins S6b and S6c have similar affinities towards the ETB receptor, whereas S6c has reduced affinity for the ETA receptor by about 100-
to 10,000-fold [11]. Between S6b and S6c, three residues are diverged (S2T, K9E, and Y13N) (Fig. 3a). Moreover, a previous study showed that [E9]S6b functions as a ETB-selective agonist [33] and is approximately equipotent with S6c, suggesting that the charge of the 9th residues in the sarafotoxins determines the subtype selectivity of the sarafotoxins. To obtain structural insight into the
Fig. 3. Interactions between S6b and ETB. a, Comparison of the amino acid sequences of ET-1, ET-3, S6b, and S6c. b, c, Comparison of the S6b structures in the complex (b) and solution (c). d, Superimposition of S6b (purple), ET-1 (pink), and ET-3 (orange). e-g, Interactions between the N-terminal region and the receptor in the S6b- (e), ET-1- (f), and ET-3- (g) bound structures. h, Superimposed structures of the S6b-, ET-1-, and ET-3-bound receptors. i-k, Superimposition of the S6b-, ET-1-, and ET-3-bound structures, focused on residues 12 (i), 17 (j), and 19 (k) of the peptide ligands.
Please cite this article as: T. Izume et al., Crystal structure of human endothelin ETB receptor in complex with sarafotoxin S6b, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.091
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TM6 and ECL3 in the S6b-bound receptor. The intracellular side of the S6b-bound receptor moves outwardly as compared with those of the endothelin-bound receptors. This observation suggests that TM5 adopt more active conformation for G-protein coupling, as in NTSR1. Moreover, the MD simulations based on the S6bbound ETB structure provides structural clues for the ETB-selectivity of S6c. Author contributions T.I. expressed, purified, and crystallized the S6b-bound ETB receptor, collected data, and refined the structures. H.M. performed the molecular dynamics simulation. W.S. designed the study and mainly wrote the manuscript with T.I. W.S. and O.N. supervised the research. Data availability
Fig. 4. Models of the sarafotoxin-bound endothelin receptor. a-d, Models of the human ETA and ETB receptors in complex with S6b or S6c, obtained by molecular dynamics simulations. Interactions between sarafottoxins and ECL2 and N-termini of the receptors are shows as sticks.
subtype selectivity of the sarafotoxins, we performed molecular dynamics simulations of the human ETB and ETA receptors in complex with S6b or S6c. We focused on the receptor interactions with the 9th residues of the sarafotoxins, which are located at the N-terminal end of the a-helical regions. In the S6b-bound ETB receptor, K9 forms a salt bridge with D246ECL2 in ECL2, anchoring the a-helical region to ECL2 (Fig. 4a). In the S6c-bound ETB receptor, D246ECL2 repels E9, displacing the a-helical region toward TM6-7 (Fig. 4b), thereby E9 forms a hydrogen bond with Y247ECL2. Nevertheless, S6b and S6c have similar affinities for the ETB receptor, suggesting that the polar interaction between the 9th residue and receptor is not critical for sarafotoxin binding. Instead, the extensive hydrophobic residues in ECL2 play an important role in sarafotoxin binding (Fig. 4a and b). However, the hydrophobic residues L252ECL2 and I254ECL2 in the ETB receptor are replaced with the polar residues H236ECL2 and T238ECL2 in the ETA receptor, respectively. These replacements would reduce the hydrophobic interactions with the sarafotoxin ahelical region. Instead, in the S6b-bound ETA receptor, R232ECL2 forms a salt bridge with E10 (Fig. 4c), and the N-terminal residues of the ETA receptor cover the a-helical region. These interactions compensate for the weaker hydrophobic interactions with the ahelical region and allow the high-affinity binding of S6b. In the S6cbound ETA receptor, E9 forms an ionic interaction with R232ECL2 together with E10 (Fig. 4d), but still repels E230ECL2. Due to the interaction, the N-terminal residues of the ETA receptor cannot cover the a-helical region of S6c, thus opening up the ligand binding pocket. Overall, E9 in S6c may rearrange the interaction over the a-helical region, consequently destabilize the interaction with the ETA receptor, and reduce the affinity.
4. Discussion Our study determined the crystal structure of the S6b-bound ETB receptor and elucidated that S6b structurally mimics the endothelins and activates the receptor. The different residues between S6b and the endothelins form the additional contacts with
Coordinates and structure factors have been deposited in the Protein Data Bank, under the accession number 6LRY. The raw X-ray diffraction images are also available at Zenodo (https://doi.org/10. 5281/zenodo.3603541). All other data are available from the authors upon reasonable request. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The diffraction experiments were performed at SPring-8 BL32XU (proposal 2016A2527). We thank the beamline staff at BL32XU of SPring-8 (Sayo, Japan) for technical assistance during data collection, and K. Yamashita for the assistance of the PDB deposition and uploading the raw data. This research was partially supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under grant number JP19am0101070 (support number 1628). This work was also supported by grants from the Platform for Drug Discovery, Informatics and Structural Life Science by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), JSPS KAKENHI grants 16H06294 (O.N.), 17J30010, 30809421 (W.S.), 19J13421 (T.I.), and 17J02425 (H.M.). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.091. References [1] M. Yanagisawa, H. Kurihara, S. Kimura, A novel potent vasoconstrictor peptide produced by vascular endothelial cells, Nature 332 (1988) 411e415. [2] J.J. Maguire, A.P. Davenport, Endothelin@25 - new agonists, antagonists, inhibitors and emerging research frontiers: IUPHAR Review 12, Br. J. Pharmacol. 171 (2014) 5555e5572. [3] H. Arai, S. Hori, I. Aramori, H. Ohkubo, S. Nakanishi, Cloning and expression of a cDNA encoding an endothelin receptor, Nature 348 (1990) 730e732. [4] T. Sakurai, et al., Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor, Nature 348 (1990) 732e735. [5] G. Remuzzi, N. Perico, A. Benigni, New therapeutics that antagonize endothelin: promises and frustrations, Nat. Rev. Drug Discov. 1 (2002) 986e1001. [6] L.J. Rubin, et al., Bosentan therapy for pulmonary arterial hypertension, N. Engl. J. Med. 346 (2002) 896e903. [7] W. Shihoya, et al., Activation mechanism of endothelin ET B receptor by
Please cite this article as: T. Izume et al., Crystal structure of human endothelin ETB receptor in complex with sarafotoxin S6b, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.091
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Please cite this article as: T. Izume et al., Crystal structure of human endothelin ETB receptor in complex with sarafotoxin S6b, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.091