Stimulated by thromboxane A2, an endogenous arachidonic acid metabolite, the thromboxane A2 receptor (TP) plays a pivotal role in cardiovascular homeostasis, and thus is considered as an important drug target for cardiovascular disease. Here, we report crystal structures of the human TP bound to two nonprostanoid antagonists, ramatroban and daltroban, at 2.5 Å and 3.0 Å resolution, respectively. The TP structures reveal a ligand-binding pocket capped by two layers of extracellular loops that are stabilized by two disulfide bonds, limiting ligand access from the extracellular milieu. These structures provide details of interactions between the receptor and antagonists, which help to integrate previous mutagenesis and SAR data. Molecular docking of prostanoid-like ligands, combined with mutagenesis, ligand-binding and functional assays, suggests a prostanoid binding mode that may also be adopted by other prostanoid receptors. These insights into TP deepen our understanding about ligand recognition and selectivity mechanisms of this physiologically important receptor.
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Atomic coordinates and structure factor files for the TP–ramatroban and TP–daltroban complex structures have been deposited in the Protein Data Bank (PDB) with accession codes 6IIU and 6IIV, respectively. All other data generated or analyzed during this study are included in this published article and its supplementary information file or are available from the corresponding authors on reasonable request.
Woodward, D. F., Jones, R. L. & Narumiya, S. International union of basic and clinical pharmacology. lxxxiii: classification of prostanoid receptors, updating 15 years of progress. Pharmacol. Rev. 63, 471–538 (2011).
Hirata, T. & Narumiya, S. Prostanoid receptors. Chem. Rev. 111, 6209–6230 (2011).
FitzGerald, G. A. Mechanisms of platelet activation: thromboxane A2 as an amplifying signal for other agonists. Am. J. Cardiol. 68, 11B–15B (1991).
Brass, L. F., Zhu, L. & Stalker, T. J. Minding the gaps to promote thrombus growth and stability. J. Clin. Invest. 115, 3385–3392 (2005).
Kinsella, B. T. Thromboxane A2 signalling in humans: a ‘tail’ of two receptors. Biochem. Soc. Trans. 29, 641–654 (2001).
Patrono, C., García Rodríguez, L. A., Landolfi, R. & Baigent, C. Low-dose aspirin for the prevention of atherothrombosis. N. Engl. J. Med. 353, 2373–2383 (2005).
Meadows, T. A. & Bhatt, D. L. Clinical aspects of platelet inhibitors and thrombus formation. Circ. Res. 100, 1261–1275 (2007).
Navarro-Núñez, L. et al. Thromboxane A2 receptor antagonism by flavonoids: structure-activity relationships. J. Agric. Food Chem. 57, 1589–1594 (2009).
Gomoll, A. W. & Ogletree, M. L. Failure of aspirin to interfere with the cardioprotective effects of ifetroban. Eur. J. Pharmacol. 271, 471–479 (1994).
Ogletree, M. L., Harris, D. N., Greenberg, R., Haslanger, M. F. & Nakane, M. Pharmacological actions of SQ 29,548, a novel selective thromboxane antagonist. J. Pharmacol. Exp. Ther. 234, 435–441 (1985).
Patscheke, H. et al. Inhibitory effects of the selective thromboxane receptor antagonist BM 13.177 on platelet aggregation, vasoconstriction and sudden death. Biomed. Biochim. Acta 43, S312–S318 (1984).
Tanaka, T. et al. Antiplatelet effect of Z-335, a new orally active and long-lasting thromboxane receptor antagonist. Eur. J. Pharmacol. 357, 53–60 (1998).
Ting, H. J., Murad, J. P., Espinosa, E. V. & Khasawneh, F. T. Thromboxane A2 receptor: biology and function of a peculiar receptor that remains resistant for therapeutic targeting. J. Cardiovasc. Pharmacol. Ther. 17, 248–259 (2012).
Rosentreter, U., Böshagen, H., Seuter, F., Perzborn, E. & Fiedler, V. B. Synthesis and absolute configuration of the new thromboxane antagonist (3R)-3-(4-fluorophenylsulfonamido)-1,2,3,4-tetrahydro-9-carbazolepropan oic acid and comparison with its enantiomer. Arzneimittelforschung 39, 1519–1521 (1989).
Francis, H. P., Greenham, S. J., Patel, U. P., Thompson, A. M. & Gardiner, P. J. BAY u3405 an antagonist of thromboxane A2- and prostaglandin D2-induced bronchoconstriction in the guinea-pig. Br. J. Pharmacol. 104, 596–602 (1991).
Okubo, K. et al. Japanese guideline for allergic rhinitis. Allergol. Int. 60, 171–189 (2011).
Yanagisawa, A., Smith, J. A., Brezinski, M. E. & Lefer, A. M. Mechanism of antagonism of thromboxane receptors in vascular smooth muscle. Eur. J. Pharmacol. 133, 89–96 (1987).
Chun, E. et al. Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors. Structure 20, 967–976 (2012).
Ballesteros, J. & Weinstein, H. Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. Methods Neurosci. 25, 366–428 (1995).
Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nat. Protoc. 4, 706–731 (2009).
Chiang, N., Kan, W. M. & Tai, H. H. Site-directed mutagenesis of cysteinyl and serine residues of human thromboxane A2 receptor in insect cells. Arch. Biochem. Biophys. 334, 9–17 (1996).
Hanson, M. A. et al. Crystal structure of a lipid G protein-coupled receptor. Science 335, 851–855 (2012).
Srivastava, A. et al. High-resolution structure of the human GPR40 receptor bound to allosteric agonist TAK-875. Nature 513, 124–127 (2014).
Chrencik, J. E. et al. Crystal structure of antagonist bound human lysophosphatidic acid receptor 1. Cell 161, 1633–1643 (2015).
Hua, T. et al. Crystal structure of the human cannabinoid receptor CB1. Cell 167, 750–762.e714 (2016).
Cao, C. et al. Structural basis for signal recognition and transduction by platelet-activating-factor receptor. Nat. Struct. Mol. Biol. 25, 488–495 (2018).
Park, J. H., Scheerer, P., Hofmann, K. P., Choe, H. W. & Ernst, O. P. Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454, 183–187 (2008).
Ulven, T. & Kostenis, E. Minor structural modifications convert the dual TP/CRTH2 antagonist ramatroban into a highly selective and potent CRTH2 antagonist. J. Med. Chem. 48, 897–900 (2005).
Ballatore, C. et al. Cyclopentane-1,3-dione: a novel isostere for the carboxylic acid functional group. Application to the design of potent thromboxane (A2) receptor antagonists. J. Med. Chem. 54, 6969–6983 (2011).
Tai, H. H., Huang, C. & Chiang, N. Structure and function of prostanoid receptors as revealed by site-directed mutagenesis. Adv. Exp. Med. Biol. 407, 205–209 (1997).
Neuschäfer-Rube, F., Engemaier, E., Koch, S., Böer, U. & Püschel, G. P. Identification by site-directed mutagenesis of amino acids contributing to ligand-binding specificity or signal transduction properties of the human FP prostanoid receptor. Biochem. J. 371, 443–449 (2003).
Audoly, L. & Breyer, R. M. Substitution of charged amino acid residues in transmembrane regions 6 and 7 affect ligand binding and signal transduction of the prostaglandin EP3 receptor. Mol. Pharmacol. 51, 61–68 (1997).
Stitham, J., Stojanovic, A., Merenick, B. L., O’Hara, K. A. & Hwa, J. The unique ligand-binding pocket for the human prostacyclin receptor. Site-directed mutagenesis and molecular modeling. J. Biol. Chem. 278, 4250–4257 (2003).
So, S. P. et al. Identification of residues important for ligand binding of thromboxane A2 receptor in the second extracellular loop using the NMR experiment-guided mutagenesis approach. J. Biol. Chem. 278, 10922–10927 (2003).
Stillman, B. A., Audoly, L. & Breyer, R. M. A conserved threonine in the second extracellular loop of the human EP2 and EP4 receptors is required for ligand binding. Eur. J. Pharmacol. 357, 73–82 (1998).
Shinozaki, K. et al. Synthesis and thromboxane A2 antagonistic activity of indane derivatives. Bioorg. Med. Chem. Lett. 9, 401–406 (1999).
Katritch, V., Cherezov, V. & Stevens, R. C. Structure-function of the G protein-coupled receptor superfamily. Annu. Rev. Pharmacol. Toxicol. 53, 531–556 (2013).
Venkatakrishnan, A. J. et al. Molecular signatures of G-protein-coupled receptors. Nature 494, 185–194 (2013).
Cimetière, B. et al. Synthesis and biological evaluation of new tetrahydronaphthalene derivatives as thromboxane receptor antagonists. Bioorg. Med. Chem. Lett. 8, 1375–1380 (1998).
Theis, J. G., Dellweg, H., Perzborn, E. & Gross, R. Binding characteristics of the new thromboxane A2/prostaglandin H2 receptor antagonist [3H]BAY U 3405 to washed human platelets and platelet membranes. Biochem. Pharmacol. 44, 495–503 (1992).
Ladouceur, G., Mais, D. E., Jakubowski, J. A., Utterback, B. G. & Robertson, D. W. Structural homologies among thromboxane (TXA2) receptor antagonists - minimal pharmacophoric requirements for high-affinity interaction with TXA2 receptors. Bioorg. Med. Chem. Lett. 1, 173–178 (1991).
McKenniff, M. G., Norman, P., Cuthbert, N. J. & Gardiner, P. J. BAYu3405, a potent and selective thromboxane A2 receptor antagonist on airway smooth muscle in vitro. Br. J. Pharmacol. 104, 585–590 (1991).
Stearns, B. A. et al. Novel tricyclic antagonists of the prostaglandin D2 receptor DP2 with efficacy in a murine model of allergic rhinitis. Bioorg. Med. Chem. Lett. 19, 4647–4651 (2009).
Sugimoto, H. et al. An orally bioavailable small molecule antagonist of CRTH2, ramatroban (BAYu3405), inhibits prostaglandin D2-induced eosinophil migration in vitro. J. Pharmacol. Exp. Ther. 305, 347–352 (2003).
Hata, A. N., Lybrand, T. P. & Breyer, R. M. Identification of determinants of ligand binding affinity and selectivity in the prostaglandin D2 receptor CRTH2. J. Biol. Chem. 280, 32442–32451 (2005).
Abramovitz, M. et al. The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs. Biochim. Biophys. Acta 1483, 285–293 (2000).
Laskowski, R. A. & Swindells, M. B. LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J. Chem. Inf. Model. 51, 2778–2786 (2011).
Kabsch, W. Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
Smart, O. S. et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. D Biol. Crystallogr. 68, 368–380 (2012).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
Sastry, G. M., Adzhigirey, M., Day, T., Annabhimoju, R. & Sherman, W. Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J. Comput. Aided Mol. Des. 27, 221–234 (2013).
Harder, E. et al. OPLS3: a force field providing broad coverage of drug-like small molecules and proteins. J. Chem. Theory Comput. 12, 281–296 (2016).
Koldsø, H. et al. The two enantiomers of citalopram bind to the human serotonin transporter in reversed orientations. J. Am. Chem. Soc. 132, 1311–1322 (2010).
This work was supported by the National Key R&D Program of China 2018YFA0507000 (B.W. and Q.Z.), the Key Research Program of Frontier Sciences, CAS, Grant no. QYZDB-SSW-SMC024 (B.W.) and QYZDB-SSW-SMC054 (Q.Z.), and the National Science Foundation of China grants 31825010 (B.W.) and 81525024 (Q.Z.). We thank R.C. Stevens, K. White, M. Audet and T. James for careful review and scientific feedback on the manuscript. The synchrotron radiation experiments were performed at the BL41XU of SPring-8 with approval of the Japan Synchrotron Radiation Research Institute (proposal no. 2016A2517, 2016A2518, 2016B2517 and 2016B2518). We thank the beamline staff members K. Hasegawa, H. Okumura, N. Mizuno, T. Kawamura and H. Murakami of the BL41XU for help on X-ray data collection.
The authors declare no competing interests.
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Fan, H., Chen, S., Yuan, X. et al. Structural basis for ligand recognition of the human thromboxane A2 receptor. Nat Chem Biol 15, 27–33 (2019). https://doi.org/10.1038/s41589-018-0170-9
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