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Structural basis for ligand recognition of the human thromboxane A2 receptor

Abstract

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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Fig. 1: Overall structures of TP–ramatroban and TP–daltroban complexes.
Fig. 2: Extracellular loops in the structures of lipid receptors in extracellular view.
Fig. 3: Binding modes of ramatroban and daltroban in TP.
Fig. 4: Docking poses of prostanoid-like ligands SQ-29548 and U46619 in TP.

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Data availability

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.

References

  1. 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).

    Article  CAS  PubMed  Google Scholar 

  2. Hirata, T. & Narumiya, S. Prostanoid receptors. Chem. Rev. 111, 6209–6230 (2011).

    Article  CAS  PubMed  Google Scholar 

  3. FitzGerald, G. A. Mechanisms of platelet activation: thromboxane A2 as an amplifying signal for other agonists. Am. J. Cardiol. 68, 11B–15B (1991).

    Article  CAS  PubMed  Google Scholar 

  4. Brass, L. F., Zhu, L. & Stalker, T. J. Minding the gaps to promote thrombus growth and stability. J. Clin. Invest. 115, 3385–3392 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kinsella, B. T. Thromboxane A2 signalling in humans: a ‘tail’ of two receptors. Biochem. Soc. Trans. 29, 641–654 (2001).

  6. 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).

    Article  CAS  PubMed  Google Scholar 

  7. Meadows, T. A. & Bhatt, D. L. Clinical aspects of platelet inhibitors and thrombus formation. Circ. Res. 100, 1261–1275 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. Navarro-Núñez, L. et al. Thromboxane A2 receptor antagonism by flavonoids: structure-activity relationships. J. Agric. Food Chem. 57, 1589–1594 (2009).

    Article  CAS  PubMed  Google Scholar 

  9. Gomoll, A. W. & Ogletree, M. L. Failure of aspirin to interfere with the cardioprotective effects of ifetroban. Eur. J. Pharmacol. 271, 471–479 (1994).

    Article  CAS  PubMed  Google Scholar 

  10. 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).

    CAS  PubMed  Google Scholar 

  11. 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).

    CAS  PubMed  Google Scholar 

  12. 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).

    Article  CAS  PubMed  Google Scholar 

  13. 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).

    Article  CAS  PubMed  Google Scholar 

  14. 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).

    CAS  PubMed  Google Scholar 

  15. 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).

  16. Okubo, K. et al. Japanese guideline for allergic rhinitis. Allergol. Int. 60, 171–189 (2011).

    Article  CAS  PubMed  Google Scholar 

  17. 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).

    Article  CAS  PubMed  Google Scholar 

  18. Chun, E. et al. Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors. Structure 20, 967–976 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 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).

    Article  CAS  Google Scholar 

  20. Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nat. Protoc. 4, 706–731 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 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).

    Article  CAS  PubMed  Google Scholar 

  22. Hanson, M. A. et al. Crystal structure of a lipid G protein-coupled receptor. Science 335, 851–855 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Srivastava, A. et al. High-resolution structure of the human GPR40 receptor bound to allosteric agonist TAK-875. Nature 513, 124–127 (2014).

    Article  CAS  PubMed  Google Scholar 

  24. Chrencik, J. E. et al. Crystal structure of antagonist bound human lysophosphatidic acid receptor 1. Cell 161, 1633–1643 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hua, T. et al. Crystal structure of the human cannabinoid receptor CB1. Cell 167, 750–762.e714 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Cao, C. et al. Structural basis for signal recognition and transduction by platelet-activating-factor receptor. Nat. Struct. Mol. Biol. 25, 488–495 (2018).

    Article  CAS  PubMed  Google Scholar 

  27. 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).

    Article  CAS  PubMed  Google Scholar 

  28. 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).

    Article  CAS  PubMed  Google Scholar 

  29. 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).

  30. 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).

    Article  CAS  PubMed  Google Scholar 

  31. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  32. 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).

    Article  CAS  PubMed  Google Scholar 

  33. 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).

    Article  CAS  PubMed  Google Scholar 

  34. 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).

    Article  CAS  PubMed  Google Scholar 

  35. 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).

    Article  CAS  PubMed  Google Scholar 

  36. Shinozaki, K. et al. Synthesis and thromboxane A2 antagonistic activity of indane derivatives. Bioorg. Med. Chem. Lett. 9, 401–406 (1999).

    Article  CAS  PubMed  Google Scholar 

  37. Katritch, V., Cherezov, V. & Stevens, R. C. Structure-function of the G protein-coupled receptor superfamily. Annu. Rev. Pharmacol. Toxicol. 53, 531–556 (2013).

    Article  CAS  PubMed  Google Scholar 

  38. Venkatakrishnan, A. J. et al. Molecular signatures of G-protein-coupled receptors. Nature 494, 185–194 (2013).

    Article  CAS  PubMed  Google Scholar 

  39. Cimetière, B. et al. Synthesis and biological evaluation of new tetrahydronaphthalene derivatives as thromboxane receptor antagonists. Bioorg. Med. Chem. Lett. 8, 1375–1380 (1998).

    Article  PubMed  Google Scholar 

  40. 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).

    Article  CAS  PubMed  Google Scholar 

  41. 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).

    Article  CAS  Google Scholar 

  42. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 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).

    Article  CAS  PubMed  Google Scholar 

  44. 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).

  45. 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).

    Article  CAS  PubMed  Google Scholar 

  46. 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).

    Article  CAS  PubMed  Google Scholar 

  47. Laskowski, R. A. & Swindells, M. B. LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J. Chem. Inf. Model. 51, 2778–2786 (2011).

    Article  CAS  PubMed  Google Scholar 

  48. Kabsch, W. Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Google Scholar 

  51. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. 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).

    Article  CAS  PubMed  Google Scholar 

  54. 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).

    Article  CAS  PubMed  Google Scholar 

  55. 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).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

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.

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H.F. optimized the construct, developed the purification procedure, purified the TP receptors for crystallization and performed crystallization trials and ligand-binding assays. S.C. performed signaling assays. X.Y. performed molecular docking. S.H. solved the structures. H.Z. collected X-ray diffraction data. W.X. helped to optimize the receptor. Y.X. oversaw molecular docking. B.W. and Q.Z. initiated the project, planned and analyzed experiments, supervised the research and wrote the manuscript with input from all co-authors.

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Correspondence to Qiang Zhao or Beili Wu.

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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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