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Crystal structure of the human angiotensin II type 2 receptor bound to an angiotensin II analog

Abstract

Angiotensin II (AngII) plays a central role in regulating human blood pressure, which is mainly mediated by interactions between AngII and the G-protein-coupled receptors (GPCRs) AngII type 1 receptor (AT1R) and AngII type 2 receptor (AT2R). We have solved the crystal structure of human AT2R binding the peptide ligand [Sar1, Ile8]AngII and its specific antibody at 3.2-Å resolution. [Sar1, Ile8]AngII interacts with both the ‘core’ binding domain, where the small-molecule ligands of AT1R and AT2R bind, and the ‘extended’ binding domain, which is equivalent to the allosteric modulator binding site of muscarinic acetylcholine receptor. We generated an antibody fragment to stabilize the extended binding domain that functions as a positive allosteric modulator. We also identified a signature positively charged cluster, which is conserved among peptide-binding receptors, to locate C termini at the bottom of the binding pocket. The reported results should help with designing ligands for angiotensin receptors and possibly to other peptide GPCRs.

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Fig. 1: Overall structure of AT2R-BRILICL3 in complex with s-AngII and Fab4A03.
Fig. 2: Structural comparison of s-AngII–AT2R with compound 1–AT2R and olmesartan–AT1R.
Fig. 3: s-AngII binding mode to AT2R.
Fig. 4: Similarity in binding mode between s-AngII and ET-1 toward each receptor.
Fig. 5: Effect of Fab4A03 on ligand binding and the interface between AT2R and Fab4A03.

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References

  1. de Gasparo, M., Catt, K. J., Inagami, T., Wright, J. W. & Unger, T. International Union of Pharmacology. XXIII. The angiotensin II receptors. Pharmacol. Rev. 52, 415–472 (2000).

    PubMed  Google Scholar 

  2. Mehta, P. K. & Griendling, K. K. Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system. Am. J. Physiol. Cell. Physiol. 292, C82–C97 (2007).

    Article  PubMed  CAS  Google Scholar 

  3. Karnik, S. S. et al. International Union of Basic and Clinical Pharmacology. XCIX. Angiotensin receptors: interpreters of pathophysiological angiotensinergic stimuli. Pharmacol. Rev. 67, 754–819 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Zaman, M. A., Oparil, S. & Calhoun, D. A. Drugs targeting the renin–angiotensin–aldosterone system. Nat. Rev. Drug Discov. 1, 621–636 (2002).

    Article  PubMed  CAS  Google Scholar 

  5. Fredriksson, R., Lagerström, M. C., Lundin, L. G. & Schiöth, H. B. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups and fingerprints. Mol. Pharmacol. 63, 1256–1272 (2003).

    Article  PubMed  CAS  Google Scholar 

  6. Oliveira, L. et al. The angiotensin II AT1 receptor structure–activity correlations in the light of rhodopsin structure. Physiol. Rev. 87, 565–592 (2007).

    Article  PubMed  CAS  Google Scholar 

  7. Shenoy, S. K. & Lefkowitz, R. J. Angiotensin II–stimulated signaling through G proteins and β-arrestin. Sci. STKE 2005, cm14 (2005).

    PubMed  Google Scholar 

  8. Whalen, E. J., Rajagopal, S. & Lefkowitz, R. J. Therapeutic potential of β-arrestin- and G-protein-biased agonists. Trends Mol. Med. 17, 126–139 (2011).

    Article  PubMed  CAS  Google Scholar 

  9. Berk, B. C. Angiotensin type 2 receptor (AT2R): a challenging twin. Sci. STKE 2003, PE16 (2003).

    PubMed  Google Scholar 

  10. Hein, L., Barsh, G. S., Pratt, R. E., Dzau, V. J. & Kobilka, B. K. Behavioral and cardiovascular effects of disrupting the angiotensin II type 2 receptor in mice. Nature 377, 744–747 (1995).

    Article  PubMed  CAS  Google Scholar 

  11. Ichiki, T. et al. Effects on blood pressure and exploratory behavior of mice lacking angiotensin II type 2 receptor. Nature 377, 748–750 (1995).

    Article  PubMed  CAS  Google Scholar 

  12. Miura, S. & Karnik, S. S. Angiotensin II type 1 and type 2 receptors bind angiotensin II through different types of epitope recognition. J. Hypertens. 17, 397–404 (1999).

    Article  PubMed  CAS  Google Scholar 

  13. Miura, S., Matsuo, Y., Kiya, Y., Karnik, S. S. & Saku, K. Molecular mechanisms of the antagonistic action between AT1 and AT2 receptors. Biochem. Biophys. Res. Commun. 391, 85–90 (2010).

    Article  PubMed  CAS  Google Scholar 

  14. Porrello, E. R. et al. Angiotensin II type 2 receptor antagonizes angiotensin II type 1 receptor–mediated cardiomyocyte autophagy. Hypertension 53, 1032–1040 (2009).

    Article  PubMed  CAS  Google Scholar 

  15. Porrello, E. R., Delbridge, L. M. & Thomas, W. G. The angiotensin II type 2 (AT2) receptor: an enigmatic seven-transmembrane receptor. Front. Biosci. 14, 958–972 (2009).

    Article  CAS  Google Scholar 

  16. Caballero, R. et al. Interaction of angiotensin II with the angiotensin type 2 receptor inhibits the cardiac transient outward potassium current. Cardiovasc. Res. 62, 86–95 (2004).

    Article  PubMed  CAS  Google Scholar 

  17. Ruiz-Ortega, M., Lorenzo, O., Rupérez, M., Blanco, J. & Egido, J. Systemic infusion of angiotensin II into normal rats activates nuclear-factor-κB and AP-1 in the kidney: role of AT1 and AT2 receptors. Am. J. Pathol. 158, 1743–1756 (2001).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Ruiz-Ortega, M. et al. Angiotensin II activates nuclear transcription factor κB through AT1 and AT2 in vascular smooth muscle cells: molecular mechanisms. Circ. Res. 86, 1266–1272 (2000).

    Article  PubMed  CAS  Google Scholar 

  19. Zhao, Y. et al. Angiotensin II induces peroxisome proliferator-activated receptor gamma in PC12W cells via angiotensin type 2 receptor activation. J. Neurochem. 94, 1395–1401 (2005).

    Article  PubMed  CAS  Google Scholar 

  20. Zhang, H. et al. Structural basis for ligand recognition and functional selectivity at angiotensin receptor. J. Biol. Chem. 290, 29127–29139 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Zhang, H. et al. Structure of the angiotensin receptor revealed by serial femtosecond crystallography. Cell 161, 833–844 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Hines, J., Fluharty, S. J. & Yee, D. K. Chimeric AT1/AT2 receptors reveal functional similarities despite key amino acid dissimilarities in the domains mediating agonist-dependent activation. Biochemistry 40, 11251–11260 (2001).

    Article  PubMed  CAS  Google Scholar 

  23. McMullen, J. R., Gibson, K. J., Lumbers, E. R. & Burrell, J. H. 125I[Sar1, Ile8]-angiotensin-II has a different affinity for AT1 and AT2 receptor subtypes in oviane tissues. Regul. Pept. 105, 83–92 (2002).

    Article  PubMed  CAS  Google Scholar 

  24. Miura, S. et al. Molecular mechanism underlying inverse agonist of angiotensin II type 1 receptor. J. Biol. Chem. 281, 19288–19295 (2006).

    Article  PubMed  CAS  Google Scholar 

  25. Bouley, R. et al. N- and C-terminal structure–activity study of angiotensin II on the angiotensin AT2 receptor. Eur. J. Pharmacol. 343, 323–331 (1998).

    Article  PubMed  CAS  Google Scholar 

  26. Le, M. T. et al. Peptide and nonpeptide antagonist interaction with constitutively active human AT1 receptors. Biochem. Pharmacol. 65, 1329–1338 (2003).

    Article  PubMed  CAS  Google Scholar 

  27. Kruse, A. C. et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101–106 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Congreve, M., Oswald, C. & Marshall, F. H. Applying structure-based drug design approaches to allosteric modulators of GPCRs. Trends Pharmacol. Sci. 38, 837–847 (2017).

    Article  PubMed  CAS  Google Scholar 

  29. Conn, P. J., Lindsley, C. W., Meiler, J. & Niswender, C. M. Opportunities and challenges in the discovery of allosteric modulators of GPCRs for treating CNS disorders. Nat. Rev. Drug Discov. 13, 692–708 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. May, L. T., Leach, K., Sexton, P. M. & Christopoulos, A. Allosteric modulation of G-protein-coupled receptors. Annu. Rev. Pharmacol. Toxicol. 47, 1–51 (2007).

    Article  PubMed  CAS  Google Scholar 

  31. Keov, P., Sexton, P. M. & Christopoulos, A. Allosteric modulation of G-protein-coupled receptors: a pharmacological perspective. Neuropharmacology 60, 24–35 (2011).

    Article  PubMed  CAS  Google Scholar 

  32. Digby, G. J., Shirey, J. K. & Conn, P. J. Allosteric activators of muscarinic receptors as novel approaches for treatment of CNS disorders. Mol. Biosyst. 6, 1345–1354 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Ballesteros, J. A. & Weinstein, H. Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. in Methods in Neurosciences Vol. 25 (ed. Sealfon, S.) 366–428 (Academic Press, San Diego, CA, 1995).

  34. Bosnyak, S. et al. Relative affinity of angiotensin peptides and novel ligands at AT1 and AT2 receptors. Clin. Sci. 121, 297–303 (2011).

    Article  PubMed  CAS  Google Scholar 

  35. Balakumar, P. & Jagadeesh, G. Structural determinants for binding, activation and functional selectivity of the angiotensin AT1 receptor. J. Mol. Endocrinol. 53, R71–R92 (2014).

    Article  PubMed  CAS  Google Scholar 

  36. Cabana, J. et al. Identification of distinct conformations of the angiotensin II type 1 receptor associated with the Gq/11 protein pathway and the β-arrestin pathway using Molecular Dynamics simulations. J. Biol. Chem. 290, 15835–15854 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Takezako, T., Unal, H., Karnik, S. S. & Node, K. Structure–function basis of attenuated inverse agonism of angiotensin II type 1 receptor blockers for active-state angiotensin II type 1 receptor. Mol. Pharmacol. 88, 488–501 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Spyroulias, G. A. et al. Comparison of the solution structures of angiotensin I and II. Implication for structure–function relationship. Eur. J. Biochem. 270, 2163–2173 (2003).

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  40. Aumelas, A. et al. Studies on angiotensin II and analogs: impact of substitution in position 8 on conformation and activity. Proc. Natl. Acad. Sci. USA 82, 1881–1885 (1985).

    Article  PubMed  CAS  Google Scholar 

  41. Zhang, H. et al. Structural basis for selectivity and diversity in angiotensin II receptors. Nature 544, 327–332 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Unal, H. & Karnik, S. S. Constitutive activity in the angiotensin II type 1 receptor: discovery and applications. Adv. Pharmacol. 70, 155–174 (2014).

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Hirata, K. et al. Achievement of protein micro-crystallography at SPring-8 beamline BL32XU. J. Phys. Conf. Ser. 425, 012002–012006 (2013).

    Article  CAS  Google Scholar 

  45. Yamashita, K., Hirata, K. & Yamamoto, M. KAMO: towards automated data processing for microcrystals. Acta Crystallogr. D Struct. Biol. 74, 441–449 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Foadi, J. et al. Clustering procedures for the optimal selection of datasets from multiple crystals in macromolecular crystallography. Acta Crystallogr. D. Biol. Crystallogr. 69, 1617–1632 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 26, 795–800 (1993).

    Article  CAS  Google Scholar 

  48. Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D. Biol. Crystallogr. 66, 133–144 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Vigan, A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Cryst. 30, 1022–1025 (1997).

    Article  Google Scholar 

  50. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D. Biol. Crystallogr. 67, 355–367 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Krissinel, E. Crystal contacts as nature’s docking solutions. J. Comput. Chem. 31, 133–143 (2010).

    Article  PubMed  CAS  Google Scholar 

  52. Paithankar, K. S. & Garman, E. F. Know your dose: RADDOSE. Acta Crystallogr. D. Biol. Crystallogr. 66, 381–388 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Karplus, P. A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030–1033 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D. Biol. Crystallogr. 62, 72–82 (2006).

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We thank Y. Yamanaka for supporting crystallization of Fab and BL32XU beamline scientists Y. Kawano for assisting with X-ray crystallographic data collection and K. Yamashita for assisting with data processing using the KAMO system. AT2R-knockout mice were kindly provided by T. Inagami (Vanderbilt University). This work was funded by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (grant no. 15J04343 (S.H.), 15J04343 (H.A.) and 22590270 (H.A.)). S.H. is a recipient of a JSPS postdoctoral fellowship. DNA sequencing analysis was performed at the Medical Research Support Center, Graduate School of Medicine, Kyoto University. Radioisotope experiments were performed at the Radioisotope Research Center, Kyoto University. This study was also supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics and Structural Life Science) and the Basis for Supporting Innovative Drug Discovery and Life Science Research from the Japan Agency for Medical Research and Development (AMED), JST/PRESTO, and the Takeda Science Foundation (H.A. and T.K). The X-ray crystallographic data were collected at SPring-8 (proposal no. 2013B1092, 2014B1355 and 2015A1044) and Photon Factory (proposal no. 2015R-62).

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Contributions

H.A. and S.I. designed the project; H.A., M.S. and T.U. performed the initial screen of AT2R; H.I., O.K.-A. and T.H. generated the anti-AT2R-expressing hybridoma cells; H.A., H.I., O.K.-A. and T.H. expressed, purified and evaluated the antibody; N.N. performed the cloning and expression of the Fab fragment; H.A., C.S. and T.U. purified and crystallized the AT2R–4A03Fab complex; H.A., T.U. and Y.S. performed the construction and binding assay for the AT2R mutants; H.A. and K.H. collected and processed the synchrotron data; H.A., S.H., T.S. and K.H. solved and refined the structure; H.A., S.H. and S.I. analyzed the data and compiled the figures for the manuscript; and H.A., S.H. and S.I. wrote the manuscript, with contributions from T.K., K.H., H.I. and T.H.

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Correspondence to So Iwata.

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Supplementary Figure 1 Snake plot diagram of AT2R.

The AT2R construct was designed as shown. Truncated positions are indicated with scissors; disulfide bonds are shown in yellow; ligand-binding residues are shown in red; and Fab4A03-recognizing residues are shown in blue.

Supplementary Figure 2 Crystallographic R values of the [Sar1, Ile8]-AngII–AT2R-BRILICL3–Fab4A03 complex.

Average R factor and free R values versus d*2 are represented as blue and green lines, respectively.

Supplementary Figure 3 Crystal packing view, overall electron density map and electron density map of [Sar1, Ile8]-AngII.

a, Crystal packing views parallel to the membrane layer. Each molecule is colored as follows: AT2R (green), BRIL (cyan), Fab4A03L (salmon) and Fab4A03H (yellow-orange). b, The electron density 2mFoDFc map was calculated from the 3.2-Å resolution dataset contoured at 1.0σ. c, The omitted mFoDFc electron density (blue mesh) contoured at 1.5σ (omit map), 3.0σ (omit map) and 3.0σ (Polder map, or improved omit map, implemented in the PHENIX software suite). AT2R is shown as a green cylindrical cartoon. [Sar1, Ile8]-AngII is shown as a licorice model with a yellow backbone, blue nitrogen atoms and red oxygen atoms. [Sar1, Ile8]-AngII is abbreviated s-AngIl.

Supplementary Figure 4 Comparison of ligand-binding sites for the peptide and compound in the AT2R structure.

a, Overall structures are shown as AT2R bound to [Sar1, Ile8]-AngII in green (this study) and AT2R (PDB ID: 5UNF) bound to compound 1 in gray. [Sar1, Ile8]-AngII is shown as a licorice model with a yellow backbone, and compound 1 is shown as a licorice model with a light blue backbone. b, Comparison of ECL2 of AT2R with (green) or without (gray) Fab4A03. c,d, Residues surrounding [Sar1, Ile8]-AngII and compound 1 (C1) in AT2R are shown in gray. Ligands are shown as licorice models, with the carbon atoms of [Sar1, Ile8]-AngII in yellow and C1 in light blue. AT2R is shown with the same orientation of transmembrane helices for ArgECL2. ArgECL2 and Lys2155.42 are residues critical for binding of both ligands (labeled in red). Thr1784.60 is a critical residue for C1 binding (labeled in blue).

Supplementary Figure 5 Hydrophobic interaction with [Sar1, Ile8]-AngII.

ECL2 AT2R is shown as a green cartoon. Ligand-binding residues are shown as licorice models, and hydrophobic residues are indicated with green surfaces. [Sar1, Ile8]-AngII (s-AngII) is shown as a licorice model figure with a yellow backbone, blue nitrogen atoms and red oxygen atoms.

Supplementary Figure 6 Binding assays of AngII and [Sar1, Ile8]-AngII for wild-type AT2R with or without Fab4A03.

a,b, Specific binding assays of 125I-labeled [Sar1, Ile8]-AngII (a) and AngII (b) for wild-type AT2R with (square) or without (circle) Fab4A03. The corresponding Kd and Bmax values were calculated by fitting a non-linear curve. Each point represents the means ± s.d. of triplicate experiments. Vertical axes indicate relative specific binding values. [Sar1, Ile8]-AngII is abbreviated s-AngIl.

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Asada, H., Horita, S., Hirata, K. et al. Crystal structure of the human angiotensin II type 2 receptor bound to an angiotensin II analog. Nat Struct Mol Biol 25, 570–576 (2018). https://doi.org/10.1038/s41594-018-0079-8

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