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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from $8.99

All prices are NET prices.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 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).

  2. 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).

  3. 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).

  4. 4.

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

  5. 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).

  6. 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).

  7. 7.

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

  8. 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).

  9. 9.

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

  10. 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).

  11. 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).

  12. 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).

  13. 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).

  14. 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).

  15. 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).

  16. 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).

  17. 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).

  18. 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).

  19. 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).

  20. 20.

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

  21. 21.

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

  22. 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).

  23. 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).

  24. 24.

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

  25. 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).

  26. 26.

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

  27. 27.

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

  28. 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).

  29. 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).

  30. 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).

  31. 31.

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

  32. 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).

  33. 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. 34.

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

  35. 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).

  36. 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).

  37. 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).

  38. 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).

  39. 39.

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

  40. 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).

  41. 41.

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

  42. 42.

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

  43. 43.

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

  44. 44.

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

  45. 45.

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

  46. 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).

  47. 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).

  48. 48.

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

  49. 49.

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

  50. 50.

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

  51. 51.

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

  52. 52.

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

  53. 53.

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

  54. 54.

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

Download references


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).

Author information


  1. Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    • Hidetsugu Asada
    • , Shoichiro Horita
    • , Tatsuro Shimamura
    • , Norimichi Nomura
    • , Tomoko Uemura
    • , Chiyo Suno
    • , Takuya Kobayashi
    •  & So Iwata
  2. RIKEN, SPring-8 Center, Hyogo, Japan

    • Kunio Hirata
    •  & So Iwata
  3. Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO), Saitama, Japan

    • Kunio Hirata
  4. Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan

    • Mitsunori Shiroishi
  5. Molecular Genetics, Institute of Life Science, Kurume University, Fukuoka, Japan

    • Yuki Shiimura
  6. Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan

    • Hiroko Iwanari
    • , Takao Hamakubo
    •  & Osamu Kusano-Arai
  7. Japan Agency for Medical Research and Development (AMED), Core Research for Evolutional Science and Technology (CREST), Kyoto, Japan

    • Takuya Kobayashi


  1. Search for Hidetsugu Asada in:

  2. Search for Shoichiro Horita in:

  3. Search for Kunio Hirata in:

  4. Search for Mitsunori Shiroishi in:

  5. Search for Yuki Shiimura in:

  6. Search for Hiroko Iwanari in:

  7. Search for Takao Hamakubo in:

  8. Search for Tatsuro Shimamura in:

  9. Search for Norimichi Nomura in:

  10. Search for Osamu Kusano-Arai in:

  11. Search for Tomoko Uemura in:

  12. Search for Chiyo Suno in:

  13. Search for Takuya Kobayashi in:

  14. Search for So Iwata in:


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.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to So Iwata.

Integrated supplementary information

  1. 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.

  2. 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.

  3. 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.

  4. 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).

  5. 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.

  6. 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.

Supplementary information

About this article

Publication history