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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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).
The authors declare no competing interests.
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Integrated supplementary information
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 2mFo–DFc map was calculated from the 3.2-Å resolution dataset contoured at 1.0σ. c, The omitted mFo–DFc 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).
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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