The Crystal Structure of Angiotensin II Type 2 Receptor with Endogenous Peptide Hormone

Article

The Crystal Structure of Angiotensin II Type 2 Receptor with Endogenous Peptide Hormone Graphical Abstract

Authors Hidetsugu Asada, Asuka Inoue, Francois Marie Ngako Kadji, ..., Chiyo Suno, Junken Aoki, So Iwata

Extracellular AngII

Correspondence Phe8

Ile8

[email protected] (H.A.), [email protected] (S.I.)

In Brief Met128

3.36

Helix8 Intracellular

AT2R (AngII) AT2R ([Sar1, Ile8]-AngII) AT2R (AT2R selective AngII inhibitor)

Highlights d

d

d

d

The structure of AT2R bound with endogenous peptide hormone was solved Interaction of Met1283.36 and Phe8 of AngII seems a key for the activation of AT2R The non-canonical coordination of helix 8 was observed Asn1113.35-Asn2957.46 internal lock looks important in the activation of ATRs

Asada et al., 2020, Structure 28, 1–8 March 3, 2020 ª 2019 Elsevier Ltd. https://doi.org/10.1016/j.str.2019.12.003

Asada et al. determined the structure of angiotensin II type 2 receptor (AT2R) bound with endogenous peptide hormone, angiotensin II. The structure reveals important features of the angiotensin peptide for the receptor activation, which provides us a molecular basis to understand the physiological role of this peptide hormone.

Please cite this article in press as: Asada et al., The Crystal Structure of Angiotensin II Type 2 Receptor with Endogenous Peptide Hormone, Structure (2019), https://doi.org/10.1016/j.str.2019.12.003

Structure

Article The Crystal Structure of Angiotensin II Type 2 Receptor with Endogenous Peptide Hormone Hidetsugu Asada,1,9,* Asuka Inoue,2,3,4 Francois Marie Ngako Kadji,2 Kunio Hirata,5,6 Yuki Shiimura,7 Dohyun Im,1 Tatsuro Shimamura,1 Norimichi Nomura,1 Hiroko Iwanari,8 Takao Hamakubo,8 Osamu Kusano-Arai,8 Hiromi Hisano,1 Tomoko Uemura,1 Chiyo Suno,1 Junken Aoki,2,4 and So Iwata1,5,* 1Department

of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan 3Advanced Research & Development Programs for Medical Innovation (PRIME), Chiyoda, Tokyo 100-0004, Japan 4Advanced Research & Development Programs for Medical Innovation (LEAP), Chiyoda, Tokyo 100-0004, Japan 5RIKEN, SPring-8 Center, Hyogo 679-5165, Japan 6Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO), Saitama 332-0012, Japan 7Molecular Genetics, Institute of Life Science, Kurume University, Fukuoka 830-0011, Japan 8Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan 9Lead Contact *Correspondence: [email protected] (H.A.), [email protected] (S.I.) https://doi.org/10.1016/j.str.2019.12.003 2Graduate

SUMMARY

Angiotensin II (AngII) is a peptide hormone that plays a key role in regulating blood pressure, and its interactions with the G protein-coupled receptors, AngII type-1 receptor (AT1R) and AngII type-2 receptor (AT2R), are central to its mechanism of action. We solved the crystal structure of human AT2R bound to AngII and its specific antibody at 3.2-A˚ resolution. AngII (full agonist) and [Sar1, Ile8]-AngII (partial agonist) interact with AT2R in a similar fashion, except at the bottom of the AT2R ligand-binding pocket. In particular, the residues including Met1283.36, which constitute the deep end of the cavity, play important roles in angiotensin receptor (ATR) activation upon AngII binding. These differences that occur upon endogenous ligand binding may contribute to a structural change in AT2R, leading to normalization of the non-canonical coordination of helix 8. Our results will inform the design of more effective ligands for ATRs.

INTRODUCTION The renin-angiotensin system (RAS) plays an important role in regulating physiological and pathological processes of the cardiovascular system to maintain homeostasis (de Gasparo et al., 2000; Karnik et al., 2015; Mehta and Griendling, 2007). RAS disorders cause a wide range of conditions, including hypertension, cardiac hypertrophy, heart failure, ischemic heart disease, and nephropathy (Zaman et al., 2002). The RAS is a major regulator of blood pressure and its effects are primarily mediated by the peptide hormone angiotensin II (AngII) (de Gasparo

et al., 2000; Karnik et al., 2015; Mehta and Griendling, 2007). In humans, endogenous AngII is composed of eight amino acid residues (1Asp-Arg-Val-Tyr-Ile-His-Pro-Phe8) and primarily binds to two subtypes of G protein-coupled receptors (GPCRs, class A g-group): AngII type-1 receptor (AT1R) and AngII type2 receptor (AT2R) (de Gasparo et al., 2000; Fredriksson et al., 2003; Karnik et al., 2015; Oliveira et al., 2007). AT1R is known to be important in blood pressure regulation (Shenoy and Lefkowitz, 2005; Whalen et al., 2011), but the physiological function of AT2R remains somewhat controversial. AT2R is more abundant in the fetus and neonate, and is believed to be involved in vascular growth (Berk, 2003; Caballero et al., 2004; Hein et al., 1995; Ichiki et al., 1995; Miura and Karnik, 1999; Miura et al., 2010; Porrello et al., 2009a, 2009b; Ruiz-Ortega et al., 2000, 2001; Zhao et al., 2005). Given the physiological and pathological significance of AngIImediated signaling pathways, a comprehensive understanding of AngII function, including its molecular recognition mechanisms, is of great value. A series of reported structures have shed light on the molecular interactions between angiotensin receptors (ATRs) and their ligands: AT1R in complex with olmesartan, angiotensin II receptor blocker, and the AT1R antagonist ZD7155 (Zhang et al., 2015a, 2015b); AT2R in complex with the quinazolinone-biphenyltetrazole derivatives 1 and 2 (compounds 1 and 2); AT2R in complex with [Sar1, Ile8]-AngII and its specific antibody fragment Fab4A03 at 3.2-A˚ resolution (PDB: 5XJM) (Asada et al., 2018); and AT1R in complex with [Sar1, Ile8]-AngII (Wingler et al., 2019b). However, structural information concerning the recognition of endogenous AngII was still lacking. Here, we present the crystal structure of the endogenous ligand-receptor complex, human AT2R bound to AngII with its specific antibody fragment Fab4A03, at 3.2-A˚ resolution. Comparison of [Sar1, Ile8]-AngII-bound and AngII-bound ATR structures reveal clear differences in several side-chain orientations, including Met1283.36 of AT2R (Leu1123.36 in AT1R) (superscripts indicate Ballesteros-Weinstein numbering for Structure 28, 1–8, March 3, 2020 ª 2019 Elsevier Ltd. 1

Please cite this article in press as: Asada et al., The Crystal Structure of Angiotensin II Type 2 Receptor with Endogenous Peptide Hormone, Structure (2019), https://doi.org/10.1016/j.str.2019.12.003

Table 1. Data Collection and Refinement Statistics AT2R-AngII Complex (PDB 6JOD) Data Collection Space group

C2221

Cell dimensions a, b, c (A˚)

102.55, 426.43, 52.70

a, b, g ( ) Resolution (A˚)

90.0, 90.0, 90.0 49.85–3.20 (3.31–3.20)a

Rpim

0.0498 (0.551)

I/s(I)

17.47 (1.04)

CC1/2

99.9 (38.3)

Completeness (%)

99.8 (99.7)

Redundancy

82.3 (81.0)

Refinement Resolution (A˚)

49.85–3.20 (3.28–3.2)

No. reflections

19,767 (1,933)

Rwork/Rfree

0.235/0.287

No. atoms AT2R

2,470

mbIIG

438

AngiotensinII

75

FabH fragment

1,643

FabL fragment

1,631

B factors AT2R

160.5

mbIIG

253.5

AngiotensinII

138.6

FabH fragment

113.0

FabL fragment

113.1

Root mean squared deviations Bond lengths (A˚) Bond angles ( )

0.003

RESULTS Structure Determination The AT2R construct used for crystallization was designed by truncating the N-terminal glycosylation sites with thermostability-improved apocytochrome b562 (mbIIG) (Kimura et al., 2019) and the putative C-terminal palmitoylation site (residue range, 35–346). In addition, the Ser208Ala mutation was introduced to improve expression (Figure S1). A Fab fragment of AT2R-specific antibody (Fab4A03) and AngII were added during purification to increase thermostability and facilitate crystallization. Needle-like crystals were obtained within a few days and X-ray diffraction patterns were resolved at 3.2-A˚ resolution. Data were collected from 470 crystals, and the structure was determined by molecular replacement using AT2R (PDB: 5XJM) and apocytochrome b562 RIL (PDB: 1M6T) as templates. The crystals contain one AT2R-mbIIGN-Term/Fab4A03 complex in the asymmetric unit and belong to the space group C2221, with unit cell dimensions a = 102.8 A˚, b = 426.8 A˚, and c = 52.6 A˚ (Table 1); crystal packing is shown in Figure S2A. Fab4A03 and the receptor, including AngII, show clear electron densities, whereas the fused mbIIG shows poor electron densities (Figures S2B and S2C). Hence, we modeled AngII into a well-defined electron density inside the ligand-binding pocket and showed that it interacts with transmembrane (TM) helices 2, 5, 6, 7, and ECL2 (Figure 1). The interaction between Fab4A03 and the receptor was observed in the same manner as the previous structure, 5XJM (Figures S3A and S3B). The intermolecular interface of AT2R and AngII is elongated and buries approximately 838 A˚2 of interface area. In the complex structure, AngII binds to the ligandbinding pocket with its C terminus oriented toward the bottom of the cavity and its N terminus exposed to the extracellular space, in a similar fashion to [Sar1, Ile8]-AngII. The N terminus of AngII (1Asp-Arg-Val3) is extended, whereas the C terminus (4Tyr-Ile-His-Pro-Phe8) is folded into a C shape.

0.61

The final dataset consists of 470 datasets, each of data corresponds to a 5 wedge. a Values in parentheses are for highest-resolution shell.

conserved GPCR residues; Ballesteros and Weinstein, 1995). These residues, which are at the deepest part of the ligand-binding pocket, interact with Phe8 of AngII. Signal assays of Met1283.36 and its mutants clearly showed that the interaction of this residue and Phe8 of AngII is a key for the activation of ATRs by AngII. For AT1R, active and inactive structures have been solved and the activation mechanism has been proposed (Wingler et al., 2019a, 2019b; Zhang et al., 2015b). In the mechanism, Asn1113.35-Asn2957.46 internal lock in AT1R has been suggested to be important for the activation control of AT1R (Miura and Karnik, 1999; Takezako et al., 2015). In our structure, there is no hydrogen bond between equivalent residues. We believe that the lock interconnects that states of the ligand and the G protein binding sites and that the difference at the site between AT2R and AT1R is the main reason of higher basal activity of AT2R. We also discuss the role of the helix 8, which has been suggested to be important for the G protein and arrestin signaling. 2 Structure 28, 1–8, March 3, 2020

Binding Mode of AngII The binding mode of AngII-AT2R was very similar to that of [Sar1, Ile8]-AngII-AT2R (Figure 2A). The ligand-binding region of AngII is important for its interaction with the receptor (Figure 2B). Arg2 has salt bridges to Asp2796.58 and Asp2977.32, which are conserved in AT1R as Asp2636.58 and Asp2817.32. The carbonyl oxygens of Ile5 form hydrogen bonds with Tyr1032.63. The carbonyl oxygens of His6 and Pro7 form hydrogen bonds with the guanidinium group of Arg182ECL2, which is conserved in AT1R as Arg167ECL2, a key residue for ligand binding. The imidazole group of His6 forms a hydrogen bond with Tyr1042.64. The C-terminal carboxyl group of Phe8 forms a salt bridge with the side chain of Lys2155.42, which is conserved in AT1R as Lys1995.42. AngII Phe8 interacts with Leu1243.32, Met1283.36, Trp2696.48, Phe2726.51, and Phe3087.43. Ligand binding and mutagenesis experiments confirmed that Tyr1042.64, Met1283.36, Arg182ECL2, Lys2155.42, Trp2696.48, Phe2726.51, Asp2977.32, and Phe3087.43 play critical roles in AngII binding (Figure 2C). Met128Leu and Phe272His, which are AT1R-type mutants, had almost the same affinity compared with wild-type AT2R. Although Fab4A03 does not affect the conformation of side chains important for ligand binding, the conformation of ECL2 on the surface is affected, which also occurs in [Sar1, Ile8]-AngII-bound AT2R

Please cite this article in press as: Asada et al., The Crystal Structure of Angiotensin II Type 2 Receptor with Endogenous Peptide Hormone, Structure (2019), https://doi.org/10.1016/j.str.2019.12.003

A

Figure 1. Structure of AT2R-mbIIGN-Term in Complex with AngII and Fab4A03

AngII

B

D1

ECL2

FabL mbIIG TM3

FabH Ext

P8

TM5

TM2

AT2R

(A) The overall structure of the AngII-bound AT2RmbIIGN-Term/Fab4A03 complex. Membrane boundaries, as defined by the OPM database (http://www.opm.phar.umich.edu), are shown as a beige box. Each molecule is colored as follows: AT2R (green), bIIG (cyan), Fab4A03L (salmon), and Fab4A03H (yellow-orange). Intracellular (Int) and extracellular (Ext) regions of the receptor are indicated. (B) Cross-sectional view of the ligand-binding pocket of AT2R (orange surface). AngII is shown as a licorice model, with carbon atoms in yellow, nitrogen in blue, and oxygen in red. The C terminus of AngII is located at the bottom of the ligandbinding cavity.

TM1

Int

TM4

TM6

TM7

(Figure S3C). The residues in AT2R: Trp1002.60, Tyr1082.68, Leu3007.35, and Ile3047.39 interact with [Sar1, Ile8]-AngII, except for Ile8 (Asada et al., 2018). Therefore, these residues will also play important roles in AngII binding. Previous studies have shown that AngII is a full agonist for AT1R and [Sar1, Ile8]-AngII is a partial agonist (Fierens et al., 2000; Inoue et al., 1997; Miura et al., 1999; Noda et al., 1996). These ligands differ at the first and eighth residues, but Arg/Sar1 does not affect binding affinity or receptor activation. Since the main chain shapes of Phe/Ile8 are the same (Figure 2A), it is reasonable to hypothesize that the receptor activation level depends on the difference of the side chain at this position. Receptor Activation Mechanism by Angiotensin II The active and inactive structures of AT1R and a possible activation mechanism based on them have been reported (Wingler et al., 2019a, 2019b; Zhang et al., 2015a, 2015b). We compare the structures of the AngII-AT2R and [Sar1, Ile8]-AngII-AT1R complexes in the context of this activation mechanism with a particular focus on the ligand-binding pocket and highly conserved motifs of class A GPCRs, including NPxxY, PIF, and DRY (Figure 3). Interaction between the ligands and the bottom of the ligand-binding cavity, including Met1283.36 (Leu1123.36 for AT1R) seems a key for the receptor activation (Figure 4). The insertion of AngII Phe8 into this hydrophobic core seems to trigger the activation of the receptor at the G protein binding site, where the conformational change is transferred though the area, including the internal lock (Asn1113.35-Asn2957.46 for AT1R; the equivalent in AT2R is Asn1273.35-Ser3117.46) (Figures 3 and 4) (Miura and Karnik, 1999; Takezako et al., 2015). This internal lock is known to be important to stabilize the inactive conformation of AT1R; in AT1R, the Asn1113.35 Gly mutation shifts the equilibrium of the receptor, making the receptor constitutively active (Balakumar and Jagadeesh, 2014; Cabana et al., 2015; Unal and Karnik, 2014). This has also been confirmed using pathway-selective bioluminescence resonance energy transfer biosensors and microsecond molecular dynamics simulation (Namkung et al., 2018; Singh et al., 2019). In the

AngII-bound AT2R structure, a hydrogen bond between Ser3117.46 (equivalent to Asn2957.46) and Asn1273.35 is not observed. It is not clear if they form a hydrogen bond in the inactive conformation, since we do not have the AT2R inactive structure. This difference of the internal lock, however, could provide a molecular basis of high basal activity of AT2R. We also found that the conformational states of the bottom part of the ligandbinding pocket and the internal lock could be connected (Figures 4A and 4B). In the AT2R structures, Met1283.36 gives away and moves toward Phe3087.43 to accommodate Phe8 side chain upon the binding of AngII. Equivalent residues of Met1283.36 and Phe3087.43 of AT2R are Leu1123.36 and Phe3087.43 of AT1R, respectively. At the bottom of the ligand-binding pocket, these AT1R residues form hydrophobic core, as observed in AT2R, and upon the binding of the full agonist AngII, Leu1123.36 and Phe3087.43 seem to need to give away to make room to accommodate Phe8, leading to the rotation of helix 7 holding Phe3087.43. In the structure of a partial agonist [Sar1, Ile8]-AngII and AT1R complex, one hydrogen bond is maintained at the Asn1113.35-Asn2957.46 internal lock, whereas two bonds are observed for the antagonist-bound inactive conformation. The rotation of helix 7, caused by the full agonist AngII binding, may disrupt this internal lock completely, leading to the full active conformation. We also observed different conformations of Arg1423.50 of AT2R in DRY motif and the equivalent of AT1R. We do not have a clear explanation, but this may not reflect the conformational states but be specific to respective receptors. Canonical Conformation of Helix 8 Shift by Angiotensin II In most GPCR structures, helix 8 lies parallel to the membrane, pointing outside of the 7TM bundle, regardless of the activation state of the receptor. For example, b2-adrenergic receptor has the same helix 8 conformation in active and inactive states (Rasmussen et al., 2007, 2011). However, in the structure of AT2R bound to compound 1 (AT2R selective AngII-binding inhibitor) and compound 2 (AT1R/AT2R dual AngII-binding inhibitor), helix 8 adopts an unusual conformation by flipping over to interact with the intracellular ends of TM3, TM5, and TM6 (Zhang et al., 2017) (Figure 5). This non-canonical shift of helix 8 suggests Structure 28, 1–8, March 3, 2020 3

Please cite this article in press as: Asada et al., The Crystal Structure of Angiotensin II Type 2 Receptor with Endogenous Peptide Hormone, Structure (2019), https://doi.org/10.1016/j.str.2019.12.003

A

AngII Y2045.31

Y2045.31 D2977.32 D2796.58 R182ECL2 K2155.42 P2726.51 W2696.48

Figure 2. Mode of AngII Binding to AT2R

s-AngII

D2977.32 Y1032.63

D2796.58

Y1032.63

R182ECL2 K2155.42

Y1042.64 L1243.32 F308

Y1042.64 L1243.32

P2726.51

7.43

M1283.36

M1283.36

F3087.43

W2696.48

B

(A) Comparison view of AngII and [Sar1, Ile8]-AngII (s-AngII) in the ligand-binding site. The AngIIbinding residues of AT2R (this study) are shown in green. The [Sar1, Ile8]-AngII-binding residues of AT2R (5XJM) are shown in orange. Hydrogen bonds are shown as black dashed lines. (B) Schematic interaction between AngII and AT2R. Hydrogen bonds are shown as blue dashed lines. Green boxes indicate residues conserved in AT1R. Residues indicated in red are critical for ligand binding (NA, not available due to weak binding when mutated to Ala). (C) Dissociation constants determined using an alanine-scanning mutagenesis assay. The coloring scheme is the same as in (B). Residues are ordered from the extracellular side to intracellular side. Error bars indicate SEM. [Sar1, Ile8]-AngII is abbreviated as s-AngII.

C AngII

KD (nM) WT

Y2045.31

D297A

D2796.58 D297

Y204A D279A Y103A

7.32

Y1032.63 Y1042.64

R182ECL2

Y104A R182A K215A F272A F272H L124A

K2155.42

L124V F308A

L1243.32 W2696.48

F308Y M128A

F2726.51 F3087.43 M1283.36

M128L W269A

Conserved residues with AT1R Hydrogen bond

that despite the active-like conformation of the AT2R 7TM bundle, downstream signaling via G proteins and b-arrestin is not transmitted by steric blocking G protein and b-arrestin binding (Zhang et al., 2017). In this study, which used the endogenous ATR peptide hormone, the AngII-bound AT2R structure revealed that helix 8 shifts to the canonical conformation of GPCRs, which can bind G protein and b-arrestin (Figure 5), and the aromatic rings of Phe3258.50 in helix 8 and Phe3167.51 and Phe3207.55 in TM7 interact with each other (Figure 5B). The p–p interactions between Phe3207.55 and Phe3258.50 are not visible in the structure of AT2R bound to compounds 1 and 2 (PDB: 5UNF). DISCUSSION In the [Sar1, Ile8]-AngII-bound AT2R structure that we previously identified, helix 8 could not be assigned due to unclear/weak electron density (Asada et al., 2018). The different conformation 4 Structure 28, 1–8, March 3, 2020

of the helix 8 in the current structure could be caused by the different ligand NA (i.e., AngII) or simply due to the crystal 9.92 ± 2.32 packing difference. Further experiments 4.07 ± 0.40 are required to clarify the importance NA of the helix in the AT2R activation NA mechanism. NA In AT2R signal transduction upon NA AngII and [Sar1, Ile8]-AngII binding, G 2.70 ± 1.16 protein and b-arrestin signaling could 4.96 ± 1.05 not be verified by transforming growth 7.90 ± 2.67 factor a (TGF-a) shedding or b-arrestin 2.70 ± 1.16 NA recruitment assays, suggesting that NA AT2R signaling requires additional ef3.75 ± 0.73 fectors such as ATIP and SHP-1 (HoriuInt NA chi et al., 2012; Nouet et al., 2004; Wruck et al., 2005). However, in the AT1R signal assay for AngII and [Sar1, Ile8]-AngII binding, AngII was a balanced agonist of both G protein and b-arrestin signaling pathways for wild-type AT1R, and [Sar1, Ile8]-AngII was a partial agonist of both G protein and b-arrestin signaling pathways (Figure S4 and Table S1). In the Leu1123.36Met mutant (AT1R to AT2R type mutation), both G protein and b-arrestin signaling pathways yielded results similar to those obtained with wild-type AT1R. On the other hand, in the AngII-bound Leu1123.36Ala mutant, G protein signaling activity was reduced to a greater extent than b-arrestin signaling (DLog RAi = 0.92). Thus, our results indicate that ATR activation starts with an interaction between 3.36 position (Leu1123.36 and Met1283.36) and AngII Phe8. Following this interaction, activation motifs (DRY, PIF, NPxxY, and CWxP), including His2566.51, Trp2536.48, Tyr2927.43, and Tyr3027.53, are induced to adopt their active conformations. In particular, the lateral rotation of TM3 and outward movement of TM6 and TM7 are essential for complete disruption of the Asn1113.35-Asn2957.46 interaction in AT1R. Double electron-electron resonance spectroscopy (Wingler et al., 2019a) and molecular dynamics 2.34 ± 0.60 9.34 ± 1.87

Ext

Please cite this article in press as: Asada et al., The Crystal Structure of Angiotensin II Type 2 Receptor with Endogenous Peptide Hormone, Structure (2019), https://doi.org/10.1016/j.str.2019.12.003

TM3

TM5

TM7

P2235.50

TM3

I1323.40

N1273.35

PIF N1113.35 N2957.46

F2656.44

N-N lock N-N lock PIF NPxxY

TM6

N1113.35 N1273.35 N2957.46

Helix 8

N-N lock

N1113.35

TM3

N3147.49

P3157.50 Y3187.53

TM7

N1273.35

N2957.46

Superposition of AngII-AT2R (green; this study), [Sar1, Ile8]-AngII-AT1R (cyan; 6DO1), and antagonist-AT1R (pink; 4YAY). The activation motifs PIF, NPxxY, and DRY are indicated by red, blue, and green boxes, respectively. The interaction between Asn111 and Asn295 in AT1R (Asn127 and Ser311 in AT2R) is shown with each box, with hydrogen bonds shown as dashed lines. Asn111-Asn295 internal lock is abbreviated as N-N lock.

N-N lock

DRY

TM6

Figure 3. Structural Comparison of Activation Motifs and N111-N295 Internal Lock in AngII-Bound AT2R, [Sar1, Ile8]-AngII-Bound AT1R and Antagonist-Bound AT1R

NPxxY

AT2R (AngII) AT1R (s-AngII) AT1R (Antagonist)

Y1433.51 D1413.49

TM3

R1423.50

DRY

simulation (Singh et al., 2019) provide valuable insights into conformational dynamics. In addition, a high-resolution structure provides an overview of a receptor’s conformation and is essential to define the structural state of key regions, including the core of the receptor. Our structural data provide important information to aid in design of novel ATR agonists. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

d

KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS B Microbes B Cell Lines METHOD DETAILS B Complete Amino Acid Sequence and Expression Construct B Protein Expression and Purification (AT2R-mbIIGN-term) B Radioligand Binding Assay B TGF-a Shedding Assay

NanoBiT-Based b-Arrestin Recruitment Assay Data Collection, Structure Determination, and Analysis QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND CODE AVAILABILITY B B

d d

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j.str. 2019.12.003. ACKNOWLEDGMENTS This work was funded by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (no. 15J04343 to H.A., no. 22590270 to H.A. and 17K08264 to A.I.). 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 (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from the Japan Agency for Medical Research and Development (AMED) under grant number JP18am0101079 (H.A. and S.I.), JP18gm5910013 (A.I.), JP18gm0010004 (A.I. and J.A.) and JP19am0101070 (K.H.); JST/PRESTO (K.H.); the Takeda Science Foundation (H.A). The synchrotron radiation experiments were performed at the BL32XU of SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute

Structure 28, 1–8, March 3, 2020 5

Please cite this article in press as: Asada et al., The Crystal Structure of Angiotensin II Type 2 Receptor with Endogenous Peptide Hormone, Structure (2019), https://doi.org/10.1016/j.str.2019.12.003

A

A H2566.51 I8 ZD7155

I1123.36 Y2927.43

AT1R (ZD7155) AT1R (s-AngII)

W2536.48

Helix 8

B

AT2R (AngII)

Helix 8

AT2R (Compound-1) F2726.51

B

F8 I8

F3167.51

F3207.55 M1283.36 W2696.48

F3087.43

AT2R (AngII) AT2R (s-AngII)

F3258.50

Figure 4. Conformational Changes of TM Helices upon Ligand Binding (A) Interaction between deep end of ligand-binding cavity of AT1R and [Sar1, Ile8]-AngII (green) or ZD7155 (magenta). [Sar1, Ile8]-AngII-AT1R and ZD7155AT1R are shown in cyan and pink, respectively. [Sar1, Ile8]-AngII is abbreviated as s-AngII. (B) Interaction between deep end of ligand-binding cavity of AT2R and AngII (cyan) or [Sar1, Ile8]-AngII (yellow). AngII-AT2R and [Sar1, Ile8]-AngII-AT2R shown in green and orange, respectively. The hydrophobic interaction network between Phe8 and each residue. Interactions are shown in red dashed lines. [Sar1, Ile8]-AngII is abbreviated as s-AngII.

(JASRI) (proposal nos. 2013B1092, 2014B1355, and 2015A1044). We thank BL32XU beamline scientists Yoshiaki Kawano for assisting with X-ray crystallographic data collection and Keitaro Yamashita for assisting with data processing using the KAMO system. AUTHOR CONTRIBUTIONS H.A. and S.I. designed the project. H.A. and T.U. performed the initial screen of AT2R. H.I., O.K.-A., and T.H. generated anti-AT2R antibody-expressing hybridoma cells. H.A., H.I., O.K.-A., and T.H. expressed, purified, and evaluated the antibody. D.I. generated the thermostability-improved BIIL mutant. N.N. performed the cloning and expression of the Fab fragment. H.A., C.S., T.U., and H.H. purified and crystallized the AT2R/4A03Fab complex. H.A., H.H., and Y.S. constructed the AT2R and AT1R mutants and performed the binding assay. H.A. and K.H. collected and processed the synchrotron data. H.A., T.S., and K.H. solved and refined the structure. A.I. and F.M.N.K. designed, performed, and analyzed the TGF-a shedding and NanoBiT b-arrestin recruitment assay under the supervision of J.A. H.A. and S.I. analyzed the data and compiled the figures for the manuscript. H.A. and S.I. wrote the manuscript, with contributions from K.H., T.H., and A.I. All authors discussed the results and commented on the manuscript. DECLARATION OF INTERESTS The authors declare no competing interests.

6 Structure 28, 1–8, March 3, 2020

Figure 5. Conformational Changes of Helix 8 upon Ligand Binding (A) Comparison of conformational changes of helix 8 upon binding of different ligands are shown. Conformation of AngII and compound 1 binding to AT2R are indicated in green and orange. (B) The details of side-chain orientation and helix 8 shifts are shown. Red dashed lines indicate p–p interactions. Red arrows indicate the predicted movement of helix 8 upon AngII binding.

Received: September 2, 2019 Revised: November 11, 2019 Accepted: December 5, 2019 Published: December 30, 2019 REFERENCES Adams, P.D., Afonine, P.V., Bunko´czi, G.b., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.-W., Kapral, G.J., GrosseKunstleve, R.W., et al. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Struct. Biol. 66, 213–221. Asada, H., Horita, S., Hirata, K., Shiroishi, M., Shiimura, Y., Iwanari, H., Hamakubo, T., Shimamura, T., Nomura, N., Kusano-Arai, O., et al. (2018). Crystal structure of the human angiotensin II type 2 receptor bound to an angiotensin II analog. Nat. Struct. Mol. Biol. 25, 570–576. Balakumar, P., and Jagadeesh, G. (2014). Structural determinants for binding, activation, and functional selectivity of the angiotensin AT1 receptor. J. Mol. Endocrinol. 53, R71–R92. Ballesteros, J.A., and Weinstein, H. (1995). [19] 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.

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STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

(Asada et al., 2018)

N/A

Invitrogen

10359-016

Antibodies Anti-human AT2R antibody (D5720-4A03) Bacterial and Virus Strains pFastBac baculovirus system Chemicals, Peptides, and Recombinant Proteins Protease Inhibitor Cocktail

Nacalai tesque

25955-11

DDM (n-Dodecyl-b-D-Maltopyranoside), Sol-Grade

Anatrace

D310S

cholesterol hemi-succinate

Sigma

C6512

Monoolein (1-Oleoyl- rac-glycerol)

Sigma

M7765

Choresterol

Sigma

C8667

Iodoacetamide

FUJIFILM Wako Pure Chemical Corporation

093-02892

Angiotensin II

Peptide Institute

4001

[Sar1, Ile8]-AngII

Peptide Institute

4016

TALON Metal affinity resin

TaKaRa Bio

Z5652N

Ni-Sepharose 6 Fast Flow resin

GE Healthcare

17531801

Angiotensin II (human), [125I]Tyr4-

PerkinElmer

NEX105

Thermo Fisher Scientific

23227

Critical Commercial Assays BCA Protein Assay Deposited Data AngII-bound AT2R/Fab4A03 complex

This study

PDB: 6JOD

[Sar1, Ile8]-AngII -bound AT2R/Fab4A03 complex

(Asada et al., 2018)

PDB: 5XJM

Soluble cytochrome b562

(Chu et al., 2002)

PDB: 1M6T

[Sar1, Ile8]-AngII -bound AT1R

(Wingler et al., 2019b)

PDB: 6DO1

ZD7155-bound AT1R

(Zhang et al., 2015b)

PDB: 4YAY

Compound1-bound AT2R

(Zhang et al., 2017)

PDB: 5UNF

Experimental Models: Cell Lines Sf9 cells

Thermo Fisher Scientific

11496-015

Human: HEK293 cells (parental HEK293)

(Inoue et al., 2012)

N/A

Recombinant DNA AT2R-mbIIGN-Term

Thermo Fisher Scientific

N/A

AT1R-SmBiT

This study

N/A

AP-TGF-a (codon-optimized)

(Inoue et al., 2012)

N/A

LgBiT-barr1EE

(Dixon et al., 2016; Shihoya et al., 2018)

N/A

SHIKA

(Ueno et al., 2016)

N/A

KUMA

(Hirata et al., 2016)

N/A

KAMO

(Yamashita et al., 2018)

https://github.com/keitaroyam/yamtbx/ blob/master/doc/kamo-en.md

ZOO

(Hirata et al., 2019)

N/A

Polder map

(Liebschner et al., 2017)

https://www.phenix-online.org/ documentation/reference/polder.html

BLEND

(Foadi et al., 2013)

http://www.ccp4.ac.uk/

XDS

(Kabsch, 2010)

http://xds.mpimf-heidelberg.mpg.de

Software and Algorithms

(Continued on next page)

Structure 28, 1–8.e1–e4, March 3, 2020 e1

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Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

XSCALE

(Kabsch, 2010)

http://xds.mpimf-heidelberg.mpg.de/ html_doc/xscale_program.html

Molrep

(Vagin and Teplyakov, 2000)

http://www.ccp4.ac.uk/

REFMAC5

(Murshudov et al., 2011)

http://www.ccp4.ac.uk/html/refmac5.html

phenix.refine

(Adams et al., 2010)

https://www.phenix-online.org/

Coot

(Emsley et al., 2010)

https://www2.mrc-lmb.cam.ac.uk/ personal/pemsley/coot/

PISA

(Krissinel, 2010)

http://www.ccp4.ac.uk/dist/checkout/ pisa/help/index.html

Cuemol2

http://www.cuemol.org

http://www.cuemol.org

PyMOL 1.8

Schro¨dinger

http://www.pymol.org/

Prism 5 & 7

Graphpad

https://www.graphpad.com/scientificsoftware/prism/

Millipore

UFC910096

Superdex 200 Increase 10/300 GL

GE Healthcare

28990944

TALON Metal affinity resin

TaKaRa Bio

Z5652N

Ni-Sepharose 6 Fast Flow resin

GE Healthcare

17531801

PD-10 column

GE Healthcare

17085101

PSFM-J1 Medium

FUJIFILM Wako Pure Chemical Corporation

160-25851

DMEM

Nissui Pharmaceutical

05900

Micro Bio-Spin Chromatography Columns

BioRad

7326204

Other Amicon Ultra-15 Centrifugal Filter Units 100,000 MWCO

LEAD CONTACT AND MATERIALS AVAILABILITY Lead Contact: Hidetsugu Asada ([email protected]). Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact. All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement. EXPERIMENTAL MODEL AND SUBJECT DETAILS Microbes E. coli cells were cultured in LB medium. Cell Lines Sf9 insect cells were cultured in PSFM-J1 medium. HEK cells were cultured in DMEM medium. The cell lines used were authenticated by suppliers. METHOD DETAILS Complete Amino Acid Sequence and Expression Construct The cloned Homo sapiens AT2R sequence contains residues 1–346 of 363 residues (UniProt accession number: P50052); AT2R was mutated with Ser208Ala for crystal structure determination as shown in bold. The underlined region represents a thermostabilized apocytochrome b562 from E. coli (M7W/R98I/H102I/R106G; bIIG) that was further improved by modifying a loop region (Gln41–Phe62 replaced with Gly-Ser-Gly-Ser-Gly linker), referred to as mbIIG (D.I. et al., unpublished data). mbIIG was inserted between residues 34 and 35. Additional N- and C-terminal residues retained after tobacco etch virus (TEV) cleavage are shown in italics. Details of the Fab4A03 fragments, including amino acid sequence, CDR, and AT2R-interacting residues were described previously (Asada et al., 2018). GADLEDNWETLNDNLKVIEKADNAAQVKDALTKMRAAALDAGSGSGDILVGQIDDALKLANEGKVKEAQAAAEQLKTTINAYIQKYGTS CSQKPSDKHLDAIPILYYIIFVIGFLVNIVVVTLFCCQKGPKKVSSIYIFNLAVADLLLLATLPLWATYYSYRYDWLFGPVMCKVFGSFLTLNM FASIFFITCMSVDRYQSVIYPFLSQRRNPWQASYIVPLVWCMACLSSLPTFYFRDVRTIEYLGVNACIMAFPPEKYAQWAAGIALMKNILGF IIPLIFIATCYFGIRKHLLKTNSYGKNRITRDQVLKMAAAVVLAFIICWLPFHVLTFLDALAWMGVINSCEVIAVIDLALPFAILLGFTNSCVNPFL YCFVGNRFQQKLRSVFRVPITWLQGKRESLEENLYFQ e2 Structure 28, 1–8.e1–e4, March 3, 2020

Please cite this article in press as: Asada et al., The Crystal Structure of Angiotensin II Type 2 Receptor with Endogenous Peptide Hormone, Structure (2019), https://doi.org/10.1016/j.str.2019.12.003

Protein Expression and Purification (AT2R-mbIIGN-term) The codon-optimized Homo sapiens AT2R-mbIIGN-Term cDNA was cloned into the pFastBac1 vector (Thermo Fisher Scientific) with hemagglutinin (HA) and FLAG epitope tag sequences followed by a TEV cleavage site attached at the N-terminus, and a TEV cleavage site followed by a His8 tag at the C-terminus. Spodoptera frugiperda (Sf9) insect cells expressing AT2R-mbIIGN-Term were grown to a density of 2.0–3.0 3 106 cells/ml (multiplicity of infection (MOI) = 0.5, 60-hour infection) using a baculovirus-mediated expression system. After harvesting the cells by centrifugation (7,000 3 g, 10 minutes, 4 C), cell pellets were flash-frozen at 80 C. The harvested cells were resuspended with hypotonic buffer [10 mM HEPES (pH 7.5), 20 mM KCl, 10 mM MgCl2, protease inhibitor cocktail (Nacalai Tesque)], followed by homogenization and centrifugation (100,000 3 g, 30 minutes, 4 C). Pellets were collected and washed twice to prepare membranes using high osmotic buffer [5 mM HEPES (pH 7.5), 0.5 M NaCl, 10 mM KCl, 5 mM MgCl2, protease inhibitor cocktail]. The washed membrane was solubilized by incubating (1 hour, 4 C) with solubilization buffer [50 mM HEPES (pH 7.5), 800 mM NaCl, 10% (v/v) glycerol, 1.0% (w/v) N-dodecyl-beta-d-maltoside (DDM), 0.2% (w/v) cholesterol] supplemented with iodoacetamide (100 mg/ml) (Wako Pure Chemical Industries) and excess amounts of AngII (Peptide Institute). After centrifugation (100,000 3 g, 30 minutes, 4 C), supernatants were incubated (12–16 hours at 4 C) with TALON Metal affinity resin (TaKaRa Bio) that had been pre-equilibrated with the same buffer. The mixture was washed with 10 column volumes (CVs) of wash buffer [50 mM HEPES (pH 7.5), 200 mM NaCl, 10% (w/v) glycerol, 0.1% (w/v) DDM, 0.02% (w/v) cholesterol, AngII (excess amount)], followed by 3 CVs of elution buffer [25 mM HEPES (pH 7.5), 200 mM NaCl, 10% (v/v) glycerol, 0.03% (w/v) DDM, 0.006% (w/v) cholesterol, AngII (excess amount)]. The collected eluates were pooled and concentrated to 2.5 ml with a concentrator (Amicon Ultra-15 100K; Millipore) and imidazole was removed using a PD-10 column (GE Healthcare). The solution was digested with a twofold molar excess of TEV protease (12–16 hours, 4 C), which was then applied to Ni-Sepharose 6 Fast Flow resin (GE Healthcare). The flowthrough fractions were collected for subsequent crystallization. Radioligand Binding Assay Radioligand binding studies were performed using Sf9 insect cell membranes expressing wild-type or mutant AT2R. The membranes were prepared as described above for protein expression and purification and stored at 80 C until needed. All experiments were performed in triplicate in a total volume of 100 ml. The membranes were solubilized with solubilization buffer [50 mM HEPES (pH 7.5), 800 mM NaCl, 10% (v/v) glycerol, 1.0% (w/v) DDM, 0.2% (w/v) cholesterol]. After centrifugation (100,000 3 g, 30 minutes, 4 C), the protein concentration of supernatants was determined using the bicinchoninic acid (BCA) method (#23227; Thermo Fisher Scientific) with bovine serum albumin (BSA) as a standard. Supernatants were diluted to 0.1 mg/ml and incubated (1 hour at room temperature) with 50 ml of TALON Metal Affinity Resin (50% slurry) that had been pre-equilibrated with the same buffer. The mixture was loaded into an empty column (#732-6204; Bio-Rad). Non-specific binding was determined in the presence of 10 mM AngII. The final concentrations of [125I]-AngII (Perkin Elmer) were typically 0.31–10 nM at saturation. The mixture was washed with a total of 40 CVs of wash buffer [50 mM HEPES (pH 7.5), 200 mM NaCl, 10% (w/v) glycerol, 0.1% (w/v) DDM, 0.02% (w/v) cholesterol, 15 mM imidazole] with gravity flow. Proteins were eluted with 20 CVs of elution buffer [25 mM HEPES (pH 7.5), 200 mM NaCl, 10% (v/v) glycerol, 0.03% (w/v) DDM, 0.006% (w/v) cholesterol, 250 mM imidazole] and collected in counting tubes. The bound 125I-labeled ligands were quantified using an AccuFLEX g 8010 (Hitachi). Data were analyzed by nonlinear curve fitting using GraphPad Prism 5. Binding data are reported as means ± SEM. TGF-a Shedding Assay Activity of the endogenous agonist (AngII) and its derivative ([Sar1, Ile8]-AngII) for AT1R mutants on G protein signaling was determined using a TGF-a shedding assay, which measures Gq/11 and G12/13 signaling as described previously (Inoue et al., 2012). Briefly, a pCAGGS plasmid (gift from Dr. Jun-ichi Miyazaki, Osaka University) encoding wild-type or mutant AT1R (human, full-length, untagged) was cotransfected with a pCAGGS plasmid encoding alkaline phosphatase (AP)-tagged TGF-a (AP-TGFa; human codon-optimized) into HEK293A cells using a polyethylenimine (PEI) transfection reagent (200 ng AT1R plasmid, 500 ng AP-TGFa plasmid, 4 ml of 1 mg/ml PEI solution per well in a 6-well culture plate). After a 1-day incubation, the transfected cells were harvested by trypsinization, neutralized with DMEM containing 10% FCS and penicillin–streptomycin, washed once with Hank’s Balanced Salt Solution (HBSS) containing 5 mM HEPES (pH 7.4), and resuspended in 6 ml of the HEPES-containing HBSS. The cell suspension was seeded into a 96-well plate at a volume of 90 ml per well (typically, 48 wells per transfected cells) and incubated for 30 minutes in a CO2 incubator. Test compounds (10X, diluted in 0.01% BSA and 5 mM HEPES-containing HBSS, 10 ml volume) were added to duplicate wells and incubated for 1 hour. After centrifugation, conditioned media (80 ml) was transferred to an empty 96-well plate. AP reaction solution (10 mM p-nitrophenylphosphate (p-NPP), 120 mM Tris–HCl (pH 9.5), 40 mM NaCl, 10 mM MgCl2) was dispensed into the cell culture plates and plates containing conditioned media (80 ml). Absorbance at 405 nm was measured before and after a 1-hour incubation at room temperature using a microplate reader (SpectraMax 340 PC384; Molecular Devices). Ligand-induced AP-TGF-a release was calculated as described previously (Inoue et al., 2012). Unless otherwise noted, vehicle-treated AP-TGF-a release signal was set as a baseline. AP-TGF-a release signals were fitted with a four-parameter sigmoidal concentration-response curve, from which EC50 and Emax values were obtained, using Prism 7 software (GraphPad Prism). Negative values of logarithmically transformed EC50 values (pEC50) were used to calculate the mean and SEM of independent experiments. The relative intrinsic activity (RAi) (Ehlert et al., 1999) was used to calculate scores for activity of mutant AT1R receptors. From a sigmoid-fitted concentration–response curve of an AT1R mutant, the maximal response (Emax) was divided by the potency (EC50) and the Emax/EC50 value was normalized against Structure 28, 1–8.e1–e4, March 3, 2020 e3

Please cite this article in press as: Asada et al., The Crystal Structure of Angiotensin II Type 2 Receptor with Endogenous Peptide Hormone, Structure (2019), https://doi.org/10.1016/j.str.2019.12.003

that of wild-type AT1R. The resultant dimensionless, relative Emax/EC50 parameter, defined as RAi, was then log10-transformed. Log(RAi) values were used to calculate the means and SEM of independent experiments, and the resultant data were used to evaluate mutant AT1R ligand responses. NanoBiT-Based b-Arrestin Recruitment Assay Agonistic activity of ligands for AT1R mutants (b-arrestin recruitment) was measured using the NanoBiT-based b-arrestin recruitment assay as described previously (Dixon et al., 2016; Shihoya et al., 2018). AT1R was C-terminally fused with a small fragment (SmBiT) of the NanoBiT complementation luciferase with a 15–amino acid flexible linker (GGSGGGGSGGSSSGG). A PCR-amplified AT1R fragment and an oligonucleotide-synthesized SmBiT were assembled and inserted into the pCAGGS plasmid using a NEBuilder HiFi DNA Assembly system (New England Biolabs). A b-arrestin construct derived from human b-arrestin1 (barr1) with R393E and R395E internalization-defective mutations and N-terminal fusion of a large fragment (LgBiT) with the 15–amino acid linker was described previously (Shihoya et al., 2018). The plasmid encoding the AT1R-SmBiT construct was cotransfected with the plasmid encoding the LgBiT-barr1 into HEK293A cells using the PEI method (400 ng AT1R-SmBiT plasmid, 200 ng LgBiT-barr1 plasmid, 8 ml of 1 mg/ml PEI solution per 6-cm culture dish). After a one-day incubation, transfected cells were harvested with EDTA-containing Dulbecco’s phosphate-buffered saline and resuspended in 4 ml of HBSS containing 5 mM HEPES and 0.01% BSA (BSA-HBSS). The cell suspension was seeded into a white 96-well plate at a volume of 80 ml per well (typically, 48 wells per transfected cells) and loaded with 20 ml of 50 mM coelenterazine (Carbosynth). After a 2-hour incubation at room temperature, the background luminescent signals were measured using a luminescent microplate reader (SpectraMax L; Molecular Devices). Test compounds (6X, diluted in BSA-HBSS) were manually added to the cells (20 ml) in duplicate. After ligand addition, the luminescent signals were measured for 15 minutes at 20-second intervals. The luminescent signal was normalized to the initial count, and the fold-change values over 5–10 minutes after ligand stimulation were averaged. The fold-change b-arrestin recruitment signals were fitted with a four-parameter sigmoidal concentration–response, and the pEC50 and Emax values were obtained as described above. Data Collection, Structure Determination, and Analysis The diffraction data were collected at 100 K at the SPring-8 micro-focus beamline BL32XU (Hirata et al., 2013) (Japan) using an EIGER X 9M detector (Dectris). A micro-focused beam with a size of 10 mm 3 10 mm and a wavelength of 1 A˚ was used for both a raster scan and data collection. A dataset with a total oscillation range of 5 and 0.1 oscillations per frame was collected from each crystal under an absorbed dose of 10 MGy. A total of 470 datasets were collected with the automated data collection system ZOO (Hirata et al., 2019) and merged, integrated, and scaled using the KAMO system (Yamashita et al., 2018). The structure of the AT2R-mbIIGN-Term/Fab4A03 complex was determined by molecular replacement with the program MOLREP (Vigan and Teplyakov, 1997), using the atomic coordinates of AT2R (PDB ID: 5XJM) and BRIL (PDB ID: 1M6T) as the search models. The model was initially refined using the ‘jelly body’ refinement function implemented in REFMAC5 (Murshudov et al., 2011), and was further rebuilt in COOT and refined with phenix.refine (Table 1). The refined structures were visualized with PyMOL (http://www.pymol.org/) and CueMol2 (http://www.cuemol.org/). The PISA server (Krissinel, 2010) was used to identify protein–protein interactions and estimate the solvent-accessible surface area. QUANTIFICATION AND STATISTICAL ANALYSIS Statistical analyses were performed using GraphPad Prism 5 or 7 software. Radioactive ligand binding data was recorded in triplicates for each conditions and respective statistical details are included in the Method Details section as well as in the figure captions of each data plot. Data were expressed as mean ± SEM. Concentration-response curves were fitted to all data by the Nonlinear Regression: Variable slope (four parameter) in the Prism 7 tool. Liner regression and representation of 90% confidence bands were performed by the Prism 7 tool. DATA AND CODE AVAILABILITY Coordinates and structure factors have been deposited in the Protein Data Bank (PDB ID 6JOD). The PDB accession codes 5XJM, 1M6T, 6DO1, 4YAY and 5UNF were referenced in this study. The UniProt accession codes P50052 for human AT2R was used in this study. All other data are available from the corresponding authors on reasonable request.

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