Structural Basis for Ligand Recognition and FunctionalSelectivity at Angiotensin Receptor*�
Received for publication, August 31, 2015, and in revised form, September 24, 2015 Published, JBC Papers in Press, September 29, 2015, DOI 10.1074/jbc.M115.689000
Haitao Zhang‡§1, Hamiyet Unal¶1,2, Russell Desnoyer¶, Gye Won Han§, Nilkanth Patel‡, Vsevolod Katritch‡,Sadashiva S. Karnik¶3, Vadim Cherezov§, and Raymond C. Stevens‡§4
From the Departments of ‡Biological Sciences and §Chemistry, Bridge Institute, University of Southern California, Los Angeles,California 90089 and the ¶Department of Molecular Cardiology, Lerner Research Institute of Cleveland Clinic,Cleveland, Ohio 44195
Background: Angiotensin receptor (AT1R) blockers are critical therapeutics used to treat cardiovascular disease.Results: We solved the AT1R-olmesartan structure and identified specific interactions for olmesartan derivatives with differentfunctions.Conclusion: Our results identified residues critical for the binding of different ligands and allosteric modulation by sodium ion.Significance: Our results provide new insights into the structural basis for ligand recognition and functional selectivity at AT1R.
Angiotensin II type 1 receptor (AT1R) is the primary blood pres-sure regulator. AT1R blockers (ARBs) have been widely used inclinical settings as anti-hypertensive drugs and share a similarchemical scaffold, although even minor variations can lead to dis-tinct therapeutic efficacies toward cardiovascular etiologies. Thestructural basis for AT1R modulation by different peptide and non-peptide ligands has remained elusive. Here, we report the crystalstructure of the human AT1R in complex with an inverse agonistolmesartan (BenicarTM), a highly potent anti-hypertensive drug.Olmesartan is anchored to the receptor primarily by the residuesTyr-351.39, Trp-842.60, and Arg-167ECL2, similar to the antagonistZD7155, corroborating a common binding mode of differentARBs. Using docking simulations and site-directed mutagenesis,we identified specific interactions between AT1R and differentARBs, including olmesartan derivatives with inverse agonist, neu-tral antagonist, or agonist activities. We further observed that themutation N1113.35A in the putative sodium-binding site affectsbinding of the endogenous peptide agonist angiotensin II but notthe �-arrestin-biased peptide TRV120027.
Angiotensin II (AngII)5 type 1 receptor (AT1R) is a G pro-tein-coupled receptor (GPCR), mainly found in heart, brain,
liver, and kidneys, regulating normal blood pressure, as well asfluid and electrolyte homeostasis (1, 2). Overstimulation ofAT1R leads to diseases such as hypertension, cardiovascularhypertrophy, and fibrosis, whereas blocking the activity ofAT1R lowers blood pressure (3, 4). Considering the critical rolesof AT1R in cardiovascular pathophysiology, AT1R blockers(ARBs) have been developed and clinically used for the treat-ment of hypertension, diabetic nephropathy, cardiac hypertro-phy, arrhythmia, and renal failure (5– 8). ARBs share similarstructural motifs and include widely used anti-hypertensivedrugs, such as losartan, candesartan, valsartan, irbesartan,telmisartan, eprosartan, olmesartan, and azilsartan (5, 6, 9, 10).Although most ARBs are considered to be neutral AT1R antag-onists, olmesartan, for example, is an established inverse ago-nist toward inositol phosphate (IP) production (11).
In addition to anti-hypertensive activity, some ARBs alsoshow variable efficacies toward protection against organ dam-age in diabetic nephropathy, cardiac hypertrophy, arrhythmia,and renal failure (5). Multiple studies suggest that such addi-tional tissue-protective benefits from treatment with ARBs maybe mediated by AT1R �-arrestin signaling, although the struc-tural basis for differential activation of this mechanism by ARBsis unknown (8, 10). AngII analogs, such as TRV120027, lackingG protein agonism but activating �-arrestin signaling, protectthe heart and vasculature in a pathological setting better thancommon ARBs (2, 8, 9), suggesting the need for a better under-standing of the mechanisms for functional selectivity of differ-ent AT1R ligands.
Recently, we determined the room temperature crystalstructure of AT1R in complex with its antagonist ZD7155 usingserial femtosecond crystallography at an x-ray free-electronlaser (12). Here, we further elucidated the function of a repre-sentative inverse agonist by solving the crystal structure of olm-esartan-bound AT1R using conventional x-ray cryo-crystallog-raphy at a synchrotron source. Comparison of the two AT1Rstructures revealed similar conformations of the receptor and
* This work was supported by National Institutes of Health Grants U54GM094618 (to V. K., V. C., and R. C. S.) and R01 HL57470 and R01 HL115964(to S. S. K.) and by Grant T32 HL007914 (to H. U.). The authors declare thatthey have no conflicts of interest with the contents of this article.
� This article was selected as a Paper of the Week.The atomic coordinates and structure factors (code 4ZUD) have been deposited
in the Protein Data Bank (http://wwpdb.org/).1 Both authors contributed equally to this work.2 Present address: Dept. of Basic Sciences, Faculty of Pharmacy and Betul Ziya
Eren Genome and Stem Cell Center, Erciyes University, Kayseri 38039, Turkey.3 To whom correspondence may be addressed: Dept. of Molecular Cardiol-
ogy, Lerner Research Institute of Cleveland Clinic, Cleveland, OH 44195.Tel.: 216-444-1269; Fax: 216-444-9263; E-mail: [email protected].
4 To whom correspondence may be addressed: Depts. of Biological Sciences andChemistry, Bridge Institute, University of Southern California, Los Angeles, CA90089. Tel.: 213-740-1992; Fax: 213-821-7854; E-mail: [email protected].
5 The abbreviations used are: AngII, angiotensin II; AT1R, angiotensin II type 1receptor; ARB, AT1R blocker; GPCR, G protein-coupled receptor; IP, inositolphosphate; DDM, n-dodecyl-�-D-maltopyranoside; CHS, cholesterol
hemisuccinate; LCP, lipidic cubic phase; HBSS, Hanks’ balanced salt solu-tion; �-OR, �-opioid receptor; SNP, single nucleotide polymorphism.
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conserved molecular recognition modes for antagonist andinverse agonist toward AT1R. Molecular docking simulationsand site-directed mutagenesis led to identification of residuesin AT1R that differentially interact with ARBs, as well as olm-esartan derivatives in which specific modifications alteredpharmacological properties from inverse agonism to neutralantagonism and partial agonism. Our results thus revealed aparadigm for pharmacological modulation of functional selec-tivity that might be generally applicable to other ARBs interact-ing with AT1R. Furthermore, mutagenesis of the conserved res-idues in the putative sodium-binding pocket suggested theirinvolvement in AT1R regulation by the �-arrestin biased pep-tide ligand TRV120027.
Experimental Procedures
Protein Engineering for Structural Studies—DNA encodingthe human AT1R was optimized for expression in insect cellsand synthesized by GenScript. The construct used for theAT1R-olmesartan structure has truncations of residues 1, 7–16,and 316 –359. The apocytochrome b562 RIL from Escherichiacoli with mutations M7W, H102I, and R106L (BRIL (13)) wasfused to the N terminus of AT1R. The BRIL-AT1R chimerasequence was subcloned into pFastBac1 vector (Invitrogen),with N-terminal hemagglutinin (HA) signal sequence, a FLAGtag, a His10 tag, and a tobacco etch virus protease cleavage site.
Protein Expression and Purification—The BRIL-AT1R pro-tein was expressed in Spodoptera frugiperda (Sf9) insect cellsusing the Bac-to-Bac baculovirus expression system (Invitro-gen). Cells expressing the BRIL-AT1R protein were lysed byrepeated washing and centrifuging with the hypotonic buffer(10 mM HEPES, pH 7.5, 10 mM MgCl2, 20 mM KCl) and the highosmotic buffer (10 mM HEPES, pH 7.5, 1.0 M NaCl, 10 mM
MgCl2, 20 mM KCl), adding the EDTA-free complete proteaseinhibitor mixture tablets (Roche Applied Science). The washedmembranes were suspended in the hypotonic buffer with 100�M olmesartan (Sigma). After a 1-h incubation at 4 °C, themembranes were then solubilized in 50 mM HEPES, pH 7.5, 500mM NaCl, 1% (w/v) n-dodecyl-�-D-maltopyranoside (DDM,Anatrace), 0.2% (w/v) cholesterol hemisuccinate (CHS, Sigma),and 20% (v/v) glycerol, for 4 h. The supernatant containing thesolubilized BRIL-AT1R protein was isolated by high speed cen-trifugation and then bound to TALON IMAC resin (Clontech)overnight. The resin was washed with 10 column volumes ofwashing buffer I (50 mM HEPES, pH 7.5, 500 mM NaCl, 10%(v/v) glycerol, 0.1% (w/v) DDM, 0.02% (w/v) CHS, 20 mM imid-azole, and 20 �M olmesartan) and 10 column volumes of wash-ing buffer II (50 mM HEPES, pH 7.5, 500 mM NaCl, 10% (v/v)glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS, 50 mM imidazole,and 20 �M olmesartan). The BRIL-AT1R protein was eluted by3 column volumes of eluting buffer (50 mM HEPES, pH 7.5, 500mM NaCl, 10% (v/v) glycerol, 0.02% (w/v) DDM, 0.004% (w/v)CHS, 300 mM imidazole, and 100 �M olmesartan). The BRIL-AT1R protein was then treated overnight with His-taggedtobacco etch virus protease and peptide:N-glycosidase F tocleave the tags and glycosylation sites and concentrated to 30mg/ml with a 100-kDa cutoff concentrator (Vivaspin) for crys-tallization. The protein yield and monodispersity were tested byanalytical size exclusion chromatography.
Lipidic Cubic Phase Crystallization—The BRIL-AT1R pro-tein was reconstituted in lipidic cubic phase (LCP) by mixingthe protein with monoolein supplemented with 10% choles-terol using a lipid syringe mixer (14). Crystallization trials wereperformed by an NT8-LCP robot (Formulatrix) in 96-well glasssandwich plates (Marienfeld) using 40 nl of protein-loaded LCPand 800 nl of precipitant solution per well. Plates were stored at20 °C and imaged using a Rock Imager 1000 (Formulatrix). Thecrystals grew in the condition of 100 mM sodium citrate, pH 5.0,400 mM KH2PO4, 25% (v/v) PEG400, and 6% (v/v) DMSO. Thecrystals were harvested using micromounts (MiTeGen) directlyfrom LCP and flash-frozen in liquid nitrogen for diffractiondata collection.
Diffraction Data Collection and Structural Determination—Crystallographic data collection was performed at 23ID-Dbeamline (GM/CA) of the Advanced Photon Source atArgonne National Laboratory. An unattenuated 10-�m mini-beam with a wavelength of 1.0330 Å was used at 1 s exposureand 1.0° oscillation. Diffraction data were collected using a Pila-tus3 6M detector from four non-overlapping spots on a singlecryo-cooled at 100K crystal of 70 � 70 � 15 �m3 in size andthen integrated and scaled using XDS (15). The molecularreplacement solution was obtained by Phaser (16) in a CCP4suite (17) using separate AT1R and BRIL molecules from theAT1R-ZD7155 structure (Protein Data Bank code 4YAY) assearch models. The resulting BRIL-AT1R chimera model wasrefined by cycling between Refmac5 (18) and manual adjust-ments in Coot (19). Because of the merohedral twinned data inthe P32 space group, twinning refinement with twin law (h,k,land k,h,�l) determined by phenix.xtriage (20) and Refmac5 wasused. The presence of twinning did not impede structure solu-tion by molecular replacement. The overall model has a goodstereochemistry with no Ramachandran outliers (94.3% infavored and 5.7% in allowed regions) as determined by Mol-Probity (21). The crystallographic data collection and refine-ment statistics are shown in Table 1.
Docking of Olmesartan Derivatives into the AT1R Ligand-binding Pocket—Olmesartan derivatives were docked into theAT1R crystal structure using the energy-based docking proto-col implemented in ICM molecular modeling software suite(Molsoft, LLC). The initial receptor docking model was gener-ated by adding missing side chains and hydrogen atoms and byoptimizing their conformations, followed by generation of softpotential maps of the receptor in a large box (30 � 30 � 30 Å3)covering the extracellular half of the receptor. Molecular mod-els of compounds were generated from two-dimensional repre-sentations, and their three-dimensional geometry was opti-mized using MMFF-94 force field (22). Molecular dockingemployed biased probability Monte Carlo optimization of theligand internal coordinates in the grid potentials of the receptor(23). To ensure convergence of the docking procedure, at leastfive independent docking runs were performed for each ligandstarting from a random conformation; Monte Carlo samplingand optimization were performed at “thoroughness” parameterset to 30. The objective energy function included the ligandinternal strain and a weighted sum of the grid map values inligand atom centers. Up to 30 alternative complex conforma-tions of the ligand-receptor complex were generated and
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where EvW, Eel, Ehb, Ehp, and Esf are van der Waals, electrostatic,hydrogen bonding, non-polar, and polar atom solvation energydifferences between bound and unbound states; Eint is theligand internal strain; �STor is the ligand conformationalentropy loss upon binding; T � 300 K, and �1,2,3,4 are all ligand-and receptor-independent constants. The results of individualdocking runs for each ligand were considered consistent if atleast three of the five docking runs produced similar ligandconformations (root mean square deviation of �2.0 Å) andBinding Score ��20.0 kJ/mol. All calculations were per-formed using 32-core AMD Opteron 2.3 GHz work stationrunning Linux CentOS 6, taking about 10 min/ligand. Theunbiased docking procedure did not use distance restraintsor any other a priori derived information for the ligand-receptor interactions.
Construction of AT1R Mutants and Cell Transfection—Com-plementary DNA (cDNA) encoding the human AT1R withN-terminal HA-tag (HA-AT1R) was originally cloned into theexpression vector pMT3 at the EcoRI and NotI sites. The singlemutants were constructed by a PCR-based site-directedmutagenesis strategy as described previously (26). COS1 cellswere grown in Dulbecco’s modified Eagle’s medium (DMEM)(Invitrogen) supplemented with 10% fetal bovine serum (FBS,Thermo Fisher Scientific) and 100 IU of penicillin/streptomy-cin (Sigma). Cells were seeded onto poly-D-lysine-treated cellculture plates at a density of 3 � 106 cells per 10-cm diameterplate. After overnight culture, the cells were transiently trans-fected with wild-type or mutant AT1R DNA using FuGENE 6transfection reagent (Roche Applied Science) according to themanufacturer’s instructions.
Membrane Preparation for Binding Assays—Ligand bindingwas analyzed using total membranes prepared from COS1 tran-siently expressing wild-type HA-AT1R and BRIL-AT1R con-structs. Transfected/infected cells were harvested in osmoticlysis buffer (25 mM Tris-HCl, pH 7.5, and 5 mM EDTA, pH 8.0)with protease inhibitor mixture (Sigma) homogenized by adounce homogenizer. The homogenate was incubated by rotat-ing for 10 min at 4 °C and centrifuged for 5 min at 200 � g. Thesupernatant was then centrifuged at 37,000 � g for 30 min at4 °C. The precipitate containing the total membranes was sus-pended in membrane binding buffer (140 mM NaCl, 5.4 mM
KCl, 1 mM EDTA, 0.006% bovine serum albumin (BSA), 25 mM
HEPES, pH 7.4). Protein concentration was determined by theBio-Rad Protein Assay. For both saturation and competitionbinding assays, 10 �g of homogeneous cell membrane was usedper well as described above.
125I-AngII Binding Assays—Saturation binding assays wereperformed under equilibrium conditions with 125I-AngII (giftfrom Dr. Robert Speth, University of Mississippi) concentra-tions ranging between 0.125 and 12 nM (specific activity, 2176Ci/mmol) as triplicates in 96-well plates for 1 h at room tem-perature as described previously (27). Nonspecific binding was
measured in the presence of 10�5 M 125I-AngII (Bachem). Thecells were harvested by filtering the binding mixture throughWhatman GF/C glass fiber filters (102 � 256 mm), which wereextensively washed with washing buffer (20 mM sodium phos-phate, 100 mM NaCl, 10 mM MgCl2, 1 mM EGTA, pH 7.2). Thebound ligand fraction was determined as the counts/min usinga scintillation counter (MicroBeta2 Plate Counter, PerkinElmerLife Sciences). The binding kinetics were analyzed by the non-linear curve-fitting program LigandR. The means � S.E. for theKd and Bmax values were calculated.
[3H]Olmesartan Binding Assays—Saturation binding assayswere performed under equilibrium conditions, with [3H]olm-esartan (American Radiolabeled Chemicals) concentrationsranging between 0.125 and 12 nM (specific activity, 16Ci/mmol) as duplicates in 96-well plates for 1 h at room tem-perature. Nonspecific binding was measured in the presence of10�5 M olmesartan (gift from Daichi-Sankyo Co., Japan). Thecells were harvested by filtering the binding mixture throughWhatman GF/C glass fiber filters (102 � 256 mm), which wereextensively washed with washing buffer (20 mM sodium phos-phate, 100 mM NaCl, 10 mM MgCl2, 1 mM EGTA, pH 7.2). Thefilter membranes were soaked in 5 ml of Ecoscint A scintillationfluid (National Diagnostics) and incubated for 1 h at room tem-perature. The bound ligand fraction was determined as the dis-integrations/min using a Beckman LS 6000 Liquid ScintillationCounter (Global Medical Instrumentation). The binding kinet-ics were analyzed by nonlinear curve fitting. The means � S.E.for the Kd and Bmax values were calculated.
Competition Binding Assays—Competition binding assayswere performed under equilibrium conditions, with 2 nM radio-ligand and concentrations of the competing ligand rangingbetween 0.04 and 1000 nM. The binding kinetics were analyzedby the nonlinear curve-fitting program GraphPad Prism 5. Themeans � S.E. for the IC50 values were calculated. Olmesartanderivatives were gifts from Daichi-Sankyo Co., Japan. Losartanwas a gift from Merck, and candesartan was a gift from AstraZeneca. Telmisartan and eprosartan were purchased fromSanta Cruz Biotechnology, and valsartan was purchased fromToronto Research Chemicals.
Fluorescent Imaging Plate Reader (FLIPR�)-based Intracellu-lar Calcium Levels in Cells—Calcium levels were measuredusing FLIPR� calcium 5 assay kit (Molecular Devices). A daybefore the calcium experiments, HA-AT1R- and BRIL-AT1R-transfected cells were seeded at a density of 100,000 cells/well in100 �l of medium onto a 96-well clear bottom black cell cultureplate that was pre-coated with poly-L-lysine. The plate wasmaintained in a cell culture incubator set at 37 °C for 26 –28 h.On the day of the experiment, cells were initially serum-starvedfor 2 h by replacing the medium with 80 �l of serum-freeDMEM. Following serum starvation, 100 �l of calcium-sensi-tive dye along with 2� (2.5 mM final concentration) probenecid(Life Technologies, Inc.) was added to the cells. During thisstep, for antagonist dose-response curves, 20 �l of desired con-centrations of antagonist from a 10� stock prepared in D-PBS(1.47 mM KH2PO4, 138 mM NaCl, 2.67 mM KCl, 8.1 mM
Na2HPO4, pH 7.3) were added to the cells. For all other wells 20�l of D-PBS was added. The cells were maintained for 30 min inthe cell culture incubator set at 37 °C and another 30 min at
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room temperature. During the incubation, ligands at 5� thedesired final concentration in D-PBS were prepared in a U-bot-tom 96-well plate. Both the cells and ligand containing 96-wellplates were loaded on to a Flexstation 3 instrument (MolecularDevices). The instrument was programmed in FLEX mode toadd ligands (50 �l at 5� concentration) to the cells and to mon-itor the fluorescence before and after adding the ligands. Dur-ing measurements of agonist dose response, AngII was added atvarious concentrations. 100 nM AngII was added for antagonistdose-response curves wherein the cells were already pre-treated with the desired concentrations of antagonist duringthe calcium dye loading step.
IP Formation Studies—COS1 cells (cultured in 60-mm Petridishes), 24 h after transfection, were labeled for 24 h with[3H]myoinositol (1.5 mCi/Petri dish), 22 Ci/mmol specificactivity (Amersham Biosciences), at 37 °C in DMEM contain-ing 10% bovine calf serum. The labeled cells were washed withserum-free medium three times and incubated with DMEMcontaining 10 mM LiCl for 20 min; specified ligands (1 �M finalconcentration) were added, and incubation was continued foranother 60 min at 37 °C. At the end of incubation, the mediumwas removed, and total soluble IP was extracted from the cellsby the perchloric acid extraction method. The amount of[3H]IP in the perchloric acid extract was counted.
FIGURE 1. Comparisons of the ligand binding and functional activities between the wild-type (HA-AT1R) and crystallized construct (BRIL-AT1R). A,binding of olmesartan to the wild-type HA-AT1R and crystallized BRIL-AT1R. Binding studies were performed using isolated membranes from transientlytransfected COS1 cells. Saturation binding curves were measured using [3H]olmesartan, and the corresponding Kd and Bmax values were obtained by non-linearcurve fitting. B, displacement of [3H]olmesartan with the agonist AngII and the antagonist [Sar1,Ile8]AngII in the wild-type HA-AT1R and the crystallizedBRIL-AT1R. Binding studies were performed using isolated membranes from transiently transfected COS1 cells. Competition binding curves for peptide agonistAngII and peptide antagonist [Sar1,Ile8]AngII were generated, and the corresponding IC50 values were calculated. The IC50 values for AngII to inhibit olmesartanbinding were 1.8 � 0.2 and 18.4 � 2.3 nM for HA-AT1R and BRIL-AT1R, respectively. The IC50 values for [Sar1,Ile8]AngII to inhibit olmesartan binding were 1.2 �0.1 and 25.5 � 4.9 nM for HA-AT1R and BRIL-AT1R, respectively. C, intracellular calcium responses of wild-type HA-AT1R and BRIL-AT1R. AngII and olmesartandose-response curves for HA-AT1R and BRIL-AT1R are shown. For the antagonist dose response, cells were treated with 0 –10 �M concentrations of olmesartanfollowed by stimulation with 100 nM AngII. The EC50 values for AngII dose response were 10.2 � 3.2 and 11.9 � 3.1 nM for HA-AT1R and BRIL-AT1R, respectively.The IC50 values for olmesartan to inhibit AngII response were between 23.9 � 8.0 and 4.4 � 0.4 nM for HA-AT1R and BRIL-AT1R, respectively. All results aboveare presented as mean � S.E. and represent three experiments performed in triplicate.
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ELISA-based Estimation of Wild Type and Mutated AT1R inCells—24 h after transfection, cells were split into 12-well platesat a density of 500,000 cells per well. After an additional 24 h,cells were washed twice with Hanks’ balanced salt solution(HBSS: 0.44 mM KH2PO4, 0.34 mM Na2HPO4, 137 mM NaCl,5.36 mM KCl, 1.26 mM CaCl2, 0.81 mM MgSO4, 0.5 mM MgCl2,4.17 mM NaHCO3, 5.55 mM D-glucose, pH 7.3) with 1% BSA,and HA-hAT1R was labeled with anti-HA antibody (1 �g/ml,Sigma) in HBSS, 1% BSA for 1 h at 4 °C. The cells were thenwashed twice with HBSS, 1% BSA, and cells were fixed with 4%paraformaldehyde in HBSS for 15 min. All steps prior to cellfixing were carried out on ice to prevent endocytosis of AT1Rduring processing of the samples. The cells were washed twicewith HBSS, 0.5% BSA and then incubated for another 1 h atroom temperature in HBSS, 0.5% BSA supplemented withhorseradish peroxidase-conjugated anti-mouse IgG (1:1000,Sigma) secondary antibody. The cells were washed twice withHBSS, 0.5% BSA. Finally, the cells were incubated with 400 �lper well of o-phenylenediamine dihydrochloride substrate (0.4mg/ml) prepared in 0.05 M phosphate/citrate buffer, pH 5.0,containing 0.03% sodium perborate (Sigma) for 10 min in thedark at room temperature. The reaction was stopped with 100�l per well of 3 N HCl, and the absorbance was read at 492 nmusing an ELISA plate reader (Molecular Devices). For detectionof total protein, 0.1% Triton X-100 was included in all the buf-fers to promote permeabilization of the cell membrane, and thecells were fixed with 4% paraformaldehyde before adding theprimary antibody. Control experiments were performed withmock-transfected cells and wild type-transfected cells to whichthe primary antibody was not added.
Statistical Analysis—Results are presented as mean � S.E.Changes in specific binding of radiolabeled ligands and cell sur-face expression of AT1R constructs were normalized to thosemeasured with the wild-type AT1R control (100%). IC50 valuesin binding assays were determined by non-linear regressionanalysis using the Prism software (GraphPad Software).
Results
Comparison of the Two AT1R Structures—Our extensiveefforts on AT1R stabilization and crystallization in LCPresulted in two different types of crystals. The first engineeredAT1R construct with N-terminally fused BRIL in complex withits antagonist ZD7155 yielded a high density of microcrystalswith an average size of 10 � 2 � 2 �m3. In our previous studies,we applied a recently developed method of serial femtosecondcrystallography using LCP as a crystal growth and carriermatrix (LCP-SFX) to deliver microcrystals to the intersectionwith an x-ray free-electron laser beam (14, 28 –30) and solved aroom temperature structure of the antagonist-bound AT1R(12). A different AT1R construct with a 4-residue shorter Cterminus was studied by the ligand binding and calcium signal-ing assays to determine its functional and pharmacologicalproperties compared with the wild-type AT1R (Fig. 1). The[3H]olmesartan binding affinities for HA-AT1R and BRIL-AT1R are similar (Fig. 1A), although the displacements of thebound [3H]olmesartan by AngII and [Sar1,Ile8]AngII are morethan 10-fold less efficient for BRIL-AT1R compared withHA-AT1R (Fig. 1B). However, the calcium-signaling activationby the bound AngII for HA-AT1R and BRIL-AT1R showed sim-ilar EC50 values (Fig. 1C). This finding suggests that N-terminalBRIL insertion may sterically hinder the entrance of largerpeptide ligands (such as AngII) into the ligand bindingpocket of AT1R. This observation is consistent with ourprevious results that mutations on extracellular loop 2 ofAT1R (such as F182ECL2A) affected the peptide ligand([Sar1,Ile8]AngII) binding, whereas F182ECL2A substitutiondid not affect the non-peptide ligand (candesartan) binding(12). This construct in complex with an inverse agonist olm-esartan was crystallized in a different space group (P32 forthe AT1R-olmesartan complex versus C2 for AT1R-ZD7155)and produced larger crystals with a maximum size of 70 �70 � 15 �m3, allowing us to use conventional synchrotronradiation to determine the olmesartan-bound AT1R struc-ture in a cryo-cooled state (Table 1).
The two AT1R structures exhibit similar conformations ofthe seven-transmembrane bundle of the receptor (root meansquare deviation of C� � 0.85 Å in 92% of the structure) andorientation of the N-terminal BRIL fusion protein, despite dif-ferent crystal packing (Fig. 2, A and B). No electron density forhelix VIII was observed in the AT1R-olmesartan structure com-pared with a 13-residue long helix VIII in the AT1R-ZD7155structure. Apparently, helix VIII was destabilized by the 4-res-idue truncation at the C terminus applied in this construct;however, lack of a stable helix VIII did not noticeably impactconformations of the other parts of receptor.
Olmesartan-binding Mode—The AT1R-olmesartan crystalstructure solved in this study confirmed our previous olmesar-tan docking simulations based on the AT1R-ZD7155 structure(12), with root mean square deviation � 1.1 Å between thepredicted and experimentally derived ligand poses. The olm-esartan-binding pocket is composed of residues from all seventransmembrane helices, as well as from two extracellular loopsECL1 and ECL2 (Fig. 2C). Conformations of the pocket resi-dues in the two structures are very close, with the largest vari-
TABLE 1Crystallographic data collection and refinement statisticsOne crystal was used for the data set. Highest resolution shell is shown in parentheses.
AT1R-olmesartan
Data collectionSpace group P32Cell dimensions
a, b, c (Å) 41.20, 41.20, 251.16�, �, (°) 90, 90, 120
ations being a rotamer change in Ile-2887.39 (superscript indi-cates residue number as per the Ballesteros-Weinstein, 1995,nomenclature (31)), as well as �1 Å shifts of Tyr-351.39 in helixI and residues Gly-1965.39 to Leu-2015.44 in helix V toward theligand pocket. These changes slightly decreased the pocket size,probably reflecting the smaller linear size of olmesartan as com-pared with ZD7155. Three residues, Tyr-351.39, Trp-842.60, andArg-167ECL2, were found to be critical for olmesartan binding,similar to our findings for AT1R-ZD7155 interactions. Arg-167ECL2 formed extensive networks of hydrogen bonds and saltbridges with the acidic tetrazole ring and the carboxyl group onthe imidazole moiety of olmesartan in the AT1R-olmesartancomplex. Mutations of Arg-167ECL2 to alanine and glutamineabolished both the AngII and olmesartan binding to AT1R(Table 2). The Arg-167ECL2 to lysine mutant did not impact
AngII binding but reduced binding affinity of olmesartan, sug-gesting that the lysine side chain in this position does not fullysubstitute for the arginine side chain that forms a salt bridgeand hydrogen bond network with both ends of olmesartan.Notably, such positioning of Arg-167ECL2 in AT1R is not con-served in other GPCRs, which may be the key for the uniquebinding specificity of ARBs to AT1R and their absence of cross-activity to other GPCRs. Tyr-351.39 and Trp-842.60 formedadditional hydrogen bond and �-� interactions, both with theimidazole moiety of olmesartan (Fig. 2). Mutation of Tyr-351.39
to alanine abolished both AngII and olmesartan binding,although larger hydrophobic isoleucine and phenylalanine sub-stitutions retained the binding of olmesartan but not of AngII(Table 2). In alanine- and isoleucine-substituted mutants forTrp-842.60, binding of both AngII and olmesartan was abol-
FIGURE 2. Crystal structure of AT1R-olmesartan. A and B, crystal packing comparison of the two AT1R structures bound to the inverse agonist olmesartan (this work)and the antagonist ZD7155 (Protein Data Bank code 4YAY). Crystal packing views perpendicularly (left) and parallel (right) to the membrane are shown for the twoN-terminal BRIL-fused AT1R structures bound to different ligands, with AT1R shown as green ribbons and BRIL shown as cyan ribbons. C, interactions between AT1R andolmesartan were determined by crystal structure. D, three SNP variations, A1634.60T, T2827.33M, and C2897.40W, located in close proximity to the AT1R orthostericbinding pocket may differentially affect efficacies of specific ARBs in individuals carrying these SNPs via changes in direct or indirect interactions with the pocket.
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ished, but olmesartan binding was preserved by the phenylala-nine mutant (Table 2).
Previously, we suggested that Lys-1995.42 at the extracellularside of helix V may form salt bridges or water-mediated inter-actions with the acidic moieties of ARBs (12). However, noapparent electron density for the Lys-1995.42 side chain waspresent in the previous AT1R-ZD7155 structure, likely due toconformational heterogeneity of this residue at room tempera-ture. In the current cryo-cooled structure of AT1R-olmesartan,clear electron density was observed for Lys-1995.42, revealing itsmore stable conformation; however, no direct interaction ofthis residue with the ligand was observed. In accord with thesestructural findings, we found little effect of the K1995.42A muta-tion on olmesartan binding (Table 2). At the same time, Lys-
1995.42 played an important role for AngII binding, as theK1995.42A mutant completely abolished AngII binding. Over-all, in both structures the residues forming the AT1R ligand-binding pocket were in similar conformations while interactingwith two different types of ligands, inverse agonist and antago-nist. Similar binding poses of ligands observed in the two crystalstructures support a conserved binding mode for most otherARBs in AT1R, as predicted based on the AT1R-ZD7155 struc-ture. The difference in functional effects between antagonistsand inverse agonists is likely due to the different stabilizingeffects of these ligands on different receptor conformations.
Molecular Recognition of Different ARBs—In our previousdocking simulations, most ARBs were predicted to be engagedin three critical interactions with Arg-167ECL2, Trp-842.60, and
TABLE 2Binding of AngII and olmesartan to various AT1R mutants
a The ELISA reading arbitrary units for HA-AT1R in COS1 total membrane prepared from transient transfections was set as 100%. The Bmax for HA-AT1R was 48.9 � 6.9pmol/mg (n � 4). Results are presented as means � S.E. and represent three experiments performed in triplicate. NB means no binding. ND means not determined.
TABLE 3Competition displacement of 3H�olmesartan binding to various AT1R mutants by different ARBsResults are presented as means � S.E. and represent three experiments performed in triplicate.
Tyr-351.39 in the AT1R ligand-binding pocket (12). Here, weused [3H]olmesartan as the radioligand and performed compe-tition displacement assays with various AT1R mutants to vali-date the docking results (Table 3). The analysis for candesartan,telmisartan, and valsartan showed that their binding modes aresimilar to olmesartan (Table 3). Losartan affinity decreased sig-nificantly more than other biphenyl-tetrazole ARBs in the Trp-842.60Phe mutation. Docking results for losartan suggested thatArg-167ECL2 does not make optimal interactions with the sub-stituted imidazole ring moiety; consequently, the ring stackingwith Trp-842.60 plays an even more important role in losartanbinding. Eprosartan lacks the tetrazole group and one of thetwo benzene rings in the common scaffold present in mostARBs. Docking simulations indicated a less optimal geometryfor salt bridges between eprosartan carboxyl groups and Arg-
167ECL2, which is probably compensated for by improved inter-actions with Tyr-351.39 and Trp-842.60. Indeed, both W842.60Fand Y351.39F mutants dramatically reduced eprosartan bindingto AT1R (�Ki � 301 � 190 and 8.2 � 2.9, respectively; Table 3).In addition, eprosartan’s alkyl tail that is attached to the imid-azole moiety was predicted to extend into the hydrophobic sub-pocket on the bottom of the AT1R ligand-binding pocket, con-sisting of Ile-2887.39 and Tyr-2927.43. The Ile-2887.39 waspredicted to form additional interactions with the thiophenegroup of eprosartan. Alanine substitution of Ile-2887.39 andTyr-2927.43 significantly decreased eprosartan binding affini-ties (�Ki � 32.5 � 7.9 and 13.4 � 3.1, respectively; Table 3).Telmisartan is another ARB with a unique chemical structure;it has two consecutive benzimidazole moieties that were pre-dicted to make additional �-� contacts with Tyr-92ECL1. Muta-
FIGURE 3. Olmesartan derivatives with different interactions and functions toward AT1R. A, IP signaling assays identified that olmesartan and R781253 areinverse agonists; R239470 is a neutral antagonist, and R794847 is a weak partial agonist of the human AT1R. Statistical analysis was performed by unpaired two-tailedt test, in which p values less than 0.05 were considered statistically significant. Olmesartan, p � 0.037; R781253, p � 0.042; R794847, p � 0.048, and AngII, p � 0.024.Results are presented as means � S.E. and represent three experiments performed in triplicate. B, docking simulations showed conserved binding modes of olmesar-tan (cyan), R239470 (blue), R781253 (green), and R794847 (red) in the AT1R ligand-binding pocket. C, schematic representations for interactions of olmesartan and itsderivatives with AT1R are shown with hydrogen bonds/salt bridges as red dashed lines. B and C, three critical residues for binding of all four ligands are highlighted inred; the residues discriminating ligand binding are labeled in green (for R239470 and R794847) and cyan (for R781253 and R794847).
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tion of Tyr92ECL1 to alanine, although mostly neutral for otherARBs tested, significantly lowered affinity for telmisartan(�Ki � 6.5 � 2.0; Table 3). Furthermore, the I2887.39A muta-tion had another discriminating effect on telmisartan bind-ing (�Ki � 11.2 � 1.7; Table 3), albeit via a different set ofcontacts compared with eprosartan.
Together, the docking results and mutagenesis data for dif-ferent ARBs reveal a molecular recognition paradigm for ARBsbinding to AT1R. The phenyl-acidic scaffold employs threecritical AT1R residues Arg-167ECL2, Trp-842.60, and Tyr-351.39;however, the relative binding energy contributions of each res-idue may vary for different variants and derivatives of the scaf-fold. Furthermore, the derivative moieties extend interaction ofspecific ARBs to additional sub-pockets of AT1R, consequentlyallowing for some wobbling of the ARB core structure in themain ligand binding pocket. Analysis of these sub-pockets sug-gests that some of the known naturally occurring mis-sensevariations in AT1R, such as L481.52V, A1634.60T, L2225.65V,A2446.39S, T2827.33M, and C2897.40W, which were reported assingle nucleotide polymorphisms (SNPs) in the AGTR1 gene,may influence ARBs binding and efficacies in humans. Thus,three of these SNP variations, A1634.60T, T2827.33M, andC2897.40W located in close proximity to the AT1R orthostericbinding pocket may differentially affect efficacies of specificARBs in individuals carrying these SNPs via changes in direct orindirect interactions with the pocket (Fig. 2D).
Functional Selectivity of Olmesartan Derivatives—Previousstudies suggested that small modifications of ligands could altertheir pharmacological properties, despite their similar bindingmodes (32–34). Olmesartan is an inverse agonist for IP produc-tion (11). In recent work with rodent AT1R (33), it was observedthat compound R781253, in which the biphenyl-tetrazole scaf-fold of olmesartan was modified with an additional 4-hydroxy-benzyl group, retained inverse agonist properties similar toolmesartan. Exchange of the carboxyl group on the imidazoleend of olmesartan by a non-acidic carbamoyl group in R239470was found to produce a neutral antagonist. Surprisingly, com-bination of these two modifications resulted in a weak partialagonist R794847; however, the structural basis of this phenom-enon remained unknown.
The pharmacological properties of olmesartan derivativesreported for rodent AT1R were confirmed in this work for thehuman AT1R (Fig. 3). Extensive co-crystallization trials ofAT1R with these three ligands were however unsuccessful.Therefore, to address the structural basis for functional selec-tivity of olmesartan derivatives, we performed docking simula-tions of R781253, R239470, and R794847 binding using the
TABLE 4Interactions of olmesartan and its derivatives with AT1R determinedby docking simulationsNumbers show the contact area (Å2) of the residue surface participating in theligand interactions; the cells are colored according to the contact area, from blue (nodirect contact) to red (maximum contact area).
TABLE 5Competition displacement of 3H�olmesartan binding to various AT1R mutants by olmesartan and its derivatives�Ki1refers to the WT response for each ligand, to evaluate the effects of each mutation. �Ki2 refers to each derivative of olmesartan, to evaluate the effects of modificationson ligand. Results are presented as means � S.E. and represent three experiments performed in triplicate.
AT1R-olmesartan structure (Fig. 3 and Table 4). Dockingresults suggested that all these ligands bind in similar orienta-tions. The additional 4-hydroxybenzyl groups of R781253 andR794847 were predicted to extend to a sub-pocket on the bot-tom of the AT1R ligand-binding pocket consisting of Leu-1123.36, Lys-1995.42, Asn-2005.43, Trp-2536.48, His-2566.51, Gln-2576.52, and Thr-2606.55 (Fig. 3). Some of the interactions of the4-hydroxybenzyl moieties in this pocket are likely to be subop-timal as they reduced the binding affinities of R781253 andR794847, as compared with olmesartan (�Ki2 � 3.4 � 0.3 and6.2 � 0.6; Table 5). Accordingly, mutations of these residues toalanine resulted in mixed effects on R781253 and R794847binding affinities (Table 5). Mutating two of the sub-pocketresidues, Trp-2536.48 and Thr-2606.55, however, had strong andunique effects on the 4-hydroxybenzyl-derivatized com-pounds, supporting their contributions to ligand binding.Although a conserved Trp-2536.48, known as the “toggleswitch,” has been implicated in activation of many GPCRs (35),previous mutagenesis studies in AT1R have also associated Lys-1995.42, His-2566.51, Gln-2576.52, and Thr-2606.55 as a clusterthat regulates the ligand-dependent activity state of AT1R (2, 5,7). Therefore, interactions of 4-hydroxybenzyl moieties in thissub-pocket, whether optimal or not, are likely to affect func-tional properties of R781253 and R794847. On the imidazoleend of the biphenyl-tetrazole scaffolds, the carbamoyl groups ofR239470 and R794847 substituted the carboxyl groups of olm-esartan and R781253. As a result, the carbamoyl moieties ofR239470 and R794847 cannot make a salt bridge to Arg-167ECL2, but instead they can form additional hydrogen bondinteractions with Tyr-872.63 and Tyr-92ECL1 (Fig. 3). Especiallyfor the agonist R794847, the Y872.63A mutant showed a dra-matically decreased binding affinity compared with the wild-type AT1R (�Ki1 � 15.8 � 2.9; Table 5). This rearrangement ofinteractions from Arg-167ECL2 to Tyr-872.63 is likely to beresponsible for reduced inverse agonism activity by R239470.Moreover, combination of the carboxyl replacement with 4-hy-droxybenzene group in R794847, apparently makes a synergis-tic contribution to the shift toward the agonistic activity of thisderivative.
Allosteric Modulation of Peptide Ligands Binding by SodiumPocket Residues—Particular interest in the functional selectivitymechanism of AT1R ligands is a potential connection betweenthe ligand-binding residues, like the toggle switch Trp-2536.48,and other functionally important features, like the sodium ion-binding site (Fig. 4). The sodium site was previously discoveredin high resolution structures of A2A-adenosine (36) and �-opi-oid receptors (�-OR). It is highly conserved in many other classA GPCRs, explaining allosteric sodium effects observed formany of these receptors (37). Reported allosteric effects ofsodium in AT1R are however varied (38). Although sodium wasshown to potentiate AngII binding in the adrenal gland (25), the
effects of sodium on ligand binding and signaling were observedonly in constitutively activated mutants of AT1R but not in thewild type (39, 40). Although no electron density for a sodiumion was determined in our structure at this resolution, struc-tural superimposition with �-OR (12) shows that 15 out of 16residues in the putative sodium pocket of AT1R are conservedand located in similar positions as in �-OR, except for Asn-2957.46 in AT1R (Ser7.46 in �-OR) (41), suggesting that sodiummay play somewhat similar roles in AT1R dynamics and func-tional selectivity of ligands compared with �-OR.
To assess the potential connections between the sodium ionand ligand binding pockets in AT1R, we examined the ability ofsodium ions to modulate receptor affinity for the full agonistAngII and �-arrestin biased agonist TRV120027 by determin-ing the IC50 values in the presence or absence of sodium ionsusing different sodium-binding site mutants. The only dra-matic shift among the sodium-coordinating mutants wasobserved for N1113.35A, which showed �300-fold higher affin-ity for AngII in the absence of sodium ions, as compared with aphysiological concentration of Na�, although this effect wasabsent for TRV120027 (Fig. 4). It was previously reported thatthe N1113.35A mutant induces constitutive activation of Gprotein-dependent signaling in AT1R, and it favors binding ofagonists over antagonists in 150 mM Na� (42). Meanwhile, asodium ion was implicated in the allosteric stabilization of aninactive conformation in many class A GPCRs (37). Therefore,a combination of the N1113.35A mutation effect with theabsence of a sodium ion and its corresponding allosteric mod-ulation may result in a dramatically increased AngII bindingaffinity. However, further studies are needed to fully under-stand the mechanism for the allosteric modulation of AT1R bysodium ion.
Discussion
Our results provided new insights into the structural basis forAT1R modulation by different types of orthosteric ligands andby an allosteric sodium ion. First, we demonstrated that com-mon scaffolds shared by ARBs utilize similar molecular recog-nition sites on AT1R. Remarkably, three residues, Arg-167ECL2,Trp-842.60, and Tyr-351.39, were found to be critical for bindingof most ARBs, as they form extensive interaction networks withthe ligands. However, binding of some ARBs involved addi-tional specific interactions with extended sub-pockets of AT1R.Second, modifications of the common biphenyl-tetrazole scaf-fold in olmesartan-derived ligands endowed different func-tional selectivity with a range of pharmacological propertiesfrom inverse to partial agonism. We observed that the sub-pocket, including Trp-2536.48 from the WXP motif served as adeterminant of the functional selectivity in this set of AT1Rligands. Interactions of the 4-hydroxybenzyl group withLeu-1123.36, Lys-1995.42, Asn-2005.43, Trp-2536.48, His-2566.51,
FIGURE 4. Allosteric modulation of AngII and TRV120027 binding by sodium ion in sodium-binding pocket mutants. A, structure of AT1R with dockedR794847 ligand shows that the toggle switch Trp-2536.48 belongs to the sodium-binding pocket and interacts with the 4-hydroxybenzyl pharmacophore ofR794847. B–D, competition binding assays were performed under equilibrium conditions, with 2 nM [3H]olmesartan and concentrations of AngII or TRV120027ranging between 0.04 and 1000 nM. The binding buffer used in the sodium containing (�Na�) experiments (red lines) was 140 mM NaCl, 5.4 mM KCl, 1 mM EDTA,0.006% BSA, 25 mM HEPES, pH 7.4. The binding buffer used in the sodium deficient (�Na�) experiments (blue lines) was 140 mM N-methyl D-glucamine, 5.4 mM
KCl, 1 mM EDTA, 0.006% BSA, 25 mM HEPES, pH 7.4. Statistical analysis was performed by unpaired two-tailed t test, in which p values less than 0.05 (asterisks)were considered statistically significant. N111A, p � 0.004. Results are presented as mean � S.E. and represent three experiments performed in triplicate.
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Gln-2576.52, and Thr-2606.55 identified a cluster of residues thatmay regulate AT1R coupling to IP signaling. It was shown pre-viously that Leu-1123.36, located next to Asn-1113.35, whenmutated to glycine induced a robust constitutive activation ofAT1R (2, 43). Thus, the observed interactions of the 4-hydroxy-benzyl group with the sub-pocket, including Leu-1123.36 andTrp-2536.48, suggested a potential pharmacophore that couldbe mimicked in the future design of small molecule AT1R ago-nists. These results allowed us to establish the rational basis fordifferent functional properties of olmesartan analogs. Struc-tural determination of AT1R uncovered that the putativesodium anchoring residues are mostly conserved betweenAT1R and �-OR. Notably, Asp-742.50, Asn-1113.35, and Asn-2957.46 in the sodium ion-binding pocket were found in previ-ous studies to be responsible for intramolecular hydrogenbonding that regulates constitutive activation of AT1R. It wasshown that the D742.50A mutation abolishes G protein signal-ing, although N1113.35A and N2957.46A mutations constitu-tively activate G protein-dependent signaling in AT1R (43).Here, we show that allosteric modulation by a sodium ion in theN1113.35A mutant affects binding of AngII but not TRV120027,suggesting different mechanisms for AT1R modulation by itsnatural ligand and �-arrestin-biased ligand.
Author Contributions—H. Z. designed, purified, characterized, andcrystallized the proteins, collected and processed diffraction data,determined the structure, analyzed the data, and wrote the paper.H. U. performed mutagenesis, signaling, and ligand binding studies,and wrote the paper. R. D. participated in mutagenesis, membraneproduction, signaling, and ELISA analysis. G. W. H. solved andrefined the structure. N. P. performed docking studies. V. K. ana-lyzed the structure, supervised docking studies, and wrote the paper.S. S. K. conceived the project, supervised mutagenesis and functionalstudies, and wrote the paper. V. C. supervised crystallization andcrystallographic data collection, processed diffraction data, analyzedthe data, and wrote the paper. R. C. S. conceived and supervised theproject and wrote the paper.
Acknowledgments—We thank J. Velasquez for help with molecularbiology, M. Chu for help with baculovirus expression, and A. Walkerfor assistance with manuscript preparation. We thank Dr. Shin-ichiro(Fukuoka University School of Medicine, Fukuoka, Japan) for helpand Daiichi Sankyo Co., Ltd., Tokyo, Japan for generous supply ofolmesartan and its derivatives. This research used resources of theAdvanced Photon Source, a United States Department of EnergyOffice of Science User Facility operated for the Department of EnergyOffice of Science by Argonne National Laboratory under ContractDE-AC02– 06CH11357. Parts of this research were carried out at theGeneral Medical Sciences and NCI Structural Biology Facility at theAdvanced Photon Source (GM/CA@APS), Argonne National Labo-ratory. GM/CA@APS has been funded in whole or in part by GrantACB-12002 from NCI and Grant AGM-12006 from NIGMS.
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Angiotensin Receptor and Anti-hypertensive Drugs
DECEMBER 4, 2015 • VOLUME 290 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 29139
Vsevolod Katritch, Sadashiva S. Karnik, Vadim Cherezov and Raymond C. StevensHaitao Zhang, Hamiyet Unal, Russell Desnoyer, Gye Won Han, Nilkanth Patel,
ReceptorStructural Basis for Ligand Recognition and Functional Selectivity at Angiotensin
doi: 10.1074/jbc.M115.689000 originally published online September 29, 20152015, 290:29127-29139.J. Biol. Chem.
10.1074/jbc.M115.689000Access the most updated version of this article at doi:
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