Mechanism of Hormone Peptide Activation of a GPCR: Angiotensin II

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Computational Biochemistry

Mechanism of Hormone Peptide Activation of a GPCR: Angiotensin II activated state of AT1R initiated by van der Waals Attraction Khuraijam Dhanachandra Singh, Hamiyet Unal, Russell Desnoyer, and Sadashiva S Karnik J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.8b00583 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 4, 2019

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Journal of Chemical Information and Modeling

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Mechanism of Hormone Peptide Activation of a GPCR:

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Angiotensin II activated state of AT1R initiated by van der Waals Attraction

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Khuraijam Dhanachandra Singh, Hamiyet Unal, Russell Desnoyer and Sadashiva S.

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Karnik*

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Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation,

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Cleveland, Ohio44195, USA

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Running Title: AngII-Induced Conformational Changes Activating AT1R

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*Correspondence: Sadashiva S. Karnik, Lerner Research Institute, Cleveland Clinic, 9500 Euclid

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Avenue, Cleveland, Ohio 44195, United States of America

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[email protected]

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1 ACS Paragon Plus Environment

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ABSTRACT:

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We present succession of structural changes involved in hormone peptide activation of a

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prototypical GPCR. Microsecond molecular dynamics simulation generated conformational

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ensembles reveal propagation of structural changes through key “micro-switches” within human

24

AT1Rbound to native hormone. The endocrine octa-peptide angiotensin II (AngII) activates AT1R

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signaling in our body which maintains physiological blood pressure, electrolyte balance and

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cardiovascular homeostasis. Excessive AT1R activation is associated with pathogenesis of

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hypertension and cardiovascular diseases which are treated by sartan-drugs. The mechanism of

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AT1R inhibition by sartans has been elucidated by 2.8 Ȧ X-ray structures, mutagenesis and

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computational analyses. Yet, mechanism of AT1R activation by AngII is unclear. Current study

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delineates an activation scheme initiated by AngII binding. A van der Waals “grasp” interaction

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between Phe8AngII with Ile2887.39 in AT1R induced mechanical strain pulling Tyr2927.43 and

32

breakage of critical inter-helical H-bonds, first between Tyr2927.43-Val1083.32 and second between

33

Asn1113.35-Asn2957.46. Subsequently changes are observed in conserved “micro-switches”

34

DRYTM3, Yx7K(R)TM5, CWxPTM6 and NPxxYTM7 inAT1R. Activating the “micro-switches” in the

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intracellular region of AT1R may trigger formation of the G-protein binding pocket as well as

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exposure of helix-8 to cytoplasm. Thus, the active-like conformation of AT1R is initiated by the

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van der Waals interaction of Phe8AngII with Ile2887.39, followed by systematic reorganization of

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critical inter-helical H-bonds and activation of micro-switches.

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Key words: AngII, AT1R, Molecular Docking, Molecular Dynamics Simulations, Hypertension,

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Cardiovascular Diseases

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INTRODUCTION

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Nearly 5% of the human genome encodes G-protein coupled receptors (GPCRs), which are targets

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of ~34% of therapeutic drugs currently in use1. GPCRs are seven-transmembrane helical proteins

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that modulate vital cell-signaling responses to a variety of natural and pharmaceutical ligands1-5.

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Extracellular agonists bind and initiate receptor conformational changes which propagate across

48

the trans-membrane (TM) alpha-helical domain to intracellular “micro-switches” formed by highly

49

conserved sequence motifs in GPCRs. These events result in the transient formation of a cytosolic

50

cleft for binding and activating a cognate G protein. By comparing the antagonist-bound inactive

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and [agonist + G protein/antibody] bound active state structures of GPCRs, the activating structural

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changes were identified6-11. Structure-function studies have shown that mutating a micro-switch

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may still allow the activating motion of TM-helices but impair cell signaling12. Intra molecular

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events that form the electro-mechanical basis of signal transduction are predominantly documented

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for amine activated GPCR prototypes. Although three agonist-peptide bound GPCR structures are

56

available (PDB ids: 6DDF, 5UNF, 5T04), ensemble view of conformational dynamics of peptide

57

hormone activation of a GPCR is absent to date13-16.

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The angiotensin type 1 receptor (AT1R) is a clinically important GPCR that binds the renin-

59

angiotensin system hormone Angiotensin II (AngII). Cell signaling by activated AT1R involves

60

stimulation of the Gq11/12 leading to intracellular production of calcium, lipid messengers and

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activation of protein kinases17-20. In addition, G protein-independent signaling pathways are also

62

activated21-25. AngII-activated AT1R is responsible for homeostatic regulation of blood pressure

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and water-electrolyte balance in the body26,

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myocardial tissue, the liver, the kidney, the adrenal cortex, and specific brain centers. Chronic

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AT1R activation produces disease states including hypertension, inflammation, endothelial

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dysfunction, atherosclerosis, congestive heart failure and end organ damage. AT1R blocking drugs

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(ARBs) such as olmesartan are used as first line therapy in fighting these diseases. Previous studies

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have suggested a transition scheme for functional states of AT1R [Figure 1A] upon antagonist and

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agonist binding28,

70

attempts made to crystalize the AngII bound AT1R did not succeed13, 14. To bridge the knowledge

71

gap with regard to the AngII-activated state of AT1R, we performed microsecond molecular

72

dynamics simulation (MDS) analysis in a membrane bilayer environment.

29.

27.

AT1R is widely expressed in vascular and

Two high-resolution inactive state structures of AT1R are reported but



73

In this first report delineating conformational ensemble of a clinically relevant peptide hormone-

74

GPCR we carried out MDS starting with the 2.8 Ȧ x-ray crystallographic structure of human

75

AT1R13, 14. We corroborate the MDS results [Figure 1B] by (i) comparing to active-state structures

76

of established GPCRs30-34; (ii) using stereo-chemical features of AngII-analogs defined by

77

structure activity relationship (SAR)35-38 and (iii) by combining with site-directed mutagenesis

78

findings on pharmacology of AT1R37,

79

functional motifs within AT1R [Figure 1C]. During AT1R-AngII signaling, these motifs may act

80

as “micro-switches” that change from an inactive to an active conformation. Hence, inspecting

81

perturbation in CWxPTM6, DRYTM3, Yx7K(R) TM5and NPxxYTM7motifs in comparison to different

82

established activated GPCRs39-42 will further validate our MDS. SAR analysis of the AngII peptide,

83

Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8 has established that the charged property of Asp1, Arg2,

84

Phe8-COOˉ and bulk-hydrophobicity of Tyr4 determine binding affinity and specificity for AT1R

85

[FigureS1A]. The aromatic Phe8 sidechain anda cumulative influence of Tyr4, His6 and

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Pro7responsible for agonist potency of AngII in AT1R activation50-52. Mutagenesis analysis showed

87

that AngII binding requires residues, Tyr351.39, Trp842.60, Arg162ECL2, Lys1995.42and Asp2817.32

88

[FigureS1B]. In addition, the critical role of Asp742.50, Asn1113.35, Arg1263.50, Trp2536.48,

89

His2566.51, Ile2887.39, Tyr2927.43 and Asn2957.46 in receptor activation are documented37, 44, 46, 48, 49,

90

53, 54. How these features of the receptor and the hormone cooperatively produce an AngII-activated

91

state of AT1R is unknown.

92

Our working hypothesis assumes that micro-switches constrain spontaneous activation in the basal

93

state of AT1R. Distinct interactions of AngII with the AT1R binding pocket break the prime

94

constraint instigating the micro-switches to change from inactive to active conformation in the

95

course of receptor activation. Understanding the details of AngII activation of AT1R could further

96

help in designing more potent ARBs to prevent AngII associated pathogenesis.

39-49.

As in all GPCRs, several conserved residues form

97 98


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RESULTS:

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Global conformational changes−In the 1µS MDS following flexible docking of AngII to

101

membrane embedded AT1R (PDB id: 4ZUD)13, the trajectory plot for AngII and the receptor

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backbone reached equilibrium (Figure S2A). Significant chnages in root mean square fluctuation

103

(RMSF) was observed in DRY, ECL2 and ICL3 motifs as shown in Figure S2B in the AT1R-

104

AngII compared to AT1R-Olmesartan and AT1R-ZD7511. High fluctuation in these motif may

105

correlate with the activation of the rceptor. The RMSD plot indicated significant movement in

106

TM-helices, ECL2, ICL3 and Helix-8 (Figure S3). The AT1R conformation ensembles produced

107

from MDS closely match the crystal structure of peptide-agonist bound ETBR (PDB id: 5GLH)16

108

(Figure 2A & B). The volume of AngII bound pocket of AT1R is ~1203.11 ± 335.83 Å3,

109

comparable to the pocket of ETBR which has 895.92Å3 (PDB id: 5GLH)16. The extracellular

110

portions of TM1, TM3, TM4, TM5 and TM7 move in the AngII-bound state (FigureS4). These

111

structural changes are associated with the orthosteric pocket adopting a compact ‘closed’

112

configuration enabling strong interactions with AngII. Together the N-terminal tail (NT) and EC-

113

loops (ECL) including the β-strands of ECL2 form a lid-like architecture that covers the orthosteric

114

pocket (Figure 2C & 2D). Mutagenesis and accessibility mapping studies have predicted ligand-

115

specific movements in NT and ECL regions of AT1R, especially in the ECL255-57. The water

116

accessible entrance in the apo-AT1R might facilitate the entry of ligands to the orthosteric pocket.

117

By contrast, in the intracellular region, the TM1, TM3, TM5, TM6 and TM7 move outwards

118

(Figure S4). The movement of ICL3 associated with TM3, TM6 and TM7 opens a crevice that is

119

supposed to bind the G protein (Figure 2C & E), but the opening is not complete as seen in the

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Gs protein bound β2AR structure40. The outward movement of TM6 is comparatively less than

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observed in the active state crystal structures of GPCRs40.Functional studies in AT1R have

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indicated that exposure of DRYTM3 motif, the membrane proximal segments of ICL3 and the

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NPxxYTM7 motif is necessary and sufficient to recruit the G protein20. A well-defined helix-8

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observed in the inactive state AT1R shows ~15Ȧ shift towards the cytoplasmic space with

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decreased helical stability in a small number of the ensemble frames (FigureS4). Observed

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conformational change in helix-8 is relevant because functional studies have shown that helix-8 of

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AT1R is a hot-spot for recruitment of non-G protein signaling molecules such as Jak2, Cam kinase

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and Filamin A20, 58. These AngII-induced receptor conformational changes are not observed when 5 ACS Paragon Plus Environment


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olmesartan is used as the ligand for MDS and the ensemble frames are indicative of agonist bound

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structures of β2AR, ETBR, NT1R and μ-opioid receptor in which structural changes linking the

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orthosteric pocket and the cytoplasmic G-protein binding pocket is observed.

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AngII Binding Interactions−Supporting the mutagenesis findings, AngII residues 3-8 are embed

133

within the orthosteric pocket (Figure 3A). Asp1AngII and Arg2AngII extend to the NT and ECL2 and

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ECL355, 56, 59, 60, 61. Binding of AngII involves ~17 AT1R residues forming ionic, H-bond, van der

135

Waals and π-π stacking interactions (Figure 3B). The conformation of bound AngII observed in

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our MDS is similar to NMR62 or flexible docking38, or modeling studies35. Superimposition of

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AngII and Olmesartan indicates overlap of their aromatic-acidic pharmacophore groups

138

surrounded by shared residues, Tyr353.91,Trp842.60, Tyr872.63, Lys1995.42, Arg167ECL2, Ile2887.39

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and Tyr2927.43which validate competitive antagonism of AngII and ARBs (Figure S5).

140

Mutagenesis studies indicate that these are critical residues for binding both AngII and ARBs (see

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TableS1).

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The octapeptide is oriented so that the Phe8AngII α-carboxyl interaction with Lys1995.42 is robust

143

(Figure 3). MDS trajectory analysis of Phe8AngII α-carboxyl H-bond and ionic interaction with

144

Lys1995.42” estimated to be >75% in multiple trials. It is known that the Phe8AngII α-carboxyl, if

145

blocked by methylation or amination causes tachphylaxis in vivo. Lys1995.42 mutation to Ala/Gln

146

in AT1R completely abolished binding of AngII and its analog [Sar1, Ile8]AngII. Noda et al.,

147

(1995)45 suggesting that Lys1995.42 interaction with Phe8AngII α-carboxyl may be important for the

148

orientation of His6-Pro7- Phe8AngII in mediating receptor agonism. After 200 ns the guanidinium

149

group of Arg167ECL2 forms multiple interactions including water mediated bonding with backbone

150

carbonyl groups of Phe8AngII, His6AngII and Ile5AngII amounting to 250% of interaction. When

151

Arg167ECL2 was mutated AngII binding is abolished. Therefore, projecting Arg167ECL2 into the

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depth of the orthosteric pocket to support interaction with AngII backbone carbonyls might be

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critical for shaping the receptor-bound conformation of AngII. We have explored every critical

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binding pocket residue of AT1R involved in AngII binding. We did not find significant change in

155

the conformation of Arg167ECL2 in AT1R bound with AngII or olmesartan. Supporting result was

156

observed in our mutagenesis and crystal structure experiments (Table S1).

157

The NH2-backbone amide of Asp1AngII interacts with Asp263ECL3 and Ser186ECL2 and the Asp1AngII

158

carboxyl group maintains interaction with Lys20NT. Deletion of Asp1AngII reduces the binding 6 ACS Paragon Plus Environment

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159

affinity slightly and substitution with sarcosine enhances the binding affinity. The Arg2AngII

160

interacts with Asp2817.32 validating the SAR studies, which established its importance for the

161

hormone’s binding affinity. The Asp2817.32 mutation was shown to reduce AngII affinity as well

162

as efficacy60. Previous modeling studies also indicate that additional stabilization of this interaction

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may involve Asp2787.29. During simulation, Arg2AngII appears to form a strong interaction with

164

Asp1AngII as well as Asp17NT. These interactions contributed ≈130% each, implicating their role

165

in binding Arg2AngII.

166

Phe8AngII reaches deep toward the core of the receptor, while Tyr4

167

extracellular entrance of the orthosteric binding-cavity.Tyr4AngII and Phe8AngII are important for

168

agonistic potency, [Ile4, Ile8]-AngII functions as a low affinity antagonist and [Ile4]AngII and

169

[Ile8]AngII are low efficacy partial agonists for AT1R. Hence, Tyr4AngII and Phe8AngII interactions

170

are critical for activation of the AT1R (Figure 3). In MDS we observed that dynamic state of

171

Tyr4AngII precludes a stable interaction of this sidechain with the receptor.

172

Phe8AngII is oriented towards Trp842.60 forming a strong π-π stacking interaction. Also Trp842.60

173

forms intra-molecular interactions with Leu812.57, Tyr872.63, Thr882.64, Ala1043.28 and Arg167ECL2.

174

Trp842.60 mutation abolished AngII binding because the smaller aliphatic residues introduced alter

175

conformational geometry and disrupt interactions with Tyr872.63 and Arg167ECL2. Tyr92ECL1

176

mutation to Ala completely abolished AngII binding. Tyr92ECL1 forms hydrophobic interaction

177

with Pro7AngII, which is important for the active conformation of the AngII. Pro7AngII substitution

178

with a positively or uncharged residue completely abolishes binding affinity of AngII.

179

The Ile2887.39 side chain is oriented towards Phe8AngII making van der Waals interaction. Mutation

180

with Ala in this position abolished AngII binding, since the shorter side chain of Ala could not

181

reach Phe8AngIItoestablish van der Waal interaction. Mutagenesis further showed that a bulkier

182

Phe2887.39 side chain does not significantly affect AngII binding. This observation is consistent

183

with modifications of Phe8AngII such as [Sar1, Ile8]AngII and des-Phe8-heptapeptide,which are

184

devoid of AT1R agonism.

AngII

is positioned near the

185 186

AngII-induced Conformational Changes−Systematic changes observed in AT1R conformation

187

upon AngII binding in our MDS mimic structures of active-like state GPCRs. In the following



188

sections, we describe the time sequence of structural changes connecting AngII binding site to

189

different cytoplasmic “micro-switches” (Table 1).

190

1. Breaking Primary Constraint (Asn1113.35-Asn2957.46 H-bond)

191

The most important agonism determinant is Phe8AngII side chain which orientates towards a cluster

192

formed by Tyr351.39, Trp842.60, Pro2857.36, Ile2887.39 and Tyr2927.43 throughout the 1000ns MDS.

193

Phe8AngII aromatic side chain “grasps” Ile2887.39 in van der Waals attraction within ~100ns (Figure

194

4), and this “grasp” pulls the aromatic side chain of Tyr2927.43, resulting in breakage of the inter-

195

helical H-bond between Tyr2927.43-OH and the backbone carbonyl of Val1083.32 in ~200ns which

196

in turn breaks inter-helical H-bonds between Asn1113.35-Asn2957.46 in ~500ns. Interestingly,

197

equivalent intra-helical H-bonds between TM3-TM7 are absent in AT2R63.The AT2R structure

198

(PDB id: 5unf) resembles active like GPCRs and it has a Phe residue at TM7.43 position that is

199

equivalent to Tyr2927.43 in AT1R. This evidence further supports the conclusion that MDS

200

produced an active-like state of AT1R.

201 202

In contrast, binding Olmesartan establishes independent interaction with both Ile2887.39 and

203

Tyr2927.43 and consequently stabilizes the TM7-TM3 inter-helical H-bonds between Tyr2927.43-

204

OH-Val1083.32 and Asn1113.35-Asn2957.46 (Figure 5, Figure S6 and S7). In support of this finding,

205

independent studies have shown that non-peptide AT1R antagonists show reduced binding affinity

206

to active like state of AT1R63, 64. MD simulations with -Ile8AngII, -Ala8AngII and -Gly8AngII indicated

207

that the aliphatic side chains replacing Phe8AngII systematically decrease interaction with Ile2887.39

208

which is also validated by the agonist-efficacy experimental data shown in Figure S8.

209

That the disruption of Asn1113.35-Asn2957.46 bond is central to AT1R activation is well-

210

documented: (i) stable inter-helical H-bonds between Asn1113.35-Asn2957.46 are confirmed in

211

ARB-bound AT1R crystal structures13, 14; (ii) Asn1113.35 mutation to smaller residues Gly, Ala, Ser

212

and Cys constitutively activate AT1R47, 60, 65, 66 (iii) Asn2957.46 to Ser mutation mimics this effect

213

[64] and (iv) long-range effects of mutations destabilizing Asn1113.35-Asn2957.46 bond also cause

214

constitutive activation57. To further examine we modeled Asn111Gly3.35 mutant of AT1R and

215

found that the model co-ordinates matched the active-like-state AT2R structure better than inactive

216

state AT1R structure. Substitution of larger side chains, such as the Trp1113.35 produced the most


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217

inactive AT1R mutant49. The residues involved are conserved in AT2R, suggesting that the same

218

mechanism may be involved in AngII activation of AT2R.

219 220

2. Mechanism of TM-bundle Entropy

221

Inter-helical polar interaction networks involving conserved residues, Asn461.50, Asp742.60,

222

Asn2957.46 and Asn2987.49 are rearranged. The Tyr2927.43 side chain forms π-π stacking with

223

Phe772.53 and the Asn1113.35-Asn2957.46 H-bond interacts with Asp742.50 in the AngII bound state.

224

When Asn1113.35-Asn2957.46 H-bond breaks, the Asn2957.46 interacts strongly with Asp742.50 and

225

Asn461.50. The weakening of TM7-TM3 interaction sways the side chain of Leu1123.36 towards the

226

Lys1995.42. In a previous MDS study, Cabana et al., (2013)49 compared constitutively active

227

Gly1113.35 mutant with the inactive Trp1113.35 mutant. Although the Asn1113.35-Asn2957.46 H-

228

bond is broken in both of these mutants, the activation switch to Asn2957.46-Asp742.50-Asn461.50

229

H-bonds was not observed in the Trp1113.35 mutant. Instead, Asp742.50 stably interacted with

230

Trp1113.35. In the constitutively active Gly1113.35 mutant, they observed H-bond between

231

Asp742.50-Asn461.50. Therefore, orderly re-arrangement of inter-helical network coordinated with

232

Asn1113.35-Asn2957.46 interaction in the AT1R are critical steps in activation. AngII binding also

233

perturbed another important hydrogen bonding network involving Asp742.50, with Ser1093.33,

234

Tyr1133.37 and Cys2967.47 that is conserved among class A GPCRs (Figure S9). Biochemical

235

experiments showed that agonist-activation of AT1R alters the orientation of TM2, reducing its

236

interaction with TM7. The Asp742.50 and Phe772.53 side chains buried in AT1R become solvent

237

exposed when AngII-binds61, 68, 69.

238

In addition to the networks in the AngII binding pocket, extensive H-bond network linking the

239

orthosteric ligand pocket and the G-protein-binding cleft also rearrange in the active-like state. In

240

this network, polar residues are conserved as documented in family A GPCR structures and may

241

involve water molecules and a sodium ion as discovered in the high-resolution inactive-state

242

structure of a few GPCRs10,11,40. Since involvement of allosteric sodium is not experimentally

243

confirmed in AT1R, we did not include a sodium ion in our MDS analysis. However, the

244

cytoplasmic H-bond networks can be traced to the side chains of Asp742.50, Asn1113.35 Trp2536.48

245

and Asn2957.46 that could be involved in coordination of a sodium ion. As discussed below the

246

polar networks extend from CWxPTM6 motif to the cytoplasmic surface involving micro-switches,

247

DRYTM3, Yx7K(R)TM5 and NPxxYTM7 sequence. 9 ACS Paragon Plus Environment


248 249

3. Motion of Trp2536.48

250

Trp2536.48 is part of the CWxP motif, a rotamer toggle switch which leads to the opening of G

251

protein cleft in class A GPCRs. This highly conserved amino acid residue is located on the floor

252

of the ligand pocket and adjacent to the conserved Pro2556.50. No significant change in orientation

253

of Pro2556.50 is observed in our MD simulation. In the AngII activated state the orientation of

254

Trp2536.48 side chain tilts intracellular towards His2566.51 and Phe2496.44, which are in close

255

interactions with the cluster of aromatic residues Phe2045.47, Phe2085.51 and Tyr2927.43 (Figure 6).

256

Of these, His2566.51 is a key coupling-residue as mutations in this residue uncouple AT1R agonist

257

binding and G protein activation44, 45. While the Cα torsion remained constant, the Trp2536.48 side

258

chain torsion was stabilized in ~200ns in AngII-bound state and in 500ns in Olmesartan-bound

259

states (FigureS10). The nature of CWxP conformational changes is not identical in eight agonist

260

or G-protein bound active state GPCR structures in PDB (PDB ids: 2YDV, 3SN6, 5GLH, 4MQS,

261

5C1M, 3PQR, 5YNF). However the motion observed in our simulation best matched with ETBR

262

and A2ARs (FigureS10B). The hydrophobic interaction between Trp2536.48 and Phe2496.44 is also

263

observed in the active-like states of NTSR1 and μ-opioid receptor.

264 265

4. Motion of Tyr2155.58

266

Tyr215TM5 is located closer to the cytoplasmic end of TM5 and is part of the Yx7KTM5 motif.

267

Highly conserved throughout the GPCR family, the Tyr side chain at this location may serve the

268

need for the polarity and the bulky hydrophobic ring system to ensure requirements for an efficient

269

stabilizing motif in the active states of GPCRs70, 71. In crystal structures of GPCRs, Tyr2155.58

270

forms an H-bond with Arg1263.50 sidechain of the DRY motif in the active state. In AT1R, this

271

bonding is not observed throughout the simulation. Instead we observed a strong and continuous

272

interaction of Tyr2155.58 with Leu1193.43 which helps TM3 and TM5 remain closer. But in the

273

active state, the Tyr2155.58 side chain moves toward the surface of the protein and forms π-π

274

interaction with Trp2195.62 which makes the intra-cellular region of TM5 less flexible (Figure 7,

275

FigureS11).

276

5. The DRY3.51 switch.


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The Asp1253.49-Arg1263.50-Tyr1273.51 located at the cytoplasmic end of TM3 in AT1R is a highly

278

conserved motif in GPCRs and often called an “ionic-lock”, restricting G protein interaction. In

279

the current simulation we observed a distinct difference in orientation and H-bond networking of

280

Arg1263.50. In the inactive state, Arg1263.50 is involved in stable intra-helical salt bridge with

281

Asp1253.49 which stabilizes the intracellular portion of TM3 to remain compact. This observation

282

was also reported by Matsoukas et al. (2013)37. Figure 8 depicts the conformation of this micro-

283

switch in the active state. The side chain of Arg1263.50 moves towards TM6 and forms an H-bond

284

with Asp2376.32 (Figure S12).These rearrangements could be the beginning of opening of the

285

groove for binding G-protein.

286

6. The NPxxYTM7.53 Motifand Helix-8

287

The Asn2987.49-Pro2997.50-Leu7.51-Glu7.52-Tyr3027.53 motif is also highly conserved in GPCRs.

288

Mutations targeting this motif reduce G protein activation by AT1R61, 72. During AngII activation,

289

the NPxxYTM7 moves towards the conserved Tyr2155.58 forming a hydrogen-bond network with

290

Asn2987.51, Tyr3027.53 and Leu1193.43 (Figure 9). A similar hydrogen-bond network was reported

291

in activated structures of rhodopsin, β2AR and M2R. The movements associated with NPxxY also

292

contribute to opening the groove for G-protein binding.

293

A ~15Å movement of helix-8 towards the membrane is observed in AngII-bound AT1R, but not

294

in the Olmesartan-bound AT1R. The secondary structure of helix-8 changes from alpha helix to

295

unstructured in AngII bound state (Figure 9). Another dynamic feature observed in most activated

296

GPCRs including AT1Ris the increase of Tyr3027.53–Tyr1128.53 distance, indicating TM7-helix-8

297

conformational rearrangement. This distance increased gradually in MD simulations after the first

298

20ns and remained stable for the rest of the trajectory. Large changes in helix-8 conformation are

299

reported in agonist-bound µ-opioid receptor73.

300 301

Discussion

302

Delineating the molecular mechanics involved in the opening of G protein binding cleft when

303

AT1R engages AngII is an important advancement. Without MDS analysis, visualizing the

304

dynamics of receptor activation would be difficult while accounting for AngII-analog SAR data,

305

molecular pharmacology data, receptor mutagenesis data and 3D-structure data. MDS analysis

306

indicated that AT1R conforms to the general paradigm of conformational modulation by ligands.

307

The agonists promote accumulation of the activated state and the inverse agonists depleted the 11 ACS Paragon Plus Environment


308

basal state with minor structural changes (Figure1). The activated state accumulated

309

spontaneously in the constitutively active Asn111Gly3.35 mutant suggesting that the wild-type

310

AT1Ractivation must involve agonist-dependent repositioning of Asn1113.35sdechain. The

311

Asn1113.35-Asn2957.46bond is the prime constraining interaction in AT1R. When AngII binds this

312

bond is broken indirectly through strain induced by the van der Waals attraction of Ile2887.39

313

toward the well-known agonism-specifying Phe8AngII (Figsure3-5). This is a novel GPCR

314

activation mechanism unlike the agonist trigger mechanisms in well-known GPCRs. For instance,

315

rhodopsin photo-activation which serves as a prototype includes two triggering events, protonation

316

breakage of the buried salt-bridge linking TM7 (retinal-Lys Schiff-base) to TM3 (counter-ion) and

317

the resulting mechanical motion of functional micro-domains12, 74. In other ligand activated GPCRs

318

different variations of a Lewis acid/base interaction chemical mechanisms operate. For instance,

319

in β-adrenergic receptor, TM3-TM7 lock is a hydrogen bond betweenTyr7.35-Asp3.29which

320

interacts with protonated quaternary nitrogen in adrenergic ligands and networked with an Asn7.32

321

residue75, 76. In activating S1P1R, the lyso-phospholipid sphingosine 1-phosphate receptor, the

322

zwitterionic phosphonate and primary amine groups respectively bond with Arg7.35 and Glu3.2977.

323

Thus release of the TM3-TM7 ionic-lock is suggested to be the first agonist activation step in

324

GPCRs74. However, a general GPCR mechanism for breaking prime constraint remains unclear

325

especially for peptide GPCRs till now.

326

The chemical nature of interactions of AngII and ARBs with the orthosteric pocket is fundamental

327

to pharmacological outcomes i.e. agonism and inverse-agonism. MDS analysis indicated that

328

inverse-agonist (Olmesartan) binding stabilizes multiple TM3-TM7 interactions and dampens

329

intrinsic motion of helices and motifs of AT1R (Table 1, Figures 6-9 and Figures S6-S12). In

330

addition, insights regarding the differing roles of critical residues are revealed. For example, in the

331

activated state Arg167ECL2 establishes multiple interactions with the backbone carbonyl groups of

332

AngII, morphing its receptor bound-conformation. However, Arg167ECL2 binds the acidic

333

pharmacophore groups at either ends of inverse-agonists, inhibiting residue rearrangements locally

334

in the orthosteric pocket as well as globally in TM and cytoplasmic domains in the inactive-state

335

(Figure 3 and Figure S5).

336 337

Receptor bound-conformation or the preferred conformation of a hormone peptide provides critical

338

structural information for designing selective pharmaceutical scaffolds for a GPCR. AngII 12 ACS Paragon Plus Environment

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339

structure in solution shows a bioactive γ-turn. Cyclic AngII analogs which restraint this

340

conformation increase potency78, 79. Specific spatial alignment of aromatic residues in AngII, Tyr-

341

hydroxyl (Y4), His-imidazole (H6), and Phe-benzyl (F8) in this conformation maybe important80,

342

81.

343

which preserve the turn-motif are capable of modulating blood pressure via activating AT1R. Pose

344

of AngII in our MDS analysis is consistent with spatial alignment of Tyr (Y4), His (H6) and

345

Phe(F8). Additionally, Phe8AngII substitution with Ile and Gly, as well as its deletion (des-Phe8AngII)

346

affect binding and activation. As shown in Figure S7, Ang1-7(DRVY4IH6P) with either unstable

347

or absence of the bioactive γ-turn conformation binds poorly and may activate AT1R at non-

348

physiological concentrations which is also consistent with its poor MM/GBSA scores from IFD

349

and MDS. Aromatic side chain of Phe8AngII helps to project towards the Ile2887.39. Substitution

350

with non-aromatic residues (Ile, Gly or Ala) produce a different conformation in which the side

351

chain moves towards the ECL2 region.

Hormone peptides, AngII (DRVY4IH6PF8), AngIII (RVY4IH6PF8) and AngIV (VY4IH6PF8)

352 353

The design of currently used ARBs to mimic the Y4IH6PF8 motif in AngII is confirmed by MDS

354

analysis as well as in previous studies19, 28, 82. This knowledge will be useful in designing better

355

AT1R-selective scaffolds. Development of next generation AT1R agonists and antagonists in terms

356

of differences in functional selectivity and tissue tropism has important clinical implications.

357

Given that a significant number of patients are currently prescribed ARBs for indications beyond

358

blood pressure reduction, it is important to know the mechanism of action of these drugs at the

359

receptor level.

360 361

To our knowledge, this is the first report detailing activating structural dynamics of a clinically

362

important peptide hormone GPCR. There are ∼135 human GPCRs responding to endogenous

363

peptide/protein ligands. Static structures of peptide-activated NTR1, ETBR, GLPR1 and opioid

364

receptors2,

365

activation of peptide GPCRs is not easy to decipher. Hence MDS analysis starting with hAT1R

366

crystal structure described here should prove a useful model for gaining insights into the activation

367

mechanism of peptide GPCRs and drug action. Energy-based models allow characterization of

368

side-chain and backbone shift in the pocket as well as provides a 3D frame work of determinants

83, 84are

documented. Despite this progress the specific interactions that trigger



369

of agonism or inverse agonism essential for rational drug discovery. Important insights into agonist

370

induced changes, establishing the path from ligand binding to G-protein activation can be gained.

371 372

In functioning GPCRs, thermodynamic motions within the protein produce a conformational

373

landscape with discernable functional sub-states85,

374

integrated view of sub-state dynamics and time evolution of receptor from inactive to basal to

375

activated states (Figures 2-9). We observed that the orthosteric pocket and cytoplasmic domain

376

where signal transducer molecules converge are semi-independent. The cytoplasmic domain

377

harbors conserved motifs DRYTM3, Yx7KTM5, NPxxYTM7 and helix-8. These “micro-switches”

378

were originally described in rhodopsin71, and functional studies in AT1R demonstrate that high

379

degree of stability of functional-motifs in inactive state prevents spontaneous receptor activation.

380

Mutagenesis of micro-switches either constitutively activate the receptor or interfere with overall

381

signaling. The impetus for activation of cytoplasmic “micro-switches” is transmitted through a

382

network of hydrogen bonds from the conserved CWxPTM6 toggle-switch motif. Our MDS study

383

shows critical positioning of this TM6-motif, sharing a hydrogen-bonded network between TM1-

384

TM2-TM3-TM7 which includes the highly conserved Asp742.50 in AT1R. The CWxP motif is part

385

of the proposed sodium-ion binding site formed with Asn1113.35 and Asn2957.4612, 13. Further, it

386

interacts with a cluster of residues, His2566.51, Phe2045.47, Phe2085.51 and Tyr2927.43 which are

387

associated with TM6 movement36, 37. Because of high level of conservation of residues at TM6.44,

388

TM6.48 and TM2.50, the role of the CWxP motif in AT1R may be same as in other GPCRs. Recent

389

experimental dynamics study in adenosine A2R found that the interplay between TM2.50 and TM6.48

390

regulated structural dynamics localized at cytoplasmic motifs associated with receptor

391

activation87which mimics similar observations made in rhodopsin activation88, 89.

86.

The MDS ensemble frames provide an

392 393

Time-evolution of changes involving residues, Ile2887.39, Tyr2927.43, V1083.32, Asn1113.35,

394

Asn2957.46, Asn461.50, Asp742.50 and Trp2536.48 suggests a sequential conformational transition

395

model in which disruption of the TM3-TM7 interaction leads to movement of TM6 followed by

396

reorientation of the TM1-TM2-TM3-TM7 inter-helical network. Alternative non-linear transition

397

model as proposed earlier27with multiple kinetic scheme to account for native cellular and system-

398

wide conditions is more realistic. Non-linear transition models do include elements of a linear

399

transition model as has been shown for rhodopsin90-92. 14 ACS Paragon Plus Environment

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400 401

Conclusion

402

Phe8AngII engages Ile2887.39 of AT1R in van der Waals interaction which by pulling Tyr2927.43 and

403

breaking H-bond with Val1083.32 initiates AT1R activation. This is a novel mechanism unlike those

404

described in prototypical GPCR activation models. Breaking Asn1113.35-Asn2957.46 bond relaxes

405

prime constraint; subsequently Asn2957.46 interacts with Asp742.50 and Asn461.50 leading to AngII

406

activation of AT1R. This change in molecular network leads to systematic change in the intra-

407

cellular domain and these changes may be a necessary for creation of a pocket for G-protein

408

binding. In the Olmesartan-bound inactive state, we observed a stable Asn1113.35-Asn2957.46H-

409

bond interaction and in addition Asn2957.46 interacts with Asp742.50. Capturing and understanding

410

the molecular details of activation of AT1R by AngII and inhibition by ARBs could lead to better

411

design of novel and more potent drugs targeting AT1R which could effectively prevent AngII

412

pathogenesis.

413 414

Materials and Methods

415

Protein Preparation

416

The starting coordinates of AT1R [PDB IDs: 4ZUD, 4YAY] were retrieved from Protein Data

417

Bank (www.rcsb.org) and further modified for Glide docking calculations (Glide, Schrodinger,

418

LLC, NY, USA)93. For these calculations, energy minimization was applied to the protein using

419

the Protein Preparation Wizard by applying an OPLS3 (Optimized Potentials for Liquid

420

Simulations) force field94. Missing segments of loops were filled using Prime, Schrodinger, LLC

421

during the protein preparation95. Progressively weaker restraints applied to only the non-hydrogen

422

atoms and structure refinement was done based on the default protocol of Schrodinger, LLC, NY,

423

USA, as Glide uses the full OPLS3 force field at an intermediate docking stage and is claimed to

424

be more sensitive to geometrical details than other docking tools. The most likely positions of

425

hydroxyl and thiol hydrogen atoms, protonation states, and tautomers of His residues, and Chi

426

“flip” assignments for Asn, Gln, and His residues were selected. Finally, energy minimizations of

427

protein were performed until the average root-mean-square deviation of the non-hydrogen atoms

428

reached 0.3 Å. 15 ACS Paragon Plus Environment


429

Ligand Preparation

430

We retrieved AngII crystal structure from Protein Data Bank [PDB ID: 1N9V] and the structure

431

was prepared using LigPrep package from Schrodinger, LLC, NY, USA (LigPrep, Schrödinger,

432

LLC, New York, NY, 2018)96 by assigning appropriate bond order. AngII PDB structure was

433

converted to .mae format (Maestro, Schrodinger, LLC, NY, USA), geometrically optimized and

434

partial atomic charges were computed. Then, at most, 32 poses were generated with different steric

435

features for the subsequent docking study. Olmesartan structure was also prepared in Ligprep using

436

the protocol described earlier28.

437

Induced Fit Docking (IFD)

438

Induced fit docking (IFD) (Schrodinger, LLC, NY, USA) was used to dock AngII and Olmesartan

439

inside the ligand binding pocket of AT1R. First, the ligand was docked into a rigid receptor model

440

with scaled-down vdW radii. A vdW scaling of 0.5 was used for both the protein and ligand

441

nonpolar atoms. A constrained energy minimization was carried out on the protein structure,

442

keeping it close to the original crystal structure while removing bad steric contacts. Energy

443

minimization was carried out using the OPLS3 force field with implicit solvation model. The Glide

444

XP mode was used for the initial docking and ligand poses were retained for protein structural

445

refinements. Prime (Schrodinger, LLC, NY, USA) was then used to generate the induced-fit

446

protein−ligand complexes (Prime, Schrodinger, LLC, NY, USA)97. Each of the structures from the

447

previous step were subjected to side-chain and backbone refinements98. All residues with at least

448

one atom located within 4.0 Å of each corresponding ligand pose were included in the Prime

449

refinement. The refined complexes were ranked by Prime energy, and the 20 receptor structures

450

within 30 kcal/mol of the minimum energy structure were passed through for a final round of Glide

451

docking and scoring. In the final step, each ligand was re-docked into top 20 refined structure using

452

Glide XP.

453

MM/GBSA

454

Prime/MM-GBSA was used to predict the free energy of binding between the receptor and the

455

AngII or Olmesartan. The binding free energy (ΔGbind) was calculated using Prime, Schrodinger,

456

LLC, NY, USA99. The binding free energy (Gbind) is then estimated using the following equation:

457

Gbind= ER:L- (ER + EL) + Gsolv + GSA 16 ACS Paragon Plus Environment

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458

where ER:L is energy of the complex, ER + EL is the sum of the energies of the ligand and the

459

apo protein, using the OPLS3 force field, ΔGsolv (ΔGSA) is the difference between GBSA solvation

460

energy (surface area energy) of complex and sum of the corresponding energies for the ligand and

461

apo-protein100-102.

462

MD Simulation

463

Molecular dynamics (MD) simulations were carried out for AT1R-AngII and AT1R-Olmesartan

464

complexes using Desmond MD code and the OPLS3 force field92 for minimization of the system.

465

We ran the MD simulation for 1µS for each of the complexes. For AT1R-AngII complex, we

466

repeated the MD simulation 6 times for both the crystal structures solved for AT1R (PDB id’s:

467

4ZUD and 4YAY) and two times for AT1R-Olmesartan complex. Using the Desmond system

468

builder, a 10Å buffered orthorhombic system with periodic boundary conditions was constructed

469

using a POPC lipid membrane103-106 and an SPC explicit water solvent. The overall charge was

470

neutralized by 0.15 mol/L NaCl. The simulations were performed in the NPT ensemble with

471

number of elements, pressure and temperature controlled. The temperature of 300K and pressure

472

of 1.013 bar were kept constant by coupling the system to a Berendsen thermostat and barostat,

473

which are normal temperature and pressure mimicking the real life environment. An integration

474

step of 2.0 was used, Coulombic interactions were calculated using a cutoff radius of 9.0 Å, and

475

long-range electrostatic interactions were calculated using the smooth particle mesh Ewald

476

method107. Before each MD simulation, a default Desmond membrane protein relaxation protocol

477

was applied108. To monitor protein stability and conformational fluctuations throughout the

478

simulations, we computed backbone RMSDs after least-squares fitting to the starting structure.

479

This was done for the entire protein and transmembrane α-helices. The trajectory was selected

480

when the system is fully equilibrated and the RMSD reached its plateau. Statistical analyses are

481

based on the structural ensemble from each cluster. Clustering of trajectory was performed using

482

RMSD clustering tool of Schrodinger software package. Five clusters were generated and average

483

structures were generated from each cluster. Additional structural analyses were further performed

484

using these average structures. All the figures were generated using PyMol (The PyMOL

485

Molecular Graphics System, Version 2.0 Schrödinger, LLC). The volume of the binding pocket

486

was calculated using SiteMap, Schrödinger Release 2018-2: SiteMap, Schrödinger, LLC, New



487

York, NY, 2018. Five representative structure of AT1R-AngII were generated from the MDS

488

trajectory and average value was reported.

489

125I-AngII

490

Detailed procedure is described in previous reports from our laboratory 12, 55.Ligand binding was

491

analyzed using membranes prepared from HEK293T cells expressing wild-type HA-AT1R as

492

described earlier and suspended in membrane binding buffer (140 mMNaCl, 5.4 mMKCl, 1mM

493

EDTA, 0.006% bovine serum albumin, 25mMHEPES, pH 7.4). 10µg of homogeneous cell

494

membrane was used per well. Competition binding assays were performed under equilibrium

495

conditions, with 2 nM radioligand (125I-AngII purchased from Dr. Robert Speth, University of

496

Mississippi) and concentrations of the competing ligand ranging between 0.04 and 1000 nM.

497

Binding reaction was terminated by filtering the binding mixture through Whatman GF/C glass

498

fiber filters, washed with buffer (20 mMsodium phosphate, 100 mMNaCl, 10 mM MgCl2,

499

1mMEGTA, pH 7.2). The bound ligand concentration was determined as the counts/min

500

(MicroBeta2 Plate Counter, PerkinElmer Life Sciences).Nonspecific binding was measured in the

501

presence of 105 M

502

curve-fitting program GraphPad Prism 5. The means ± S.E. for the IC50 values were calculated.

Competition Binding Analysis

127I-AngII

(Bachem).The binding kinetics were analyzed by the nonlinear

503 504

Comparative Agonism by Intracellular Calcium Levels

505

Calcium levels were measured using FLIPR_ calcium 5 assay kit (Molecular Devices) as described

506

previously12, 13, 55. The HA-AT1R expressing cells were seeded at a density of 100,000 cells/well

507

in a 96-well clear bottom black plate. Following serum starvation, calcium-sensitive dye was added

508

to the cells. The Flipper instrument was programmed in FLEX mode to add ligands (0.04 and 1000

509

nM concentration) to the cells and to monitor the fluorescence before and after adding the ligands.

510

The dose-response curves plotted and % agonism calculated assuming 100% stimulation by AngII.

511 512


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782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825


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Ghosh, E.; Kumari, P.; Jaiman, D.; Shukla, A. K., Methodological Advances: The Unsung Heroes of the GPCR Structural Revolution. Nat Rev Mol Cell Biol 2015, 16, 69-81. Ramanathan, A.; Savol, A.; Burger, V.; Chennubhotla, C. S.; Agarwal, P. K., Protein Conformational Populations and Functionally Relevant Sub States. AccChem Res 2013, 47, 149-156. Shukla, D.; Hernandez, C. X.; Weber, J. K.; Pande, V. S., Markov State Models Provide Insights into Dynamic Modulation of Protein Function. AccChem Res 2015, 48, 414422. Eddy, M. T.; Lee, M. Y.; Gao, Z. G.; White, K. L.; Didenko, T.; Horst, R.; Audet, M.; Stanczak, P.; McClary, K. M.; Han, G. W.; Jacobson, K. A.; Stevens, R. C.; Wuthrich, K., Allosteric Coupling of Drug Binding and Intracellular Signaling in the A2A Adenosine Receptor. Cell 2018, 172, 68-80 e12. Lin, S. W.; Sakmar, T. P., Specific Tryptophan UV-Absorbance Changes are Probes of the Transition of Rhodopsin to its Active State. Biochemistry 1996, 35, 11149-11159. Crocker, E.; Eilers, M.; Ahuja, S.; Hornak, V.; Hirshfeld, A.; Sheves, M.; Smith, S. O., Location of Trp265 in Meta rhodopsin II: Implications for the Activation Mechanism of the Visual Receptor Rhodopsin. J MolBiol 2006, 357, 163-172. Szundi, I.; Lewis, J. W.; Kliger, D. S., Deriving Reaction Mechanisms from Kinetic Spectroscopy. Application to Late Rhodopsin Intermediates. Biophys J 1997, 73, 688702. Zaitseva, E.; Brown, M. F.; Vogel, R., Sequential Rearrangement of Interhelical Networks Upon Rhodopsin Activation in Membranes: The Meta II(A) Conformational Sub state. J Am ChemSoc2010, 132, 4815-4821. Altenbach, C.; Kusnetzow, A. K.; Ernst, O. P.; Hofmann, K. P.; Hubbell, W. L., HighResolution Distance Mapping in Rhodopsin Reveals the Pattern of Helix Movement Due to Activation. Proc Natl AcadSci U S A 2008, 105, 7439-7444. Glide; Schrödinger Release 2018-1, Schrödinger, LLC: New York, NY, 2018. Harder, E.; Damm, W.; Maple, J.; Wu, C.; Reboul, M.; Xiang, J. Y.; Wang, L.; Lupyan, D.; Dahlgren, M. K.; Knight, J. L.; Kaus, J. W.; Cerutti, D. S.; Krilov, G.; Jorgensen, W. L.; Abel, R.; Friesner, R. A., OPLS3: A Force Field Providing Broad Coverage of Drug-like Small Molecules and Proteins. J Chem Theory Comput 2016, 12, 281-296. Jacobson, M. P.; Pincus, D. L.; Rapp, C. S.; Day, T. J.; Honig, B.; Shaw, D. E.; Friesner, R. A., A Hierarchical Approach to All-Atom Protein Loop Prediction. Proteins 2004, 55, 351-367. Schrödinger Release 2018-1: LigPrep, Schrödinger, LLC, New York, NY, 2018. Schrödinger Release 2018-1: Prime, Schrödinger, LLC, New York, NY, 2018. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S., Glide: A New Approach for Rapid, Accurate Docking and Scoring. 1. Method and Assessment of Docking Accuracy. J Med Chem 2004, 47, 1739-1749. Dhanachandra Singh, K.; Karthikeyan, M.; Kirubakaran, P.; Nagamani, S., Pharmacophore Filtering and 3D-QSAR in the Discovery of New JAK2 inhibitors. J Mol Graph Model 2011, 30, 186-197.



826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850

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851

Supplementary Information. Supporting information contains: Tables showing the effect of

852

amino acid mutation on AngII binding, structure of AngII, snake plot of AT1R showing important

853

residues for AngII, RMSD of TM helices throughout MDS, superimposition of TM helices with

854

active and inactive structures, superimposition of AngII and Olmesartan in the orthosteric site of

855

AT1R, time scale of breakage of V1083.32-Y2927.43 H-bonding, time scale of breakage of N1113.35-

856

N2957.46 H-bonds, experimental binding affinity of Ile8-AngII, Ala8-AngII, Gly8-AngII and its

857

binding pose, comparison of W2536.48 torsion angle of AT1R-AngII and AT1R-Olmesartan,

858

movement of Tyr2155.58 in the AT1R activation and movement of Arg1263.50 in the AT1R

859

activation, MDS trajectory videos 1, 2, 3 and 4, 2D display of interactions of AngII with AT1R,

860

PDB file of AT1R bound with AngII.

861 862

FUNDING SOURCES


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863

This work was supported by National Institutes of Health Grants, HL132351 and R01HL142091

864

and LRI Chair’s Innovative Research Award to S.S.K.

865 866

Competing Interests

867

All authors have declared that no competing interests exist.

868



869

Figure Legends

870

Figure 1. (A) Scheme of proposed conformational states governing AT1R function. (B) 3D-

871

structure of AT1R showing orthosteric pocket and important motifs in G-protein and non-

872

G-protein signaling. (C) Multiple sequence alignment of AT1R with Class A GPCRs

873

highlighting conserved motifs.

874

Figure 2. A) Representative conformational ensembles from 1 µs MDS of AngII bound AT1R.

875

(B) Superimposed structure of AT1R-AngII and ETBR-Endothelin. Superimposed structure

876

of AT1R-AngII (from MDS) and AT1R-Olmesartan (crystal structure) bound states

877

showing Trans-membrane view (C); Extra-cellular view (D) and Intra-cellular view (E).

878

Figure 3. (A) Binding mode of AngII in the orthosteric pocket of AT1R. (B) Interaction of AngII

879

with AT1R residues >25% in at least 3 out of 6 independent MDS experiments are shown.

880

A single residue can have >100% value due to multiple interactions (eg. weak, ionic,

881

hydrophobic etc.).

882 883 884 885

Figure 4. (A) Superimposed Phe8AngII orientation changes at time intervals indicated. Orientation of Phe8AngII captured at 0ns (B) 500ns (C) and 1000ns (D). Figure 5. Representation of Phe8AngII induced motion of sidechains leading to breakage of N1113.35-N2957.46 H-bond.

886

Figure 6. Conformation of Toggle Switch Trp2536.48 of AT1R at different time interval.

887

Figure 7. Conformation of Tyr2155.58 projecting inward in AngII bound AT1R and outward in

888

Olmesartan bound AT1R (A). The same pattern is also observed in active and inactive states

889

of β2-AR (B).

890

Figure 8. Breakage of H-bond between D1253.49-R1263.50 and formation of H-bond between

891

R1263.50-D2376.32 in AngII bound AT1R. D1253.49-R1263.50 H-bond is retained in

892

Olmesartan bound state.

893

Figure 9. Secondary structure of H8 region of AT1R-AngII (A) and AT1R-ZD7511 (B). Loss of

894

alpha helix (red color) from residue

number 302 to 316 in panel (A). Ensembles of

895

structures of AT1R-AngII and AT1R-ZD7511 (C).


Page 28 of 39

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896


Figure 1

897 898



899

Figure 2

900 901


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902


Figure 3

903 904



905

Figure 4

906 907


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908


Figure 5

909 910



911

Figure 6

912 913


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914


Figure 7

915 916



917

Figure 8

918 919


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920


Figure 9

921



923 924

Table 1. Time course of molecular events observed in MD simulation. All these events are captured and shown in MDS trajectory videos 1 to 4. Time 0 ns ~200 ns

Molecular Events AT1R-AngII

AT1R-Olmesartan

Strong interaction of

Strong interaction of alkyl tail of

Phe8AngII-Ile2887.39.

omesartan-Ile2887.39.

Phe8AngII pulls up Ile2887.39.

Position of Ile2887.39 remain same, strong interaction with alkyl tail of omesartan.

~400 ns

Breakage of Val1083.35 Cα-

Val1083.32 Cα-OH-Tyr2927.34

OH-Tyr2927.34 H-bond

(TM3-TM7) H-bond is consistent.

(TM3-TM7). ~600 ns

Breakage of Asn1113.32

Asn1113.32 N2957.46 H-bond

N2957.46 H-bond (TM3-

(TM3-TM7) is consistent.

TM7). ~700

Opening of intracellular

Intracellular domain are closed for

domain for Gprotein

Gprotein binding.

binding. ~1000 ns

Open intracellular domain

Closed intracellular domain with

with active like

inactive conformation.

conformation. 925 926 927 928 929 930 931 932 38 ACS Paragon Plus Environment

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933


Graphical Abstract

934 935 936


Mechanism of Hormone Peptide Activation of a GPCR: Angiotensin II

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