Towards Understanding the Impact of Dimerization Interfaces in

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Towards Understanding the Impact of Dimerization Interfaces in Angiotensin II type 1 receptor (AT1R) Ismail Erol, Bunyemin Cosut, and Serdar Durdagi J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.9b00294 • Publication Date (Web): 20 Aug 2019 Downloaded from pubs.acs.org on August 21, 2019

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Journal of Chemical Information and Modeling

Towards Understanding the Impact of Dimerization Interfaces in Angiotensin II type 1 receptor (AT1R) Ismail Erol1,2,*, Bunyemin Cosut1, Serdar Durdagi2,3,* 1Department

of Chemistry, Gebze Technical University, Kocaeli, Gebze 41400, Turkey Biology and Molecular Simulations Laboratory, Department of Biophysics, School of Medicine, Bahcesehir University, Istanbul, 34746, Turkey 3Neuroscience Program, Graduate School of Health Sciences, Bahcesehir University, Istanbul, 34746, Turkey 2Computational

Abstract Angiotensin II type 1 receptor (AT1R), is a prototypical class A G protein-coupled receptor (GPCR) that has an important role in cardiovascular pathologies and blood pressure regulation as well as in the central nervous system. GPCRs may exist and function as monomers, however, they can assemble to form higher order structures and as a result of oligomerization, their function and signaling profiles can be altered. In the case of AT1R, the classical Gαq/11 pathway is initiated with endogenous agonist angiotensin II (ANG-II) binding. A variety of cardiovascular pathologies such as heart failure, diabetic nephropathy, atherosclerosis and hypertension are associated with this pathway. Recent findings reveal that AT1R can form homodimers and activate the non-canonical (β-arrestin mediated) pathway. Nevertheless, the exact dimerization interface and atomic details of AT1R homodimerization have not still elucidated. Here, six different symmetrical dimer interfaces of AT1R are considered and homodimers were constructed using other published GPCR crystal dimer interfaces as template structures. These AT1R homodimers were then inserted into the model membrane bilayers and subjected to all-atom molecular dynamics (MD) simulations. Our simulations results along with the principal component analysis (PCA) and water pathway analysis suggest four different interfaces as the most plausible; symmetrical transmembrane (TM)1,2,8; TM5, TM4, and TM4,5 AT1R dimer interfaces that consist of one inactive and one active protomer. Moreover, we identified ILE2386.33 as a hub residue in the stabilization of inactive state of the AT1R.

*Emails: [email protected] (IE), [email protected] (SD)

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Introduction GPCRs are the largest membrane signaling proteins induced by various types of ligands (e.g. hormones, peptides, cytokines, and neurotransmitters) and photon of light. Since almost all physiological processes in the human body are regulated by these cell surface receptors, most of the drugs available on the market target GPCRs (~40% of the FDA approved drugs).1, 2 Recent findings reveal that, GPCRs can function not only as monomers3 but also as homo/heterooligomers.4, 5 Several GPCR crystal structures are reported in dimeric forms, including β2 adrenergic receptor (β2AR)6, ligand free native opsin7, C-X-C motif chemokine receptor 4 (CXCR4)8, μ opioid receptor (μOR)9, nociceptin/orphanin FQ receptor (N/OFQR)10, β1 adrenergic receptor (β1AR)11 and the smoothened receptor (SMOR)12 with distinct dimerization interfaces.13 Dimerization can affect different stages of GPCR cell cycle which includes receptor maturation, ligand induced dynamic regulation, pharmacological diversity (positive or negative cooperativity), signal transduction (potentiating or attenuating) and internalization.14 In this context, one of the most fascinating ability of GPCRs to regulate signal transduction is mediated by its dimerization. AT1R is a prototypical GPCR that present in numerous tissues such as endothelium, brain, vascular smooth muscle, heart, adrenal gland, and kidney which modulates most of the effects of ANG-II in the renin-angiotensin-aldosteron system.15 With ANG-II binding, AT1R can couple to heterotrimeric G protein subtypes (G12/13, Gi, and Gq/11), which enables activation of downstream effectors and second messengers.16 Over stimulation of AT1R by ANG-II causes several pathologies such as, hypertrophy, fibrosis, inflammatory response, cardiovascular remodeling, endothelial dysfunction, kidney diseases and hypertension.17 Heterodimerization of AT1R with other receptor types such as the Bradykinin 2 receptor (B2R), β2AR, apelin and Mas receptor, and Dopamine D2 receptor (D2R) were reported and the functional consequences of heterodimerization questioned by several studies have been reviewed, recently.18 Last but not least, AT1R homodimerization has also been demonstrated using different experimental setups.19-25 Two different mutants of AT1R, namely EEA(A) in DRY/M motif and K199A in binding pocket, which lacked the signaling and ligand binding, were used to demonstrate AT1R homodimer formation.21 When two mutants (i.e., K119A and K102A, defective in ANG-II binding) were co-expressed together, while high affinity ANG-II binding was regained, there was no recovery in G protein coupling.19 Hansen et al.21 successfully showed that, mutant and wild type (WT)-AT1R dimers dampened Gαq activation. However, these dimers were shown to support both β-arrestin 2 and ERK phosphorylation. The authors indicated that AT1R homodimerization occurs prior to surface expression and it is ligand independent.21 Similarly, in combination of GPCRheteromer identification technology (GPCR-HIT) and protein complementation assays, AT1 homodimers have been shown to exist and function in live cells.23 To test the homodimer formation of AT1R, Karip et al.22 constructed an AT1R homodimer system by co-expressing a candesartan (an AT1R blocker) binding resistant mutant S109Y-AT1R and a G protein coupling impaired mutant DRY(AAY)-AT1R. They observed a cross-inhibition in S109Y-AT1R – AAY-AT1R dimer system, when candesartan binds to the latter protomer in COS-7 cell line.22 Another AT1R homodimer formed by cross-linking of two protomers with factor XIIIa, a transglutaminase at Q315 position in the Cterminal end of AT1R, and they observed three-fold increment in inositol phosphate (IP) levels (improved signaling) and increased internalization in the case of cross-linked AT1R dimers.20 Szalai et al.24 investigated AT1R homodimer formation, using bioluminescence resonance energy transfer (BRET) method, and successfully showed that, negative cooperativity was observed in their experimental setup. They showed that the stimulation of one protomer caused altered ligand binding and receptor conformation, as well as β-arrestin binding to conformationally altered protomer.24 More recently, a mutation study involving residues on transmembrane (TM) 4,5,6 and 7 helices performed to identify contribution of these helices in AT1R homomer formation, and they found different effects of each TMs on oligomerization phenomena of the AT1R.25 In the mutation study of Young et al.25, several hydrophobic residues were mutated to reveal their effect on homodimer formation. However, they only considered TM4-7 helices in their mutation study, although several studies reported TM1,2,8 dimerization interface such as in β1AR11, rhodopsin26, κ-opioid27, μ-opioid9, M3 muscarinic ACS Paragon Plus Environment

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receptor.28 TM1 of AT1R which is important in dimerization has recently been investigated in AT1R/secretin receptor (SCTR) heterodimer (a cross class heterodimer, AT1R belongs to class A, and SCTR belongs to class B) formation.29 Although TM4 reduced the BRET signals, authors did not claim that TM4 was involved in the dimerization interface.29 Despite the accumulation of experimental evidences on AT1R dimerization, the contributing TMs in the dimerization interface are not exactly known at the atomic resolution level. In this study, by the aid of molecular dynamics (MD) simulations, we investigated AT1R homodimerization phenomena, and suggested four dimer interfaces as symmetrical TM1,2,8; TM5; TM4 and TM4,5 that consist of one inactive and one active protomer. MATERIALS AND METHODS System Preparation. The inactive X-ray structure of AT1R (PDB ID, 4YAY30) was retrieved from protein data bank (PDB) and prepared as described in our previous papers.31, 32 However, when this project was initiated there was no available active structure of AT1R, so we used the homology model of AT1R. Homology model of active AT1R is obtained from GPCRdb33, where it was modeled based on active structure of angiotensin II type 2 receptor (AT2R) (PDB ID, 5UNG34). However, this active homology model begins with 21st amino acid residue and ends with 325th residue. In order to maintain similarity with the inactive structure, we added 6 amino acid residues to the N-terminal side and trimmed 6 amino acids from the C-terminal end. Active homology model was prepared (hydrogen atoms were added and protonation states were calculated at pH 7) using PROPKA.35 Obtained structure was energy minimized using Protein Preparation module in Schrodinger’s Maestro molecular modeling package with default settings.36 Grid files were prepared with Glide module of Schrodinger’s Maestro, by enabling generate grid suitable for peptide docking option. Ligand Preparation. Octapeptide ANG-II (ASP-ARG-VAL-TYR-ILE-HIS-PRO-PHE, DRVYIHPF) was sketched in Avogadro37 and pKa Calculator Plugin was used for the prediction of protonation states (Marvin 19.13.0 (ChemAxon (http://www.chemaxon.com)). Then, ANG-II submitted to Protein Preparation module of Schrodinger’s Maestro36 molecular modeling package and a positively charged N-terminal and negatively charged C-terminal was obtained.35 ConfGen module of Maestro molecular modeling package was used to obtain ANG-II conformers prior to molecular docking. The acquired ANG-II conformers (~800 different conformations) were then minimized with default settings.38 Peptide Docking. Obtained ANG-II conformers were docked both to inactive AT1R crystal structure and active AT1R homology model using Glide SP-Peptide docking module of Maestro.39 Default settings were used in docking simulations and following complexes were obtained: (a) ANGII/inactive AT1R crystal structure and (b) ANG-II/active AT1R homology model. Dimer Construction. As of April 1st, 2018; all available 245 GPCR structures were downloaded from GPCRdb33, and used as templates to generate AT1R dimer pairs. Their crystal mates were generated in Maestro. To obtain AT1R dimer structures, two monomer complexes were aligned to the respective chains of the templates (e.g. for the TM1,2,8 AI dimer system, ANG-II/active AT1R homology model was aligned to chain A of the 4DKL PDB-coded template, and ANG-II/inactive AT1R crystal structure was aligned to chain B of the 4DKL PDB-coded template structure). Both the ANG-II/active AT1R homology model and the ANG-II/inactive AT1R complexes were aligned to these templates and to minimize any minor steric clashes in the dimerization interface, both structures were optimized by the the Prime module of Maestro40 and the overlapping regions in the constructed dimers were alleviated. 6 different dimer templates were selected out of 245 structures: (i) TM1,2,8 (PDB ID, 4DKL9), (ii) TM4,5 loose (PDB ID, 4GPO11), (iii) TM5 (PDB ID, 4RWS41), (iv) TM4 (PDB ID, 5LWE42), (v) TM4,5 tight (PDB ID, 5O9H43), and (vi) TM6,7 (PDB ID, 5WIU44). The ANG-II/inactive and ANG-II/active monomer complexes were aligned to the dimer crystal structures as mentioned above to establish three different dimer systems: two inactive protomers (II dimer), an active and an inactive protomer (AI dimer) and two active protomers (AA).


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Due to severe steric clashes between TM6s, TM6,7 AA dimer pair was not considered. Eventually, we constructed 17 different dimer systems (see Figure S1 at the Supporting Information, 6 different interfaces, and for each interface 3 pairs; II, AI, and AA, respectively). Residue Naming. In our study, we used both sequential numbering and Ballesteros-Weinstein nomenclature, where the first number in superscript represent TM number, and the second represents the position of amino acid respect to the most conserved residue within GPCR family.45 Molecular Dynamics (MD) Simulations. All constructed dimer systems were first oriented in membrane using OPM server46, and then inserted into 512 POPC (1-palmitoyl-2oleoylphosphatidylcholine) lipid bilayer. It must be noted that all GROMACS inputs were generated using CHARMM-GUI web server.47-49 Since our AT1R sequences were trimmed both from the N and C-terminal sites, acetylated N-terminus and methylamidated C-terminus patching were applied. Systems were solvated with TIP3 water model with 20 Å thickness, neutralizing number of Na+ and Cl- ions were added (0.15 M NaCl). All details about simulation replicas and atom numbers in each systems were given in Table S1. All simulations were performed with GROMACS version 5.0.50 CHARMM36m forcefield was used in the MD simulations.51 Particle Mesh Ewald (PME) method52 for electrostatics (cutoff of 1.2 nm) and LINCS algorithm53 for bond constraints were used. Before production runs, systems were relaxed. One minimization and six equilibration protocols were applied. In total 4 nanoseconds (ns) equilibration simulations were run with decreasing stepwise restraints in 6 steps (0.25 ns, 0.25 ns, 0.5 ns, 1 ns, 1 ns, and 1 ns, respectively). In equilibration steps, Berendsen barostat and thermostats were used. At least two independent production simulations (200 ns and 500 ns) were performed at 310 K and 1 bar using Nosé-Hoover thermostat54, 55 and ParrinelloRahman barostat56, in NPT ensemble, totaling 12.5 microsecond (μs) for all studied systems. Simulation Analyses. Trajectories of all simulations were visualized using Visual Molecular Dynamics (VMD) package.57 Root mean square deviation (RMSD) calculations, distance analyses, principal component analysis (PCA) were done using rms, distance, covar, and anaeig tools of GROMACS version 5.1.2, respectively. In analyses, the first 25 ns of collected trajectory frames were trimmed from all trajectories and remaining (i.e., 175 ns and 475 ns) parts were concatenated (MD simulations of following systems were repeated by three times (i.e., a 500-ns MD simulations and 2x200-ns MD simulations): TM4,5 Loose II; TM4,5 Tight II and TM6,7 II. Thus, while for these systems 16500 trajectory frames were used, for other 14 systems a 200-ns and a 500-ns MD simulations were conducted for each (i.e., 13000 frames were used in the trajectory concatenation). For the representative structure determination, cluster tool of GROMACS was used with gromos algorithm.58 In the clustering, 2.0 Å cutoff was applied. Concatenated trajectories were used in the clustering and in PCA. Dimerization interface analysis and contact area calculations were performed with default settings using PDBePISA server59. By interface analyses, we compared starting and representative structures to monitor changes within the dimerization interface. Water pathway analysis was performed with Hollow code60 with the default grid-spacing of 0.25 Å. All figures were generated using PyMol v. 1.7.61 RESULTS AND DISCUSSION Docking pose selection criteria for the MD simulations. 800 conformers of ANG-II were docked into binding pocket of both active and inactive structures of AT1R. Glide SP-Peptide docking resulted in 237 successful docking poses in inactive state and 137 successful poses in active state. All docking poses were evaluated with the reported interactions between ANG-II and AT1R as highlighted in the literature. The selected docking poses (two monomer complexes were selected: one pose for the inactive and one pose for the active structures) were used in MD simulations. The following interactions were considered in the selection of docking poses of both ANG-II/inactive AT1R and ANG-II/active AT1R complexes: (i) ASP1 of ANG-II interaction with ARG23 of AT1R62, 63, (ii) ARG2 of ANG-II interactions with ASP263 and ASP281 of AT1R64-66, (iii) LYS199 interaction or pose location nearby -COOH group of ANG-II’s PHE867-69, and (iv) vertical binding mode of ANG-II into the binding pocket of AT1R.65 Figure 1 represents ANG-II binding conformations at the orthosteric site of the active (A) and inactive (B) structures of AT1R. ACS Paragon Plus Environment

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Figure 1. ANG-II binding poses at the binding pocket of active (A); and inactive (B) AT1R. While active AT1R is depicted in cyan, inactive AT1R is depicted in green. ANG-II at the active receptor is depicted in magenta and ANG-II at the inactive receptor is depicted in yellow. Interacting residues are depicted as sticks and receptor structures depicted as cartoon. For the sake of clarity, cartoon representations transparency is set to 60%. Interactions are depicted as yellow dashed lines. 4 Å surrounding residues of I8 of S1I8 (purple) in crystal structure were represented in (C). Alignment of active crystal structure (salmon) and model structure (cyan); PHE8 of ANG-II (magenta) was moved upwards, since the space is occupied by LEU112 and TYR113 sidechains in both structures (D). The predicted bound conformation of ANG-II is given in Figure 1 (A, active AT1R/ANG-II complex, B, inactive AT1R/ANG-II complex). In the active model (Figure 1, A), mainly polar interactions were observed at the N-terminal domain of the ANG-II. ALA21, ARG23 and ASP281 established a hydrogen bonding network with ASP1 of ANG-II. ARG2 at the ANG-II also formed binding interactions with ASP263. Another important residue that contributed to the angiotensin receptor blocker (ARB) binding, namely ARG167, was also found to be important in ANG-II binding. ARG167 formed hydrogen bond with TYR4 from its backbone. In the ligand binding at the inactive AT1R, similar residues were found to be important, but other residues also contributed to the binding. Such as LYS20, ALA21, ARG23, and ASP281 established a strong bonding network with ASP1 of ANG-II. ARG2, also formed interactions with ASP263 and ASP281 residues. ARG167 formed two hydrogen bonding interactions with the backbone atoms of TYR4 and ILE5 of the ANG-II. LYS199, another important residue at the orthosteric binding pocket of AT1R, formed the hydrogen bonding interaction with backbone atoms of HIS6 and carboxy terminal of ANG-II. Both ASP1 and ARG2 were identified as important residues for ANG-II binding70. Another study by Feng et al.64 identified the interactions between ARG2 and ASP281 as an essential interaction for full agonism. This interaction was also observed in our docking simulations. In high resolution structures of both active ACS Paragon Plus Environment

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AT1R and AT2R, ARG2 were found to form interactions with ASP263 and ASP281.66, 71 Similarly, in previous studies it was shown that ARG167 established strong polar interactions with the backbone of the ANG-II.72 Overall, our predicted binding poses that used in MD simulations in line with the previous mutagenesis studies and crystal structures. Moreover, we compared our predicted binding poses in inactive and active targets with recently published crystal structure of the active AT1R-S1I8 complex. We observed that, ILE112 and TYR113 blocked the required space for PHE8, since in crystal structure PHE is mutated with ILE which the space requirement is smaller for this residue (Figure 1, C and D). In our binding mode of ANG-II at the active structure, PHE8 moved towards to the extracellular side due to steric hindrance, and formed an U shape. However, ANG-II conformation in inactive AT1R had a distinct conformation at the C-terminal side, where PHE8, is located between TM5 and TM6, and satisfied the interaction between LYS199 and carboxy terminal end.67 MD Simulations of Dimer Systems with Different Interfaces. All systems were run at least two times with different initial velocity distributions (at least 2.1 μs for each dimer interface), in total 12.5 μs trajectory frames were obtained. Side views of different dimer pairs and interfaces are given in Figure S1 at the Supporting Information. AT1R domains TM1-7, helix 8, intracellular (ICL1-3) and extracellular loops (ECL1-3) and ANG-II were analyzed separately and compared within three dimer pair systems (inactive-inactive (II), active-inactive (AI), and active-active (AA)) based on their backbone atoms (Figure S2-S16). RMSD graphs showed that, major differences were observed in loop regions, as expected. In particular, ICL2 and ICL3 were more flexible compared to other parts of the receptor (Figures S11 and S12). Overall, in the RMSD analysis of each topological unit of the GPCR (e.g., TM1, H8, ICL1 and ECL3), it was seen that some topological units were more mobile, while others have more rigid profiles, but all units were stable in certain time courses proving that used simulation timescales were sufficient. It is not an easy task to distinguish the dimer interfaces based on RMSD values of topological units in AT1R. Thus, we performed a detailed post-processing MD analyses such as distance calculations between certain residue pairs, principal component analysis (PCA), and water pathway analysis for better understanding of the AT1R dimerization interfaces. In the following section, details of these analyses were highlighted. TM3-TM6 (ARG1263.50 - PHE2396.34) Distance Analysis. In TM3 – TM6 distance analyses, we used concatenated trajectories. One of the most important structural changes observed in the activation of GPCRs, is the outward movement of the TM6 in order to make the required space for G protein coupling. To monitor changes that occur at the intracellular halves of the TM6, we calculated the distances between Cαs of highly conserved residues among the GPCR superfamily, ARG1263.50 in TM3 and PHE2396.34 in TM6. This distance was calculated as 6.78 Å and 11.53 Å, in the crystal structure of inactive AT1R and in the active AT1R model, respectively. Thus, we tracked ARG1263.50 - PHE2396.34 Cα distances in all studied systems along the obtained trajectory frames throughout the MD simulations. In the TM1,2,8 interface, in II dimer system, ARG1263.50 - PHE2396.34 distances were not changed in both protomers and distance stabilized as it is started. A similar story was observed in AI dimer system, where active protomer was stabilized at the active form, and the inactive protomer conserved its inactive form. However, if the dimer systems consist of two active protomers, we observed a decreased distance in ARG1263.50 - PHE2396.34 in both protomers, but still this distance is higher than the measured distance in inactive crystal structure, (Figure 2, first panel). Thus, we can conclude that if the dimer system consists of two active protomers in TM1,2,8 interface, both protomers were induced to have inactive-like structural features by each other. In the TM4,5 loose interface, we have observed active-like structure in one of the protomers of II dimer system, whereas the other protomer conserved its inactive structure. Surprisingly, in AI dimer system, the active protomer remained in its active form, and also ARG1263.50 - PHE2396.34 distance was also increased in inactive protomer and enabled it to form active-like structure. In chain A of TM4,5 loose AA interface, we observed active-like structures between 200-325 ns time period, where chain B also showed higher values than inactive state (Figure 2, second panel).


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In the TM5 interface, both inactive protomers in the II dimer system, maintained their inactive structures throughout the simulations. In the AI dimer system, inactive protomer conserved its inactive form, also dimerization at the TM5 interface changed the conformation from the active to the inactive-like structure. In the TM5 interface AA dimer system, chain B conserved its active state in first 200 ns time period, where protomer A exhibited inactive-like structure (Figure 2, third panel). In the TM4 interface, interestingly, we observed an increased distance in protomer B of II dimer system to form an active conformation in the last 300 ns of the MD simulations, where protomer A maintained its inactive form. In AI dimer system, inactive protomer conserved its inactive form, and negative cooperativity was observed in active protomer, which tended to fit inactive-like structure. In AA dimer system, between 200-300 ns of the trajectories chain A exhibited active-like features where chain B changed its conformation from active to inactive-like (Figure 2, fourth panel). In the TM4,5 tight dimer interface, protomer A of II dimer system, it conserved its inactive form, but in other inactive protomer, ARG1263.50 - PHE2396.34 distance was increased slightly. In the AI dimer system, active protomer tended to change towards the inactive-like conformation, and ARG1263.50 PHE2396.34 distance in inactive protomer was increased slightly. Unlike the previous AA dimer systems, we observed a different pattern in TM4,5 tight interface, where protomer A maintained its active conformation, but second active protomer changed its conformation to inactive-like structure (Figure 2, fifth panel). In the TM6,7 II dimer system, protomer A maintained its inactive conformation along with decreasing ARG1263.50 - PHE2396.34 distance, in the other protomer, corresponding distance was increased slightly. In AI dimer system, while the inactive protomer conserved its inactive form, the measured distance in the active conformer is decreased. However, it must be noted that in the first 200 ns period of the trajectories chain A conserved its active-like structure (Figure 2, sixth panel). TM3 – TM6 distance together with directional motion of TM6 can provide useful information about the state of the protomer. Thus, we also checked direction of movement of TM6 in all systems. In TM4,5 loose II and TM6,7 AI dimer systems, TM3 – TM6 distance showed that these two systems had one active and one inactive protomer in certain time periods of the simulation trajectories. (Table S2) However, when we checked the movement direction of TM6 in TM4,5 loose II dimer system, it is clear that TM6 conserved its inactive-like conformation (i.e., instead outward movement, inward movement of TM6 was observed) while TM3 was moving towards to the dimerization interface and thus increased the TM3 – TM6 distance (Figure S17). Thus, TM4,5 loose II dimer pair was not considered in further analysis. In the TM6,7 AI dimer system, although TM3 – TM6 distances satisfy active-like structure, it is observed that the lower part of TM6 (residues 229-242) moved towards to the G protein coupling site (Figure S18). Thus, TM6,7 AI dimer system was excluded from further analysis list. Interaction stoichiometry of GPCR dimers with their interaction partners inside the cell was identified as 2:1 for AT1R homodimer, where one protomer is activated and binded to its G protein, and other is in inactivated form in previous studies.73-77 Interestingly, Parker et al.78 proposed a 2:2 interaction stoichiometry in neuropeptide Y2 receptor. However, in our simulations none of the dimer systems were fitted well to the 2:2 interaction stoichiometry. Therefore, we hypothesized that AT1R homodimers should consist of one active and one inactive protomer in parallel with the previous reports. Based on this hypothesis, along with the TM3 – TM6 distance analysis and proper position of TM6, following 7 systems were selected: TM1,2,8 AI; TM4,5 loose AI and AA; TM5 AA; TM4 II and AA; and TM4,5 tight AA.


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Figure 2. ARG1263.50 - PHE2396.34 distance changes during all MD simulations. ARG1263.50 PHE2396.34 distance was calculated as 6.78 Å and 11.53 Å, in the crystal structure of inactive AT1R and in the active AT1R model, respectively. Interfaces are given in following order: (i) TM1,2,8; (ii) TM4,5L; (iii) TM5; (iv) TM4; (v) TM4,5T; (vi) TM6,7. ACS Paragon Plus Environment

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The distances between ARG1263.50 - PHE2396.34 were measured as 6.78 Å and 11.53 Å in the corresponding inactive AT1R crystal structure and the active AT1R model, respectively. It must be noted that measured distances are in monomer cases and when they coupled with another protomer they may induce each other and corresponding distances may change slightly. In the distance analyses, we used these values to not the specify the compulsory distances for active and inactive forms in AT1R, but to give initial distance values in the active and inactive monomers and to provide a clue as to which values should be expected in each form in dimers. Representative Structures from Clustering. Using gromos algorithm with 2.0 Å of cutoff distance, we clustered similar structures from our concatenated trajectories for each dimer interface and pairs. Based on TM3 – TM6 distance, we combined our findings with cluster analysis. To do so, from each cluster, we selected the best representative structures that delineate TM6 movement and one active and one inactive pair. Selected representatives resemble 53.70%, 51.57%, 16.63%, 15.35%, 12.31%, 11.90%, and 9.46% of all frames in TM4,5 tight AA, TM1,2,8 AI, TM5 AA, TM4 II, TM4,5 loose AA, TM4,5 loose AI, and TM4 AA, respectively. Since TM4,5 loose AI and AA, and TM4 AA do not cover enough frames (i.e., lower than 15%) to represent simulated trajectories, we excluded TM4,5 loose AI and AA, and TM4 AA dimer systems for further analyses. While Figure 2 represents time-dependent trajectory-based data, Table S2 represents static information generated using the clustering method, thus no absolute overlap between Figure 2 and Table S2 may be expected. Here, we compared the ANG-II interactions in selected cluster representatives. First, we aligned chain A of TM1,2,8 AI dimer representative structure to the crystal structure of S1I8-AT1R complex, we obtained RMS value of 2.048 Å (only TM domains RMS: 1.976 Å), (Figure 3, A). In the cluster structure of TM1,2,8 ANG-II/active protomer complex (Figure 3, B), we observed two new interactions between ASP17 and ASP1 of ANG-II, which was not presented at the initial conformation (see Figure 1, A). Interactions of ARG23 with ASP1 of ANG-II, and ASP263 with ARG2 of ANG-II, were conserved. The former ASP1-ARG23 interaction was proposed by Santos et al.62, showing that R23A mutation impaired both ANG-II activity and binding, however did not altered Sar1-ANG binding and activity. ARG2 of ANG-II, has shown to be essential for full agonism when it interacts with ASP281.64 The interaction of ARG2 with two aspartates, 263 and 281, previously presented in the literature.65 In our representative pose, ASP281 also interacts with VAL3 from its backbone. The critical requirement of ARG167 both in ARB and ANG-II binding was shown previously, as its mutation leads to impaired binding of ANG-II .79-81 We observed two interactions of ARG167 with ANG-II, one of them from the backbone of the TYR4, and the second one from carboxy terminal of PHE8, a similar observation shown by other groups.72 We also observed an interaction between LYS199 and PRO7, reported to be one of the crucial residues for agonist potency70 and the mutation of this residue was observed to cause complete elimination of binding affinity of ANG-II.81 Prior studies highlighted the importance of PHE8-LYS199 ionic interaction64, 68, 69 and we also observed this salt bridge formation between side chain of the lysine and C-terminal of phenylalanine in our representative pose. A couple of studies reported that C-terminal of PHE8 may interact with TYR113. This interaction was also presented in our cluster conformation.70, 82, 83 Alignment of chain B of the TM5 AA dimer to the active crystal structure, yielded 1.494 Å of RMS (only TM domains RMS: 1.460 Å), (Figure 3, C). In cluster pose of TM5 dimer chain B, ASP1 and ARG2 of ANG-II, interacted with ALA21, ARG23, ASP263, and ASP281, respectively. The number of formed interactions was found to be much more and stronger. ARG167 was involved in three interactions with backbone atoms of TYR4 and HIS6 (two interactions). Another important interaction was detected between C-terminal of ANG-II and LYS199 (Figure 3, D). Alignment of chain B of the TM4 II dimer to the active crystal structure, yielded 1.982 Å of RMS (only TM domains RMS: 1.952 Å), (Figure 3, E). In cluster conformation of TM4 interface dimer’s chain B, we observed similar interactions of ASP1 and ARG2 of ANG-II with the AT1R. Interestingly, we observed an interaction between oxygen atom of phenol group of TYR87 and VAL3 backbone atoms. Also, TYR113 formed an hydrogen bonding interaction with the backbone atom of ACS Paragon Plus Environment

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the PRO7 of ANG-II. ARG167 and LYS199, found to form similar interactions reported above (Figure 3, F). Alignment of chain A of the TM4,5 tight AA dimer to the active crystal structure resulted 2.537 Å (only TM domains RMS: 2.319 Å), (Figure 3, G). In the cluster conformation interaction between ASP1 and backbone of the ASP281 was observed. ARG2 of ANG-II formed four interaction with ASP263 and ASP281 (two interactions with each aminoacid). TYR4 of ANG-II interacted with backbone atom of ARG167. Two ionic interactions between C-terminal of ANG-II and LYS199 were also observed (Figure 3, H).


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Figure 3. Alignment of selected protomers of different interfaces with the active crystal structure of AT1R-S1I8 (PDB ID, 6DO166). Protein structures were represented as ribbon, and ANG-II was represented as sticks. (A, TM1,2,8 interface AI dimer: crystal, salmon; cluster representative chain A, red; ANG-II, limegreen), (C, TM5 interface AA dimer: cluster representative chain B, deepteal; ANG-II, limon), (E: TM4 interface II dimer: cluster representative chain B, forest; ANG-II, aquamarine), (G, TM4,5 tight interface AA dimer: cluster representative chain A, chocolate; ANGII, yellow). ANG-II conformation at the orthosteric binding pocket of AT1R of selected interfaces, (B: TM1,2,8 interface AI dimer, D: TM5 interface AA dimer, F: TM4 interface II dimer, H: TM4,5 tight interface AA dimer).


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NPxxY Motif. The NPxxY motif that resides at TM7 is another important and highly conserved structural motif in GPCRs, and consists of ASN298, PRO299, LEU300, PHE301, and TYR302 in AT1R. Alanine mutations of highly conserved ASN, PRO, and TYR residues in AT1R resulted in altered IP responses.84, 85 Also upon receptor activation, NPxxY motif shows conformational changes to adopt a different structural rearrangement for the intracellular partner binding.86 Thus, RMSD values of this highly conserved motif across the GPCR family are used to discriminate active and inactive structures. Here, we plotted RMSD values of NPxxY motif together with the TM3-TM6 distance. The graph is given in Figure S19. Mainly the graph is divided into four parts. Regions I and III represent inactive-like and active-like intermediates, respectively, regions II and IV represent active and inactive conformations. Chain A of TM1,2,8 interface AI dimer exhibited conformations mostly between active and active-like states (regions II and III) and chain B mainly keeps conformations in inactive or inactive-like states (regions I and IV). Our simulations results suggest four different plausible interfaces that consist of one active and one inactive pair. This result is supported by the analysis in Figure S19. When we check starting structures either with active-active (AA) or inactive-inactive (II), (e.g., TM5 and TM4,5 tight AA, and TM4 II), it is clear that while one protomer prefers to stay in started conformation, other one changes its initital conformation throughout the simulation. For example, although initial conformations are active in both protomers in TM5 AA and in TM4,5 tight AA interfaces, a significant portion of the trajectory frames are shifted to regions I and IV representing inactive or inactive-like states while rest keep their initial active-like or active conformations (regions II and III). Similar situation was also observed in TM4 II. Although initial conformations are inactive in both protomers, an important amount of the trajectories were shifted to regions II and III (active-like and active states), while rest stays as they started in the regions I and III (inactive-like and inactive). Intraprotomer Interactions that Stabilize Inactive States. To get more insights into why the structural stabilities were observed in these selected four dimer systems, we measured residue-residue distances within intraprotomer systems. In the TM1,2,8 interface AI dimer system, we measured three different distances individually in each protomer: MET57 CE – ILE238 CD, ALA63 CB – ILE238 CG2, and LEU122 CD2 – ILE238 CG2. In all these three atom-atom distances were markedly different in active and in inactive chains (Figure S20). We observed that these interactions were played a substantial role in stabilizing the inactive protomer around 5 Å. However, in active chain, these distances were found higher than ~15 Å. In TM5 interface, another distance was found to be distinctive, ALA129 CB – ILE238 CD. Similar to the previous inactive structure, these distances were measured as around 5 Å in the inactive chain. The measured distances between ALA129 CB and ILE238 CD were higher than 15 Å in the active chain. (Figure S21). In the TM4 interface II dimer, four distances were considered for inactive protomer. These distances were MET57 CG – ILE238 CD, ALA63 CB – ILE238 CG2, ALA63 CB – ILE242 CD, and LEU119 CD1 – VAL246 CG2. In all distances, inactive chain was stabilized around 5 Å, however all distances substantially differed in active chain B, having a value over 10 Å (Figure S22). For the case of TM4,5 tight AA dimer interface, distinctive distances in active protomer that differ substantially than the inactive protomer were not observed. Among these distances ILE238 was the common residue in the TM1,2,8 AI; TM5 AA, and TM4 II dimer systems operating as a hub point between the functional states of AT1R. Previously, it was reported that ILE238 mutation abolishes IP production.87 We conclude that ILE238 from TM6 can interact with a nonpolar residue from TM1, TM2, or TM3 to stabilize the inactive conformation. However, all these distances were increased markedly in active protomers (Figures S20-S22). Phenylalanine Ratchet Rearrangement (FFF Motif) (PHE2085.51, PHE2496.44, and PHE2506.45). By the determination of active crystal structure of the partial agonist S1I8 bonded AT1R, revealed a phenylalanine ratchet that consists of PHE208, PHE249 and PHE259. ILE3.40, PRO5.50 and PHE6.44 side chains were rearranged in active structures of GPCRs (namely in μOR, β2AR, and M2R), outlining that these hydrophobic core plays important role in class A GPCR activation.88 The importance of the residues at 5.5189, 90 and 6.4491-93 positions in the activation mechanism was also highlighted in the literature. However, in AT1R, instead of IPF motif, FFF motif was argued to play ACS Paragon Plus Environment

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a crucial role in the activation mechanism, having discrete conformations in inactive and active states of the receptor.66 Here, we compared the active protomers obtained throughout the MD simulations with the crystal structure of the active state of the AT1R. For this aim, we used representative structures from clustering analysis and aligned them to the active crystal structure. The FFF motif in the crystal structure of active-state human AT1R bound to an ANG-II analog indicates that while PHE208 and PHE250 have outward movement, the movement of PHE249 is inward toward the binding cavity. This structural movement has been observed in all of the selected active state of our models. (Figure 4, A-D)

Figure 4. Phenylalanine ratchet alignment in cluster representatives and crystal structure is compared by aligning chain A of TM1,2,8 interface AI dimer (A), chain B of TM5 interface AA dimer (B), chain B of TM4 interface II dimer (C), and chain A of TM4,5 tight AA dimer (D) to the crystal structure of active AT1R (PDB ID, 6DO166, 94). TYR2155.58 – TYR3027.53 (YY Motif) Distance Analysis. Two tyrosine residues residing at TM5 and TM7, play an important role, by interacting directly with each other or by water mediated interactions.89, 95 In crystal structures of M2R96, β2AR97, and rhodopsin98, TYR2155.58 – TYR3027.53 interaction was suggested as water mediated, since the distance between hydroxyl groups were found to be distant to form hydrogen bond. Similarly, in the crystal structure of active AT1R, the distance between TYR2155.58 and TYR3027.53 is 4.2 Å66, likely capable of forming interaction via a bridging water. In our cluster poses, we also examined this distance. In cluster conformation of both chain B of TM5 interface AA and TM4 interface II dimers, we calculated the distance between these structurally important tyrosine residues as 4.2 and 4.6 Å, respectively. Alignment of these distances were shown in Figure 5. These two tyrosine residues in TM5 and TM7 stabilize the active structure of the receptor by interacting with each other directly or with the help of the water molecule.95


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Figure 5. YY motif in cluster representatives and crystal structure is compared by aligning chain A of TM1,2,8 interface AI dimer (A), chain B of TM5 interface AA dimer (B), chain B of TM4 interface II dimer (C), and chain A of TM4,5 tight AA dimer (D) to the crystal structure of active AT1R (PDB ID, 6DO166, 94). Change in YY motif distance in chain B of TM5 AA and chain B of TM4 II dimer system along the simulation trajectories (E). In chain A of TM1,2,8 AI dimer and chain A of TM4,5 tight AA dimer systems, distances between hydroxyl groups were found to be distant to form hydrogen bond similar to AT2R active structures.34, 71


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VAL1083.32 – TYR2927.43 and ASN1113.35 – ASN2957.46 Distance Analysis. Recently, Singh et al.81 investigated activation of AT1R with its endogenous ligand; ANG-II, and proposed the activation mechanism, where the TM3-TM7 distance plays an important role. They claimed that activation sequence first started with the interruption of the interaction between VAL1083.32 and TYR2927.43, and then, the interaction between ASN1113.35 and ASN2957.46 was interrupted.

Figure 6. Alignment of the active AT1R crystal structure (PDB ID, 6DO166) with active protomer of TM1,2,8 interface AI dimer, A and C (crystal, salmon; cluster structure, red). B: VAL1083.32 – TYR2927.43 distance change over time along the trajectory (starting distance: 5.92 Å). D: ASN1113.35 – ASN2957.46 distance change over time along the trajectory in active (green) and inactive (blue) structures, (two distances were calculated, ASN1113.35 OD1 and ASN2957.46 ND2, and ASN1113.35 ND2 and ASN2957.46 OD1). In order to compare our results with these findings, VAL1083.32 – TYR2927.43 and ASN1113.35 – ASN2957.46 distance analyses were performed. (Figure 6) VAL1083.32 Cα – OH TYR2927.43 distance was stabilized in starting distance, resembling active crystal structure (Figure 6, B). In both independent runs, similar stabilized structures were observed. ASN1113.35 – ASN2957.46, can interact in two ways; ASN1113.35 OD1 and ASN2957.46 ND2, or ASN1113.35 ND2 and ASN2957.46 OD1, where these two distances were measured as 2.9 and 3.0 Å in crystal structure, respectively. In cluster pose, these distances were calculated as 7.5 and 8.8 Å, respectively. We plotted fluctuations of these two distances over time in Figure 6, D. As proposed by Singh et al.81, we conclude that these two distances are the prerequisites that should be satisfied in the chain fashion in the activation of AT1R by ANGII. G Protein Coupling with the Active States of Selected Dimer Pairs. The selected dimer pairs were aligned with the recently crystallized µ opioid receptor (µOR)– Gi protein (PDB: 6DDF99) and also to the rhodopsin – arrestin (PDB: 5W0P100) complexes, (Figure S23). It is clearly seen in Figure S23 that, all selected structures have available space for the intracellular binding partner (i.e., Gi or arrestin). Interestingly, in Figure S23 we have observed that helix 8 (H8) of TM4 II chain B (Figure S23, C and G), has an unusual conformation compared to other structures. TM4 interface II dimers with the Gi protein and arrestin complexes were highlighted in Figure S24. Although H8 in these complexes have unusual conformation, no major clashes were observed with G protein (Figure S23, C and Figure S24, A) or arrestin (Figure S23, G and Figure S24, B). It must be noted that unusual ACS Paragon Plus Environment

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conformations of H8 and its quite dynamic behavior in different GPCRs have been previously analyzed and discussed.34,101,102 For example, US28101 and AT2R34 were reported that H8 of these GPCRs have a non-canonical conformation. It is discussed by Tate102 and named the observed phenomenon as “a receptor that might block itself” due to unusual conformation of H8 that moved towards to the intracellular binding partner site. However, when we compared the structures of US28, AT2R, and our representative structure from clustering of TM4 II, we clearly observed that, H8 of TM4 II chain B, has a conformation that is distinct from US28 and AT2R, where it does not clash with either arrestin or G protein (Figure S25). Interface Analyses. Interface analysis in GPCR dimerization is also important for assessing interface stability. To do this, we used the Pisa server to analyze our six different dimer interfaces. We used MD simulations initial and representative structures from the cluster analysis, and compared them to evaluate the changes. Since we have three different dimer pairs, namely II, AI, and AA, we have compared the initial structure and the representative structure in each system, individually, (Figures S26-S31). Following color scheme was used to highlight the changes at the dimer interface: yellow color shows the amino acid is still at the interaction interface; green color means that this is the new established interaction and it is not presented in starting structure however it is gained in the representative one, and red color represents that this interaction is presented at the initial conformation but it is lost in representative structure. In the TM1,2,8 interface AI dimer system, in the initial conformation we have observed TM1 and TM2 interactions between the protomers, however in the representative structure we have observed ECL1, ECL2, and H8 interactions in addition to TM1 and TM2 (Figure S25). In TM5 interface AA dimer, initial conformation, consisted of interfacing residues from TM3, ICL2, TM4, ECL2, TM5, ICL3 and TM6. ICL3 and TM6 contacts were lost, while other interactions were maintained at the representative structure (Figure S28). In TM4 interface II dimer, residues from TM3, ICL2, TM4, and ECL2 in the initial conformation were contacted at the interface. In the representative structure, ICL2 interactions were lost, and TM5 interaction were gained (Figure S29). In TM4,5 tight AA dimer, residues from ICL2, TM4, ECL2, and TM5 in the initial conformation were contacted, however in cluster structure some of ICL2 and ECL2 interactions were lost and TM3 contributed to the dimerization interface (Figure S30). Center of Mass (COM) Distance Between Protomer Analysis. We also monitored COM of protomers, and compared the obtained values with simulation starting distances to gain insight into protomer-protomer interactions. In TM1,2,8 interface AI dimer system, first distance between protomers was increased but after a while, it returned to the simulation starting distance (Figure S32, first panel, middle column). In TM5 interface AA dimer pair, the COM distance were maintained in both runs (Figure S32, third panel, last column). In TM4 interface II dimer, during two runs, the distance between protomers fluctuated between 38-40 Å (Figure S32, fourth panel, first column). In TM4,5 tight interface AA dimer, the distance was stabilized throughout around 36 Å (Figure S32, fifth panel, last column). The largest change was observed in the TM4,5 loose II dimer system where the protomers approached around 4 Å to each other (Figure S32). Principal Component Analysis (PCA). It is difficult to distinguish functionally relevant motions from local fluctuations. Thus, we also applied PCA method to extract collective motions from our simulation trajectories. To filter out global-collective motions from local-fast motions, we concentrated on backbone atoms and built covariance matrix of the atomic fluctuations. And this matrix diagonalized to yield set of eigenvalues and eigenvectors, which describes collective motions in our system.103,104 Similarly with the previous part, we concentrated on four systems: TM1,2,8 interface AI dimer, TM5 interface AA dimer, TM4 interface II dimer, and TM4,5 tight AA dimer. In the active protomer of TM1,2,8 interface AI dimer system, we observed a large movement in H8, and this movement caused a significant change in the lower part of TM1 and a slight movement in the lower part of TM2. In the inactive protomer of TM1,2,8 interface AI dimer system, most mobile parts were detected as ICL3 and H8. Alongside with ICL3 and H8, N-terminal, ECL2, ECL3, and TM7 moved mainly towards to the TM1 (Figure S33). In the AA dimer system of TM5 interface, we only observed small movements in the loop regions of protomer A, in ICL2 region. Unlike protomer B, had distinct movements in H8 and ICL3 together with intracellular part of TM6. N-terminal moved ACS Paragon Plus Environment

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towards to the extracellular portion of TM7, and also ECL2 moved significantly towards N-terminal (Figure S34). In the II dimer system of TM4 interface, in protomer A, N-terminal, ECL2, intracellular parts of TM5 and ICL3, TM7 and H8 regions were more mobile. In protomer B, a large movement of H8 to the G protein coupling side, was observed. TM6 opening was captured perfectly alongside with the ICL3 motion. No motion was observed in TM4, which constituted the dimer interface (Figure S35). In the chain A of the TM4,5 tight AA dimer, N terminal moved towards to the ECL2, and very slight movement was observed in ICL3 that moved away to initiate movement of TM6 towards more open form. However, in chain B, compared to the chain A, we only observed a slight movement of ECL1 towards to the ECL2 (Figure S36). Water Pathway Analysis. Representative structures from clusters of all systems were subjected to water pathway analysis. In chain A of TM1,2,8 interface AI dimer system, water penetrations were observed from upper and lower sides of the protomer, but the continuity of the water pathway was discontinued by the blocking of PHE77, VAL108, TRP253, and HIS256 residues, just below the orthosteric binding site in active protomer (Figure 7, A). However, we observed a different story in inactive protomer of TM1,2,8 interface AI dimer, where continuous water pathway from the extracellular site, weakened at the ANG-II binding pocket by HIS256, THR260, MET284, and ILE288, and blocked by the VAL49, LEU67, ALA71, LEU119, ILE241, ILE245, ASN298, and PHE301 (Figure 7, B). In TM5 interface AA dimer system, while a continuous water pathway was interrupted at the binding region of ANG-II by VAL108, LYS199, THR260, and ILE288 in one protomer (Figure 7, D), a continuous water pathway in other protomer was observed (Figure 7, E). In chain A of TM4 interface II dimer, while a continuous water pathway was blocked by VAL49, MET57, LEU67, LEU122, ILE238, PHE301, and LEU305 at the bottom site of the chain A, a continuous water pathway was observed in protomer B (Figure 7, F, G). In the chain A of TM4,5 tight interface AA dimer, just below the ANG-II binding site, continuous pathway was weakened by PHE77, VAL108, TRP253, and ILE288 (Figure 7, H). In chain B, water pathway disrupted in two distinct regions (Figure 7, I). First interruption was observed around the ANG-II binding site by PHE77, LEU112, LYS199, TRP253, THR260, ILE288, and ALA291. Second blockage was observed around G protein binding region, LEU67, LEU122, and TYR302 were contributed. There are contradictory results in the literature for the formation of continuous water pathway inside the GPCRs. Bai et al.105 reported that in liganded active state of the β2AR a close state of water channel was observed. However, in parallel with our observations, several other studies reported a continuous water pathway formation in the active state of the GPCRs.106-108 In our water pathway analysis in Figure 7E, a continuous water pathway was observed in inactive chain A. However, when intracellular binding partner region carefully examined, it can be observed that the exposure of intracellular site of the receptor distinctly different in chains A and B. The solvent accessible surface area of chain B is much larger than the chain A, as it is clearly depicted in Figure 7E. Also, when we consider TM4,5 tight interface AA dimer (Figure 7, J), similarities between chains A and B were found. Despite, chain A is in active and chain B is in inactive forms based on distance and TM6 conformation analyses, similar water pathways were observed. We speculate that, when the dimer consists two active protomers at the beginning of the simulation, water molecules penetrate inside to the cavity, and a continuity of water pathway can be seen in also in inactive-like structures (i.e., conversion from active to inactive conformation may not expel water molecules completely). Overall, in our water pathway analysis, we observed more water exposure from the intracellular cavity in active protomers of each dimer systems; TM1,2,8 interface AI dimer, TM5 interface AA dimer, TM4 interface II dimer, respectively.


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Figure 7. Chain A of TM1,2,8 interface AI dimer (A), chain B of TM1,2,8 interface AI dimer (B), and side view of water pathways in TM1,2,8 interface AI dimer (C). Chain B of TM5 interface AA dimer (D). Although a blockage of water pathway at the extracellular vestibule was observed, there was a continuous pathway in other chain (E). Side view of water pathways in TM5 interface AA dimer (E). Chain A of TM4 interface II dimer (F), although a blockage of water pathway at the intracellular vestibule of chain A, there was a continuous pathway in other chain. Side view of water pathways in TM4 interface II dimer (G). Chain A (H) and chain B of TM4,5 tight interface AA dimer (I), side view of the water pathways in the TM4,5 tight AA dimer system (J).


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CONCLUSIONS In this study, we investigated dimerization event of the AT1R in aid of the MD simulations using six different interfaces to suggest a possible dimer interfaces in atomic details. We proposed four possible interfaces (TM1,2,8; TM5; TM4; and TM4,5). One of the earliest reports on AT1R dimerization was investigated by chemical crosslinking at the GLU315 of helix 8, highlighting that H8 should be involved in the dimerization interface.20 All suggested interface systems were composed of one active protomer and one protomer in inactive or intermediate conformation, in line with previous reports on GPCR dimerization.109-112 Contradictory to our and previous results, Lyngsø proposed that in the light of Karip et al.22 findings, AT1R homodimers should constitute of two active components, since one inactive form of AT1R can inhibit the second’s activation.94 However, in these suggested interfaces, we observed stable forms, especially in TM1,2,8 interface, where both chains were stabilized in two independent runs. Recently published crystal structures of GPCRs suggested more than one interface can be involved in receptor dimerization, which is in agreement with our data.9,11 In addition, ILE2386.33 was found as a hub residue in stabilization of the inactive state of the AT1R, where it interacted with residues from TM1, TM2, or TM3. This enables a hydrophobic cluster just above the G protein binding site, and blocks the water penetration inside to the receptor, thus preventing its activation. In conclusion, we performed a detailed study to better understand of the dimerization of GPCRs and its functional outcomes. Collectively, we aimed to investigate a class A prototypical GPCR, AT1R dimerization with the aid of MD simulations and revealed interfaces will pave the way to develop better therapeutics with desired efficiencies and efficacies.

ASSOCIATED CONTENT Supporting File AUTHOR INFORMATION Corresponding Authors: *Emails: [email protected] (IE), [email protected] (SD) ORCID Ismail Erol: 0000-0001-8256-6283 Bunyemin Cosut: 0000-0001-6530-0205 Serdar Durdagi: 0000-0002-0426-0905 Funding This work was supported by the Scientific and Technological Research Council of Turkey (TÜBİTAK); Project No: 214Z122. Acknowledgment The numerical calculations reported in this paper were fully performed at TUBITAK ULAKBIM, High Performance and Grid Computing Center (TRUBA resources). This work was supported by the Scientific and Technological Research Council of Turkey (TÜBİTAK); Project No: 214Z122. ACS Paragon Plus Environment

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Author Contributions I.E., B.C. and S.D. designed the research. I.E. built models, performed and analyzed MD simulations under the guidance of SD. I.E, B.C and S.D. wrote the manuscript. Notes The authors declare no competing financial interest. References 1. 2. 3.

4. 5. 6.

7. 8.

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