Trans and Cis Conformations of the Antihypertensive Drug Valsartan

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Computational Biochemistry

Trans and Cis Conformations of the Antihypertensive Drug Valsartan Respectively Lock the Inactive and Active-Like States of Angiotensin II Type 1 Receptor: A Molecular Dynamics Study Lingyun Wang, and Feng Yan J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.8b00364 • Publication Date (Web): 13 Sep 2018 Downloaded from http://pubs.acs.org on September 14, 2018

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Journal of Chemical Information and Modeling

Trans and Cis Conformations of the Antihypertensive Drug Valsartan Respectively Lock the Inactive and Active-Like States of Angiotensin II Type 1 Receptor: A Molecular Dynamics Study

Lingyun Wang1, Feng Yan2,3* 1

Division of Nephrology, Department of Medicine, The University of Alabama at Birmingham,

Birmingham, Alabama 35294, United States 2

School of Environmental and Chemical Engineering, Tianjin Polytechnic University, Tianjin

300387, P. R. China 3

State Key Laboratory of Separation Membranes and Membrane Processes, Tianjin Polytechnic

University, Tianjin 300387, P. R. China

* Corresponding e-mail: [email protected]

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Abstract Angiotensin II type 1 receptor (AT1R) is the principal regulator of blood pressure in humans. The overactivation of AT1R by the stimulation of angiotensin II would result in high blood pressure. To prevent hypertension, non-peptide ‘sartan’ drugs, such as valsartan (VST), have been developed to competitively block the access of angiotensin II to the receptor. Nuclear magnetic resonance spectroscopy and molecular modeling studies have identified that VST in solution and in lipid micelles (a mimic membrane environment) has two distinct trans/cis conformations (VSTtrans/VSTcis) which can be transformed into each other through the isomerization of the amide bond. To date, it is still not known whether the two conformations of VST can affect the binding of AT1R with VST. To this end, the binding of AT1R with VSTtrans or VSTcis was modeled based on the recently determined crystal structures of AT1R. Molecular dynamics simulations were then performed to study the structural and dynamical differences of AT1R caused by the two conformations of VST. Simulation results show that AT1R with VSTtrans has higher structural and dynamical stabilities compared to that with VSTcis. Binding energy analysis indicates that AT1R bind more strongly with VSTtrans, and the energy difference mainly results from the contribution of van der Waals and non-polar interactions. Detailed analyses reveal that unlike AT1R with VSTtrans, AT1R with VSTcis displays an activate-like state, which is characterized by a small outward movement of transmembrane helix 6. Due to the altered interaction with the butyl group of VST, residue Tyr87 undergoes a conformational change that causes a contraction of the pocket for VST binding. The rearrangement of AT1R is then propagated to the intracellular side of the receptor through the conformational change of residue Trp253 (the toggle switch), which results in an expansion of the pocket for G protein binding and the breakage of the hydrogen bond containing the conserved residue Arg126. These data provide

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insights into the activation mechanism of AT1R caused by the binding of VSTcis, which may help to design new drug to inhibit AT1R and prevent high blood pressure.

Keywords Hypertension; angiotensin II type 1 receptor; active-like state; antihypertensive drug valsartan; trans/cis conformation; molecular dynamics simulation

1. Introduction High blood pressure, or hypertension, is one of the most prevalent diseases worldwide, and affects 85.7 million people in the United States1. Hypertension is a risk factor for many diseases such as myocardial infarction, heart failure, stroke and end-stage renal disease2. In humans, the arterial blood pressure is regulated by a coordinated hormonal cascade called the rennin-angiotensin system (RAS), whose activation is related to the pathogenesis of hypertension3. The principal effecter of RAS is angiotensin II (an octapeptide hormone). It acts as a circulating vasoconstrictor, and mainly performs its function through stimulating the angiotensin II type 1 receptor (AT1R)4. AT1R belongs to the superfamily of G-protein coupled receptor (GPCR) and is the key regulator of blood pressure and body-fluid homeostasis5. It plays vital roles in cardiovascular and renal pathophysiology. Effective control of the overactivation of AT1R would avoid the high blood pressure. To prevent hypertension, non-peptide drugs have been developed to competitively block the access of angiotensin II to AT1R. These drugs, named angiotensin receptor blockers (ARBs), include losartan, valsartan, olmesartan, candesartan, eprosartan, telmisartan, irbesartan, and azilsartan6. Among these ‘santan’ drugs, valsartan (VST) is the second orally active drug available in the market of the United States. The treatment of patients with VST could decrease

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the ratio of major adverse cardiovascular events, the incidence of new onset diabetes, and the rate of renal function decline7. Though the ‘santan’ drugs share a common molecular scaffold consisting of biphenyl-tetrazol and imidazole groups, they have slight structural differences. The main structural difference between VST and other ARBs is that the heterocyclic group in the ARBs is replaced by an alkylated amino acid in VST8. Recently, the crystal structures of human AT1R in complex with an antagonist ZD7155 (PDB ID: 4YAY)9 and with an ARB drug olmesartan (PDB ID: 4ZUD)10 have been determined. The crystal structures exhibit that the transmembrane (TM) domain of AT1R consists of seven TM α-helices (TM1-7), three intracellular loops (ICL1-3), three extracellular loops (ECL1-3), and an amphipathic helix 8 (Fig. 1A). Comparison of the two structures reveals that ZD7155 and olmesartan share a similar binding mode with AT1R, in which three residues Tyr351.39, Trp842.60, and Arg167ECL2 of AT1R play an important role in binding. (The superscript after each residue indicates Ballesteros–Weinstein numbering for the residues of GPCRs11). Molecular docking simulation and site-directed mutagenesis experiment9, 10 also indicate that ARBs utilize conserved orientation and recognition mode to bind with AT1R. These data provide a molecular basis to study the interaction of AT1R with other ARBs such as VST.

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Fig. 1. Initial simulation structure and structural deviations as a function of time. (A) Trans (τ = 180º) and cis (τ = 0º) conformations of valsartan (VST) are modeled in the orthosteric site of angiotensin II type 1 receptor (AT1R). The simulation systems of AT1R with the trans and cis conformations of VST are denoted as AT1R-VSTtrans (I) and AT1R-VSTcis (II), respectively. The transmembrane helices 1-7 and helix 8 of AT1R are shown in red, orange, yellow, green, cyan, blue, purple, and pink, respective. The gray dashed lines indicate the surfaces of membrane. (B) Root mean square deviations (RMSDs) for Cα atoms of AT1R (black lines) and for the non-hydrogen atoms of VST (red lines) as a function of simulation time. Three 400 ns simulation trajectories were performed for each system.

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The conformation and binding mode of VST in solution and in lipid micelles (a mimic membrane environment) have been explored by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling studies12, 13. The results demonstrate that VST has two distinct conformations which can be transformed into each other by the isomerization of the amide bond (Fig. 1A). As sartan drugs bind to AT1R after passing through the membrane14, it is still not known whether the two distinct conformations of VST could affect their binding with the receptor. To this end, molecular dynamics (MD) simulation, a useful tool to study the functional and dynamical changes of GPCRs15, was performed in this work. Results show that the trans conformation of VST has greater binding energy with AT1R, and locks AT1R in an inactive state. However, the cis conformation of VST changes its interaction with AT1R and increases the structural and dynamical flexibilities of AT1R, which ultimately induces an activelike state of the receptor. These data give an example of how the distinct conformations of a ligand affect the active/inactive states of GPCRs. The activation mechanism of AT1R identified in this work may provide new strategy to design potent drugs to prevent hypertension.

2. Material and methods 2.1. Simulation systems. The structure of AT1R in complex with VST was modeled based on the crystal structures of AT1R in complex with an antagonist ZD7155 (PDB ID: 4YAY)9 and with an ARB drug olmesartan (PDB ID: 4ZDU)10. In both of the crystal structures, AT1R adopts an inactive state. To get the structure of the transmembrane domain of AT1R, the missing residues in the loops of ECL2 (residues 186 to 188) and ICL3 (residues 225 to 234) were modeled using MODELLER 9.1316. The modeled structure with the lowest DOPE energy was chosen as the best model. The structure of b562RIL (a thermostabilized apocytochrome used to facilitate

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crystallization) was removed before structure modeling; while the disulfide bonds Cys18-Cys274 and Cys101-Cys180 were retained in the modeled structure. The modeled transmembrane domain of AT1R (residues 17-317) contains TM1-7, ICL1-3, ECL1-3, and helix 8. Since crystal structure data and molecular modeling results demonstrate that sartan drugs have a similar orientation to interact with AT1R 9, 10, the location and orientation of VSTtrans in AT1R was determined by superimposing the biphenyl-tetrazol group of VSTtrans with that of olmesartan (Fig. S1). The conformation of VSTcis was obtained by manually rotating the amide bond of VSTtrans by 180º (Fig. S1). This resulted in a change of hydrophobic interaction between AT1R and VST: the butyl chain of VSTtrans is surrounded by Phe772.53, Val1083.32, Ile2887.39 and Tyr2927.43; whereas the butyl chain of VSTcis is surrounded by Ile311.35, Tyr92ECL1, and Tyr872.63 (Fig. S1). For simplification, the simulation systems of AT1R with VSTtrans and VSTcis are denoted as AT1R-VSTtrans and AT1R-VSTcis, respectively. To model the membrane environment, the modeled AT1R was inserted into a palmitoyloleoyl-phosphatidyl-choline (POPC) bilayer composed of 130 lipid molecules using CHARMMI-GUI membrane builder17. Then TIP3P water molecules were added to both sides of the lipid bilayer (Fig. S2), which resulted in a simulation box of 74 Å × 74 Å × 115 Å along the x, y, and z directions, respectively. The parameters of FF99SB force field18 were assigned to the protein, ions, and water molecules, the parameter for VST is the same as in the previous work12, and a FFlipid14 force field19 was used for POPC lipids. 2.2. Molecular dynamics simulations. For each system, three independent simulations were performed starting from different initial velocities. All the molecular dynamics (MD) simulations were performed using AMBER16 simulation package20. The simulation protocol is similar to our previous studies21-26. Briefly, each system was first minimized using steepest descent and

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conjugate gradient methods. Then the whole system was gradually heated from 0 to 300 K under NVT (constant volume and temperature) condition with the constrains of AT1R and VST. After that, the constraints were gradually switched off and 400 ns production simulations were performed in NTP (constant temperature and pressure) condition. The time step of the simulations was 2 fs and the trajectories were collected every 1 ps. 2.3. Data analyses. Simulation data analyses were mainly performed using the CPPTRAJ program of AMBER1620. The secondary structure (mainly α-helix) of AT1R was evaluated by DSSP method27. The occupancy of each residue in an α-helix was calculated on the basis of the percentage of time that the residue existed in the α-helix over the equilibrated simulations. The dynamical flexibility of AT1R was identified by computing root mean square fluctuation (RMSF) on a residue-by-residue basis and then averaged over the equilibrated simulations. The volumes for the pocket of VST-binding and pocket on the intracellular side of AT1R were calculated by POVME 2.028. The binding free energy was calculated using the molecular mechanics/generalized Born surface area (MM/GBSA) approach29. The calculation procedure is similar to our previous work30-32. In brief, the binding free energy (∆Gbinding) was computed by summing the molecular mechanics energy including the electrostatic energy (∆Eele) and the van der Waals energy (∆EvdW), the solvation free energy including a polar part (∆Gpol) and a non-polar part (∆Gnonpol), and the entropy contribution to the binding (-T∆S). To determine which residue of AT1R plays an important role in the binding of VST, the interaction energy was calculated by decomposing the binding free energy based on each residue of the receptor. For comparison, average values and standard errors for distance, pocket volume and binding free energy were calculated. Significant differences for the studied variables were

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determined using the Student’s t test33 with 95% confidence. Visualization of the structure was presented by VMD software34.

3. Results and discussion To investigate whether the trans and cis conformations of VST affect their binding with AT1R, two systems were set up: (I) AT1R-VSTtrans, AT1R bindings with the trans conformation of VST, and (II) AT1R-VSTcis, AT1R bindings with the cis conformation of VST (Fig. 1A and Fig. S1). Starting from different initial velocities, three simulation trajectories of 400 ns were performed for the two systems (I-1 to I-3 and II-1 to II-3). Before data analyses, the equilibrations of the simulations were determined by checking the root mean square deviations (RMSDs) of Cα atoms of AT1R and the non-hydrogen atoms of VST (Fig. 1B). It shows that the RMSDs for both AT1R and VST reached stable state before 200 ns in all the simulations, thus the last 200 ns trajectories were used for data analysis to get the averaged data values. 3.1. AT1R with the trans conformation of VST has higher structural and dynamical stabilities, especially for transmembrane helix 6. First, secondary structure analyses were performed to investigate whether the distinct conformations of VST could cause the structural change of AT1R (Fig. 2A and Fig. S3). To give a quantitative comparison of the secondary structure, the occupancy for each TM helix was calculated by summing the occupancy of all the residues in the helix and then dividing it by the residue number. Results show that the occupancies of TM1-TM5 in AT1R-VSTtrans are similar with those of AT1R-VSTcis (the detailed occupancies for these TM helices are shown in Fig. 2A). However, the occupancy of TM6 in AT1R-VSTtrans (91.4%) are much higher than that in AT1R-VSTcis (84.5%), while the occupancy of TM7 in AT1R-VSTtrans (82.6%) is lower than that in AT1R-VSTcis (90.6%). Moreover, the

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occupancy of helix 8 is low in both systems (51.9% vs. 35.3%).

Fig. 2. Structural and dynamical changes of AT1R caused by the two conformations of VST. (A) Secondary structure analysis for AT1R. The α helices in AT1R-VSTtrans are shown in black, while those in AT1R-VSTcis are shown in red. The occupancy percentage for each α helix was calculated by summing the occupancy of all the residues in the helix and then divided by the residue number. The regions for transmembrane helices (TM) 1-7 and helix 8 are shaded in gray. (B) Root mean square fluctuation (RMSF) for each residue of AT1R. The secondary structure regions are also shaded. The structural and dynamical stabilities of AT1R with VSTtrans are increased, especially for those of TM6. Furthermore, helix 8 shows low helix occupancy and high flexibility in both systems.

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The dynamical stability of AT1R was studied by calculating the root mean square fluctuation (RMSF) that measures the averaged fluctuation amplitude of each residue over the equilibrated trajectories (Fig. 2B and Fig. S4). To assess how RMSF was changed for the TM helices, the regions of the secondary structures were also shaded in Fig. 2B. It shows that all the TM helices (TM1-TM7) are stable compared to the loop regions. Furthermore, the RMSF values for TM1-TM4 in AT1R-VSTtran are similar with those in AT1R-VSTcis. However, the RMSF values for TM5, TM6 and TM7 in AT1R-VSTtran are lower than them in AT1R-VSTcis. The results indicate that VSTtrans stabilizes the structure and dynamics of AT1R, especially for those of TM6. 3.2. AT1R has greater binding free energy with the trans conformation of VST. Binding free energy between AT1R and VST was calculated using MM/GBSA method (Table 1 and Fig. S5). The total binding free energy (∆Gbinding) of AT1R-VSTtran (-10.36 ± 1.00 kcal/mol) is much greater than AT1R-VSTcis (-4.68 ± 0.99 kcal/mol). To further analyze the energetic details, the components of binding free energy were computed and presented in Table 1. Among the energy components, the electrostatic energy (∆Eelec), van derWaals energy (∆Evdw) and non-polar solvation energy (∆Gnonpol) are favorable for binding, whereas the polar solvation energy (∆Gpol) and entropy contribution are unfavorable for binding. The entropy contribution is the same between AT1R-VSTtran (25.48 ± 0.99 kcal/mol) and AT1R-VSTcis (25.51 ± 0.97 kcal/mol). The effect of electrostatic energy (∆Eelec) is counteracted by that of polar solvation energy (∆Gpol), and the sum of these two polar energy contributions (∆Gelec+pol) shows similar values between the two systems (30.30 ± 0.31 kcal/mol for AT1R-VSTtran, and 30.62 ± 0.34 kcal/mol for AT1RVSTcis). However, the sum of the two non-polar energy contributions (∆Gvdw+nonpol) shows that AT1R-VSTtran has a lower value (-66.04 ± 0.12 kcal/mol) than AT1R-VSTcis (-60.81 ± 0.15

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kcal/mol). Thus, the nonpolar and van derWaals interactions mainly contribute to the difference of the binding free energy between AT1R and the two conformations of VST. Table 1. Binding Free Energies (kcal/mol) of AT1R with the trans and cis conformations of VST. Component AT1R-VSTtrans AT1R-VSTcis ∆Eelec -26.93 ± 0.23 -34.63 ± 0.24 ∆Evdw -58.46 ± 0.12 -53.36 ± 0.15 ∆Gpol 57.13 ± 0.21 65.25 ± 0.24 ∆Gnonpol -7.58 ± 0.01 -7.45 ± 0.01 -T∆S 25.48 ± 0.99 25.51 ± 0.97 ∆Gelec+pol 30.30 ± 0.31 30.62 ± 0.34NS ∆Gvdw+nonpol -66.04 ± 0.12 -60.81 ± 0.15* ∆Gbinding -10.36 ± 1.00 -4.68 ± 0.99* ∆Eele, electrostatic energy in the gas phase; ∆Evdw, van der Waals energy; ∆Gpol, polar solvation energy; ∆Gnonpol, non-polar solvation energy; ∆Gbinding = ∆Eele + ∆Evdw + ∆Gpol + ∆Gnonpol -T∆S. * indicates the difference is significant, while NS means the difference is non-significant.

3.3. Trans and cis conformations of VST present different interaction patterns with AT1R. The difference in the binding free energy suggests the interaction between AT1R and the two conformations of VST may be varied as well. To investigate which residues in AT1R play key roles in the binding of VST, energy decomposition analysis was performed to get the interaction energy of each residue (Fig. 3A and Fig. S6). To give a clear view of the interactions, representative simulation snapshots are presented in Fig. 3B. From Fig. 3A we can see there are more than twenty residues involved in binding. We first focused on analyzing the three residues Y351.39, W842.60 and R167ECL2 (label in blue in Fig. 3) which have been identified as the key residues for AT1R binding of its ligands 9, 10. In both systems, these three resides have a similar interaction pattern with the trans and cis conformations of VST. R167ECL2 interacts with the tetrazole and carboxyl groups of VST, W842.60 interacts with the amide and butyl groups of VST, and Y351.39 interacts with the butyl group of VST (Fig. 3B). Energy decomposition shows that W842.60 and R167ECL2 have the greatest interaction energies in both systems, while the interaction

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energy for Y351.39 is small and similar in the two systems (Fig. 3A). Compared to AT1R-VSTtrans, the interaction energies for these three residues in AT1R-VSTcis are significantly reduced (sum of the interaction energies for these three residues is -7.17 kcal/mol in AT1R-VSTtrans, while it is 5.51 kcal/mol in AT1R-VSTcis).

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Fig 3. Trans and cis conformations of VST present different interaction patterns with AT1R. (A) Interaction energy for each residue of AT1R. Three key residues for AT1R binding of its ligands are labeled in blue, the residues that have greater interaction energies in AT1R-VSTtrans are labeled in cyan, the residues that have greater interaction energies in AT1R-VSTcis are labeled in red, and the residues that have similar interaction energies in the two systems are labeled in green or black. (B) Visualization of the interactions between AT1R and VST. VST is shown in black, and the butyl group of VST is labeled in orange dotted circle. The residues of AT1R are labeled in the same colors as in figure A. For clarity, only the side chain of each residue is shown. Besides W842.60 and R167ECL2, there are mainly six residues that have greater interaction energies in AT1R-VSTtrans than in AT1R-VSTcis. These residues are F772.53, L812.57, A1634.60,

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P2857.36, I2887.39 and Y2927.43 (label in cyan in Fig. 3). All these residues, except for A1634.60, form a hydrophobic pocket to accommodate the butyl group of VST in AT1R-VSTtrans (left figure in Fig. 3B, residue Y2927.43 is not shown for clarity). However, residues F772.53 and I2887.39 lose their contacts with the butyl group of VST in AT1R-VSTcis (right figure in Fig. 3B). This leads to a reduced interaction energies of these residues in AT1R-VSTcis (sum of the interaction energies is -4.59 kcal/mol in AT1R-VSTtrans, while it is -2.60 kcal/mol in AT1RVSTcis). Accompanied with the interaction change of the five residues, residue A1634.60 alters its interaction with the tetrazole group of VST in AT1R-VSTcis which also results in a reduced interaction energy (-1.13 kcal/mol for AT1R-VSTtrans vs. -0.66 kcal/mol for AT1R-VSTcis). Next, we focused on analyzing the residues that have large interaction energies (energy value smaller than -1.0 kcal/mol) in the two systems. These residues are S1053.29, V1083.32, and S1093.33 (label in green in Fig. 3). They are all located in TM3 and interact with the biphenyl group of VST (Fig. 3B). The sum of the interaction energies of these residues exhibits no difference between AT1R-VSTtrans and AT1R-VSTcis (-4.70 kcal/mol vs. -4.51 kcal/mol). Therefore, though the interaction of each residue with the two conformations of VST is a little bit different, the interaction energies for these residues are similar in the two systems. We also found there are six residues that have greater interaction energies in AT1RVSTcis than in AT1R-VSTtrans (label in red in Fig. 3). To test whether the increased interaction energies of these residues directly come from the conformational difference of VST, the contacts between the residues and the butyl group of VST were calculated. A contact is considered to be formed if the distance between a carbon atom of the residues and that of the butyl group of VST is within 5.4 Å. It reveals that residues Y872.63 and H2562.63 directly contact with the butyl group of VST in AT1R-VSTcis, whereas they have no contact in AT1R-VSTtrans (Fig. S7). For the other

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four residues, no contact is found in both AT1R-VSTcis and AT1R-VSTtrans (data not shown). Compared to H2562.63, residue Y872.63 has larger number of contacts, suggesting its importance in interacting with VST in AT1R-VSTcis. Simulation snapshots also reveal that residues Y872.63 and H2562.63 undergo conformational changes to interact with the butyl group of VSTcis (Fig. 3B), which indicates that the interaction difference of VST could alter the structural and dynamical properties of the residues in AT1R. (It is noted that the interaction energy of W2536.48 in AT1RVSTcis is also greater than it in AT1R-VSTtrans. The role of this residue in the activation of AT1R is discussed in part 3.4. Other residues like I311.35, Y92ECL, L1123.36, Y1133.37, and F182ECL2 have small interaction energies and are similar in the two systems. Thus, they are not further discussed.) 3.4. Cis conformation of VST induces an active-like state of AT1R. We have demonstrated that the distinct conformations of VST result in structural and dynamical differences of AT1R. To test whether the dynamical changes of AT1R can alter the inactive/activate state of AT1R, several elements that are thought to be involved in the activation of GPCRs were accessed. These featured elements include the outward movement of TM6, the contraction of the binding pocket for VST, the expansion of the binding pocket on the intracellular side, conformational change of toggle switch residue W2536.48, and the breakage of the hydrogen bond containing residue R1263.50. Comparison of the inactive and active structures of GPCRs reveals that the most striking and conserved feature for the activation of GPCRs is the outward movement of TM6 relative to the helical bundle, which creates a cavity on the intracellular side of the receptor to bind with G protein35. To access the displacement of TM6, the inter-helical dihedral angle calculating the rotation degree of the intracellular end of TM6 relative to that of TM3 was calculated (Fig. 4A

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and Fig. S8). In addition, the distance between residues N2356.30 and Y541.55 representing the displacement of the intracellular end of TM6 relative to that of TM1 was also computed (Fig. 4D and Fig. S9). Result shows that the inter-helical angle of TM3-TM6 in AT1R-VSTcis (25.01 ± 0.10º) is significantly decreased compared to that in AT1R-VSTtrans (28.16 ± 0.06º), and the distance between residues N2356.30 and Y541.55 in AT1R-VSTcis (22.37 ± 0.04 Å) is significantly increased compared to that in AT1R-VSTtrans (19.00 ± 0.02 Å). Conformational analysis for the crystal structures of GPCRs has indicated that the inter-helical angle of TM3-TM6 in the active state decreases 9º compared to the inactive state36, and the displacement of TM6 is about 14.0 Å in the active state37. The small outward movement of TM6 in AT1R-VSTcis indicates AT1R is in an active-like state.

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Fig. 4. Cis conformation of VST induces an active-like state of AT1R. (A) Inter-helical dihedral angle calculating the rotation degree of the intracellular end of TM6 (in blue) relative to that of TM3 (in yellow). * indicates the difference is significant when comparing the data of AT1R-VSTcis with that of AT1R-VSTtrans. (B) Comparison of the volume for the VSTbinding site between AT1R-VSTtrans (in purple) and AT1R-VSTcis (in pink). (C) Distribution of χ1 angle of W2536.48 named “toggle switch”. The residue in AT1R-VSTcis sampled the angle range around 190º that was not visited in AT1R-VSTtrans. (D) Comparison of the distance between N2356.30 and Y541.55 and of the volume for the pocket on the intracellular side of AT1R between AT1R-VSTtrans (in purple) and AT1R-VSTcis (in pink). (E) The hydrogen bond between R1263.50 and N2356.30 is maintained in AT1R-VSTtrans, whereas it is broken in AT1R-VSTcis. To clarify how the binding of VSTcis causes the conformational change of AT1R at the VST-binding site and how the change is propagated to the intracellular G protein-binding site,

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the volumes for the VST- and G protein-binding pockets were calculated (Fig. 4B,D and Fig. S10). Results show that the volume for the VST-binding pocket of AT1R-VSTcis (697.18 ± 6.84 Å3) is reduced compared to that of AT1R-VSTtrans (797.03 ± 10.08 Å3), while that for the G protein-binding pocket of AT1R-VSTcis (3283.40 ± 32.11 Å3) is extended compared to that of AT1R-VSTtrans (2372.09 ± 37.60 Å3). Further detailed analysis was performed for the residues that have direct interactions with the butyl group of VSTcis. We found that the volume contraction for the VST-binding site of AT1R-VSTcis mainly results from the aforementioned conformational change of Y872.63 (Fig. 4B). Accompanied by the interaction change of H2566.51 with the butyl group of VST (Fig. 3B), residue W2536.48 in AT1R-VSTcis also undergoes a conformational change compared to it in AT1R-VSTtrans (Fig. 4B). W2536.48 is a highly conserved residue in GPCRs and is called “toggle switch” since it serves as a signal transducer in the activation of GPCRs. It has been reported that this residue changes its rotation state, i.e. from ‘g+’ to ‘trans’ state, in the activation of cannabinoid receptor38. The ‘g+’ state corresponds to the rotation state that the χ1 angle of the residue is between 240 º and 360º, whereas the ‘trans’ rotation state corresponds to the state that the χ1 angle is between 120º and 240º. The calculation of the χ1 angle of W2536.48 show that the ‘trans’ state is sampled by the residue in AT1R-VSTcis while the residue is restricted in the ‘g+’ state AT1R-VSTtrans (Fig. 4C), indicating the regulatory role of W2536.48 in triggering the active-like state of AT1R. In the inactive state of GPCRs, an “ion lock” is always formed between residue R3.50 and D/E6.30 to lock the state. Due to the lack of an acidic residue at position 6.30, the ion lock is not formed in AT1R. Instead, residue R1263.50 forms a hydrogen bond with the side chain of N2356.30. The hydrogen bond between these two resides is maintained in all the simulations of AT1RVSTtrans, indicating AT1R is locked in an inactive state. However, the hydrogen bond is disrupted

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in two of the three simulations of AT1R-VSTcis (Fig. S11). The breakage of the hydrogen bond would further facilitate the active-like state of AT1R. 3.5. Helix 8 reveals different dynamic behaviors in the two states of AT1R. The structural and dynamical analyses have showed that the helix occupancy of helix 8 is low in both AT1R-VSTcis and AT1R-VSTtran, and helix 8 is more flexible as compared to the TM helices (Fig. 2). This is consistent with the simulation result that helix 8 in the active-like state of neurotensin receptor 1 is intrinsically unstable and does not forms a helix structure39. Structural comparison of the active-like AT2R (a close homologue of AT1R) with the inactive AT1R shows that helix 8 in AT2R is in a conformation preventing the recruitment of G protein40. To test whether the conformation of helix 8 in AT1R-VSTcis is different from that of AT1R-VSTtran, the mass density describing the location of helix 8 along the bilayer normal (z axis) was calculated (Fig. 5A). To give a clear view of the positions of helix 8 relative to the membrane surface, final simulation structures are represented in Fig. 5B. The black dashed lines in Fig. 5A represent the density peaks of the phosphate (P) atoms of POPC lipids, which indicate the surfaces of the membrane. Results indicate that helix 8 in AT1R-VSTcis moves closer to the membrane surface compared to it in AT1R-VSTtran. This implies that helix 8 in AT1R-VSTcis is less likely to prevent the recruitment of G protein. The conformation of helix 8 in AT1R-VSTcis (the active-like state) is not similar with that in the active-like structure of AT2R. This may result from the distinct regulatory roles of helix 8 in AT1R and AT2R, or from the different inactive/active states of AT1R/AT2R caused by the varied ligands (agonist, neutral antagonist, or inverse agonist). To fully address this question, further experimental and computational works are need.

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Fig. 5. Helix 8 reveals different dynamic behaviors in the two states of AT1R. (A) Comparison of the mass density of helix 8 between AT1R-VSTtrans (in magenta) and AT1RVSTcis (in red) systems. The ranges of helix 8 along the Z-axis are shaded in gray. The black dashed lines indicate the density peaks of the phosphate (P) atoms (black lines) of POPC lipids. To give a clear view for the density of helix 8, the density values of P atoms of POPC are reduced 10 times. (B) Final structures showing the position of helix 8 relative to membrane surface.

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4. Conclusion In this work, molecular dynamics simulations were performed to investigate whether the trans and cis conformations of VST affect the binding of VST to AT1R. Results reveal that the trans conformation of VST stabilizes the structure and dynamics of AT1R, especially for that of TM6. With the favorable binding energy, the trans conformation locks AT1R in an inactive state. However, the cis conformation of VST alters its interactions with AT1R and increases the hydrophobic contacts between the butyl group of VST and residues Y872.63 and H2562.63. This causes a conformational change of Y872.63, which leads to a contraction for the VST-binding pocket of AT1R. Furthermore, accompanied with the interaction change, residue W2536.48 (the toggle switch) alters its rotation state and triggers the outward displacement of TM6. This in turn results in an extended pocket on the intracellular side of AT1R, which might facilitate the binding of G protein. With the breakage of hydrogen bond between R1263.50 and N2356.30, AT1R with the cis conformation of VST could adopt an active-like state. Our results give an insight into the molecular mechanism of how the cis conformation of VST induces the active-like state of AT1R. These data may provide new strategy for designing potent drug to prevent hypertension.

Supporting Information Environment of VST in the initial simulation structure of AT1R (Figure S1), the whole simulation system (Figure S2), secondary structure analysis for AT1R in each simulation system (Figure S3), RMSF for AT1R in each simulation system (Figure S4), time-dependent binding free energy (without entropy) for each simulation system (Figure S5), interaction energy for AT1R in each simulation system (Figure S6), comparison of the hydrophobic contacts containing the butyl group of VST (Figure S7), time-dependent inter-helical angle of TM6 relative to TM3 (Figure

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S8), distance between residues N2356.30 and Y541.55 as a function of time (Figure S9), timedependent volumes of VST-binding pocket and of G protein-binding pocket (Figure S10), and comparison of the hydrogen bond between R1263.50 and N2356.30 (Figure S11). This information is available free of charge via the Internet at http://pubs.acs.org

Acknowledgements We thank the Alabama Supercomputer Center and Supercomputer facility at the University of Alabama at Birmingham for providing computational resources. This work was supported by the National Natural Science Foundation of China (Grant No. 51303130).

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Trans and Cis Conformations of the Antihypertensive Drug Valsartan

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