Microsecond Molecular Dynamics Simulations Provide Insight into the

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Microsecond Molecular Dynamics Simulations Provide Insight into the Allosteric Mechanism of the Gs Protein Uncoupling from the # Adrenergic Receptor 2

Xianqiang Sun, Hans Ågren, and Yaoquan Tu J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 02 Dec 2014 Downloaded from http://pubs.acs.org on December 3, 2014

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The Journal of Physical Chemistry

Microsecond Molecular Dynamics Simulations Provide Insight into the Allosteric Mechanism of the Gs Protein Uncoupling from the β2 Adrenergic Receptor Xianqiang Sun, Hans Ågren, and Yaoquan Tu* Department Division of Theoretical Chemistry and Biology, School of Biotechnology KTH Royal Institute of Technology, S-106 91 Stockholm, Sweden AUTHOR INFORMATION Corresponding Author Dr. Yaoquan Tu Department Division of Theoretical Chemistry and Biology, School of Biotechnology KTH Royal Institute of Technology S-106 91 Stockholm, Sweden E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT. Experiments have revealed that in the β2 adrenergic receptor (β2AR)-Gs protein complex the α subunit (Gαs) of the Gs protein can adopt either an “open” conformation or a “closed” conformation. In the “open” conformation the Gs protein prefers to bind to the β2AR, while in the “closed” conformation an uncoupling of the Gs protein from the β2AR occurs. However, the mechanism that leads to such different behavior of the Gs protein remains unclear. Here, we report results from microsecond molecular dynamics simulations and community network analysis of the β2AR-Gs complex with Gαs in the “open” and “closed” conformations. We observed that the complex is stabilized differently in the “open” and “closed” conformations. The community network analysis reveals that in the “closed” conformation there exists strong allosteric communication between the β2AR and Gβγ, mediated by Gαs. We suggest that such high information flows are necessary for the Gs protein uncoupling from the β2AR.

KEYWORDS. Uncoupling mechanism • Gs protein • β2 adrenergic receptor • G-proteincoupled receptors • Allosteric network


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Membrane proteins, together with their cognate proteins, are required for sophisticated signaling from the extracellular side to the cytoplasmic region of the cell to maintain cellular homeostasis. G-protein coupled receptors (GPCRs) are membrane proteins that sense a wide range of stimuli, including photons, ions, small organic molecules and peptides. After sensing a stimulus, a GPCR undergoes conformational changes and activates its cognate protein to turn on the intracellular signaling cascade and to modulate downstream effector proteins.1-4 The cognate protein of a GPCR is usually the heterotrimeric G protein composed of a nucleotide bound α-subunit (Gαs) and an obligate dimer of β- and γ-subunits (Gβγ).5 The Xray crystal structure of the β2 adrenergic receptor (β2AR) bound to its G protein, referred to as the Gs protein, is the only structure available for GPCR-G protein complexes and provides us with essential information for studying the signaling from the GPCR to the G protein. In this structure, the alpha helix (AH) domain of Gαs has rotated 127° to form an “open” conformation as compared to the same domain in the crystal structure of the GTPγS-bound Gαs protein which corresponds to the “closed” conformation.6 Although artificial effects could inevitably arise due to the inclusion of a nanobody and T4 lysozyme to facilitate crystallogenesis, the displacement of the AH domain has been verified experimentally. Electron microscopy analysis has revealed that the placement of the AH domain in the absence of the nucleotide leads the complex to adopt mainly the “open” conformation and in the presence of the nucleotide, the complex adopts predominantly the “closed” conformation. 7

Double electron-electron resonance spectroscopy studies have also revealed that upon the

Gi protein binding to rhodopsin, there exist up to 20 Å changes in the distance between the nitroxide probes in the Ras-like GTPase domain and the AH domain of the Gi protein. 8 It has been suggested that the rotation of the AH domain facilitates the exchange of GDP for GTP in Gαs

5, 8, 9

and results in the reformation of the “closed” conformation which allows the

uncoupling of the Gs protein from the receptor.10, 11 However, the molecular mechanism that


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leads to such distinct behaviors of the Gs protein in the “open” and the “closed” conformations has remained elusive. Molecular dynamics (MD) simulations have proved to be an effective approach on such processes.12, 13 To understand this mechanism, we carried out a combined microsecond molecular dynamics simulations and community network analysis of the β2AR-Gs protein complex in both the “open” and the GTP-bound “closed” conformations (Figure 1).

Figure 1. Structure of the β2AR-Gs protein complex. The “open” and “closed” conformations of the complex are shown in A and B, respectively. The ligand and GTP molecule are represented by pink spheres and the β2AR is displayed as a yellow cartoon. The skeletal formula of the ligand is also included with the tolyl group colored in red. Gαs, Gβ, and Gγ subunits composing the Gs protein are displayed in cartoon and colored in green, cyan, and pink, respectively. The AH domain of Gαs is highlighted by the cyan ellipse.


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Methods: Protein structure preparation: The structure of the β2AR-Gs protein complex was obtained from the protein data bank (PDB ID: 3SN66, 14). The nanobody-35 and T4 lysozyme used to facilitate the crystallogenesis of the complex were removed from the crystal structure. The unresolved residues on ECL2 of the β2AR in this crystal structure were filled using the program Prime (version 3.3).15, 16 The missing loop of 16 residues around the α4 helix on Gαs was built with Prime using the protein structure with PDB ID 1AZT as the template. The third intracellular loop (ICL3) of the β2AR is known to be disordered in the complex. Experimental results also revealed that removing the bulk ICL3 within the β2AR does not prevent the receptor from coupling to the Gs protein.17 Besides, omitting ICL3 on the muscarinic receptors M2 and M3 does not likely affect the selectivity and efficacy of the receptor from binding to the G protein.18, 19 We thus did not consider ICL3 in our simulations. The structure thus prepared corresponds to the “open” conformation. This structure, together with the protein structure with PDB ID 1AZT,20 was further used to build the “closed” conformation. In both structures, the conformation of the Ras-like GTPase domain of Gαs (GαsRas) is conserved. To build the “closed” conformation, we aligned the GαsRas domain in 1AZT to 3SN6. Thereafter, the atomic coordinates of the alpha helix (AH) domain in 3SN6 were replaced with those in 1AZT and the GTP molecule and Mg2+ in 1AZT were transferred to it, resulting in the structure for the “closed” conformation. In both structures, the disulfide bond Cys184-Cys190 and Cys106-cys191 on the β2AR were reserved. Protonation states for the titratable residues were set according to the protein preparation results obtained from the Schrödinger software. Glu122 on the β2AR was left protonated, because this residue is stretched into the hydrophobic region of the membrane.


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Membrane preparation: In order to minimize the biases induced by the membrane model, we built a membrane system and equilibrated it before inserting the β2AR-Gs protein complex into it. A POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) bilayer with the surface area of 130 Å×130 Å on the X-Y plane was constructed using VMD.21 Water molecules were then added to both sides of the bilayer along the Z axis to form a 10 Å water layer in each side. This system was first subject to a 50000-step energy minimization, followed by a 100 ps NVT molecular dynamics (MD) simulation. Then, a 20 ns NPT MD simulation was performed to equilibrate the system and the bilayer structure from the last snapshot of the simulation was used for the following studies.

System setup: Both the “open” and the “closed” conformations of the β2AR-Gs protein complex were first embedded into the equilibrated POPC bilayer using our in-house program according to the orientations provided by the OPM database.22 A box of 132×132×200 Å3 with water molecules was then used to solvate the β2AR-Gs complex-POPC systems. Lipid molecules within 0.8 Å of the protein complex and water molecules in the bilayer were removed. Thereafter, sodium or chloride ions were added to produce the neutral systems of 0.15 M NaCl. The resulting system for the “open” conformation consists of 410 lipid molecules, 322 chloride ions, 318 sodium ions, and 89079 water molecules, with 339575 atoms in total; the resulting system for the “closed” conformation consists of 410 lipid molecules, one Mg2+ ion, 320 chloride ions, 318 sodium ions, and 89020 water molecules, with 339441 atoms in total.

Molecular dynamics simulation: MD simulations were performed using Gromacs 4.523 with CHARMM27 parameters for the proteins, lipids, and ions, and the TIP3P model for water. Force field parameters for the ligand molecule were generated with the CHARMM General


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Force Field (version 2b7) interface (version 0.9.6 beta).24 Dihedrals with the penalty score larger than 50 were recalculated according to the CHARMM parameterization philosophy with the quantum chemistry calculations carried out at the HF/6-31G* level with Gaussian 09.25 The force field parameters for the ligand were listed in Appendix 1. Three steps were used to equilibrate the systems corresponding to the “open” conformation and the “closed” conformation. In the first step, each system was subject to a 50000-step energy minimization with 1000.0 kJ/mol/nm as the force threshold. Then, each system was relaxed by an MD simulation of 100 ps with 1 fs as the time step using the NVT ensemble. In the last step, each system further underwent an NPT MD simulation for 1 ns with the time step of 2 fs for equilibration.

After the equilibration runs, each system was simulated for 1100 ns using the NPT ensemble with the temperature and pressure set to 300 K and 1 bar, respectively. Nose-Hoover thermostat and Parrinello-Rahman pressure coupling were applied during the simulations. The bonds containing hydrogen atoms were constrained with the LINCS algorithm and a time step of 2 fs was used. Periodic boundary conditions were applied, and the cut-offs for the electrostatic and van der Waals interactions were set to 12 Å, with the long range electrostatic interaction recovered by the Particle Mesh Ewald summation.

Metadynamics simulations: Well-tempered metadynamics simulations26-28 for the complex in the “open” and “closed” conformations with the agonist in the binding pocket were carried out, using plumed 2.02 implemented in Gromacs 4.6.5. For each of the complex conformations, a metadynamics simulation was preformed for 50 ns, with the RMSD value of the agonist used as the collective variable. The biasing potential was added every 500 steps,


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with the width and height of the Gaussian hills set to 0.003 and 0.3 kJ/mol, respectively, and

ΔT = 2700.

RMSD matrix analysis: The ‘g_rms’ module in Gromacs/4.6.5 was used to build the RMSD matrix. The RMSD values were calculated between each given snapshot in the trajectory. Thus, a matrix based on the RMSD values can be generated.

Helix movement analysis: The averaged Cα coordinates for residues 32-36, 95-91, 106-110, 169-165, 200-204, 295-291, and 307-311 on the β2AR were used to represent the helixes H1 to H7 in the extracellular side, respectively, for analyzing their movements in the extracellular side of the receptor. In the same way, the averaged Cα coordinates for residues 55-59, 68-72, 136-132, 149-153, 233-229, 268-272, and 325-321 on the β2AR were used to represent the helixes H1 to H7 in the intracellular side, respectively, for analyzing their movements in the intracellular side. The structure with the coordinates averaged over the last 10 snapshots of a system was defined as the final structure of the system.

Free energy landscape analysis: The free energy landscape was built based on the equation

߂‫ ܩ‬ሺ‫ݍ‬ଵ , ‫ݍ‬ଶ ሻ = −‫ܭ‬஻ ݈ܶ݊ܲሺ‫ݍ‬ଵ , ‫ݍ‬ଶ ሻ where ‫ܭ‬஻ is the Boltzmann constant, T is the temperature, and ܲሺ‫ݍ‬ଵ , ‫ݍ‬ଶ ሻ is the normalized probability distribution. In this work, q1 and q2 are the angle Gly310(Gβ)-Asp311(H8)Glu338(H8) and the distance between the backbone carbon atoms of Glu338(H8) and Gly310(Gβ), respectively. Calculations involving free energy landscape graphs were carried out using Python with modules Matplotlib and Numpy.


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Community network analysis: In the community network analysis, each residue in a protein complex is defined as a node. The “in-contact” nodes are defined as having any heavy atom within 5 Å for more than 75% of the simulation time. Edges are then added to the network by connecting the pairs of “in-contact” nodes, weighted by the correlation values of the two end nodes with ‫ݓ‬௜௝ = −log(|‫ܥ‬௜௝ |), where ‫ݓ‬௜௝ is the weight and ‫ܥ‬௜௝ the correlation value. Weaker cross-correlations result in longer edges, representing that the edges are less potential for information transfer. The network is divided into communities of highly intra-connected but loosely inter-connected nodes using the Girvan-Newman algorithm.29 The number of shortest paths that cross a given edge is defined as “betweenness” and is calculated for all the edges. Iterative removal of the edge with the largest betweenness is carried out. A modularity score is tracked to identify the division that results in an optimal community network.

Community network analysis for the “open” and “closed” conformations of the β2AR-Gs complex was carried out using the NetworkView plugin in VMD 1.9.1. Communities, edges, and intercommunity betweenesses were derived from the correlation of the nodes obtained from the MD simulations. Figure S3 shows the distribution of the communities in the β2ARGs protein complex derived from the correlation for the complex (Figure S4). Results and Discussion: From MD simulations, we observed that the agonist behaves differently in the conformational space in the “open” and “closed” conformations (Figure 2). The root-mean-square-deviation (RMSD) matrices reflect that the agonist adopts four conformational clusters in the “open” conformation (Figure 2A, Figure S1) and two conformational clusters in the “closed” conformation (Figure 2B, Figure S1). The difference in the conformations that the agonist adopts in the “open” conformation is mainly due to the rotation of the tolyl group (TG), as reflected by the change of the π-π stacking interactions between TG and the aromatic residues


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around it. The π-π stacking interactions are not explicitly expressed in the force field we used in the simulations. Instead, they are recovered through the non-bonded interaction terms, such as those for the Lennard-Jones potential and electrostatic interactions, in the force field. In 060 ns and 180-720 ns intervals, there are only weak π-π interactions between TG and Tyr308 as shown in Figure 2C. Trp313 forms π-π stacking interactions with TG in 60-180 ns (Figure 2D). In 720-830 ns, TG forms weak T-shaped π-π stacking interactions with Phe193 and Tyr308 (Figure 2E). The T-shaped π-π stacking interactions between TG and Phe193 evolve into face to face π-π stacking interactions while the π-π stacking between TG and Tyr308 disappears in 830-1100 ns (Figure 2F). On the other hand, the agonist in the “closed” conformation was found to adopt two conformational clusters, as indicated in Figure 2B. No obvious π-π stacking interactions were observed between TG and residues adjacent to it in the 0-990 ns interval (Figure 2G), while face to face π-π stacking forms between TG and Tyr308 after 990 ns (Figure 2H).


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Figure 2. RMSD matrices and representative structures of the ligand in the “open” and “closed” conformations. A, the RMSD matrix of the ligand in the “open” conformation; B, the RMSD matrix of the ligand in the “closed” conformation; C-F, representative ligand structures in the “open” conformation as indicated in the corresponding regions in A; G-H, representative ligand structures in the “closed” conformation as indicated in the corresponding regions in B.


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It is of interest to make it clear if the conformational space of the ligand is indeed correlated with the “open” or “closed” conformations of the Gs protein or not. In order to minimize the effects from the sampling problem, we used an enhanced sampling technique - metadynamics simulation - to study the conformational changes of the ligand in the “open” and “closed” conformations of the complex. From the metadynamics simulations, we found that the conformational spaces of the ligand in the “open” and “close” conformations are different, as indicated by the free energy profiles shown in Figure 3. In the “open” conformation, the agonist can be stabilized in four conformations as observed in the unbiased MD simulation (see Figure 2C-2F). In contrast, in the “closed” conformation the agonist is predominantly stabilized in the conformation shown in Figure 2G. The conformation shown in Figure 2H can also be reached, but it is much less stable than the conformation shown in Figure 2G. Thus, the metadynamics simulations confirmed the result observed from the unbiased MD simulations, that is, in the “open” and “closed” conformations, the conformational spaces of the agonist are different. From these studies, we conclude that the conformational space of the ligand is correlated with the “open” or “closed” conformation of the Gs protein.

Figure 3. Change of the free energy profiles with the RMSD value of the agonist. The labels C-G correspond to the conformations shown in Figure 2C-2G, respectively.


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From the structure of the β2AR-Gs protein complex (Figure 1), we can see that the signaling from the agonist binding pocket to Gαs goes via the helix bundle of the β2AR. We consequently analyzed the helix motion of the β2AR and observed significant movements of helices 5 and 6 (H5, H6) (Figure 4). In the “open” conformation, a significant motion of H6 has been observed in the intracellular region, and H5 was found to move in a concerted way with H6 (Figure 4B). The motions of H5 and H6 can be considered as a part of the concerted rotation of the helices in the intracellular domain of the receptor, as indicated by the Normal Mode Analyses.

30, 31

The motions of H5 and H6 upon receptor activation are also shown in

Figure 4B as blue arrows. As we can see, the motions of H5 and H6 in the “open” conformation are in the direction almost perpendicular to the blue arrows. We also observed a movement of H4 in the extracellular region (Figure 4A) which could facilitates the conformational changes of the agonist in the “open” conformation. In the “closed” conformation, H5 and H6 move apart in the intracellular region, making the coupling between H5 and H6 become weak (Figure 4B and Figure S2). Previous MD simulations of the β2AR with the agonist Isoprenaline revealed an inward motion of H5.

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This inward

motion was not observed in our simulations with the inclusion of the Gs protein. It has been confirmed that the activation of GPCR is characterized by the collaborated outward tilt motion of H5 and H6 as shown by the blue arrows in Figure 4B.6, 32-37 The outward motion of H5 and H6 was also observed in the simulations of the Cannabinoid CB2 receptor-Gi protein complex.38 Our simulation results suggest that in the β2AR-Gs protein complex, these two helices also contribute to the allosteric communication in both the “open” and the “closed” conformations. Inspection of the last snapshots from the simulations of the “open” and “closed” conformations indicates that compared with the “open” conformation, the uncoupling motion between H5 and H6 in the “closed” conformation leads to a 0.6 Å


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movement of the C-terminal of Gαs towards the β2AR and a 2.2 Å shift of the short helix (from residues 231 to 238) on Gαs away from the Gβγ subunits.

Figure 4. Helix movements of the β2AR during the simulations. A, The helix movements on the extracellular side; B, The helix movements on the intracellular side. The motions of the helices of β2AR on the intracellular side upon the receptor activation are also shown in the lower left corner of 3B. The structure for the β2AR pre-coupling to the Gs protein (PDB ID: 2RH1) is shown in cyan cartoon and the structure of the β2AR with the Gs protein coupled (PDB ID: 3SN6) is shown in green cartoon. Experimental studies have suggested that in many GPCRs, the helix 8 (H8) facilitates G protein coupling39,

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and influences the rate of receptor activation.41 We also observed a

distinct interaction pattern between H8 on the β2AR and the Gβ subunit in the “open” and “closed” conformations (Figure 5). Here, we describe this interaction pattern through tracking the evolution of the local structure, as characterized by the angle Gly310 (Gβ)-Asp311(H8)Glu338 (H8) and the distance between the backbone carbon atoms of Glu338 (H8) and Gly310 (Gβ) (Figure 5A). In both conformations, the angle decreases dramatically in the first 60 ns. This observation is most likely caused by the effect of the removal of the nanobody in


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our simulations. Thereafter, the angle decreases gradually during the simulations until 600 ns. However, the angle behaves differently in the two conformations after 600 ns of the simulations. In the “closed” conformation, it increases sharply from about 35° to 55° during 600 ns to 700 ns and remains around 55° in the following simulation while in the “open” conformation it remains around 35° after 600 ns. Investigation of the distance d between the backbone carbon atoms of Glu338 (H8) and Gly310 (Gβ) indicated that this distance increases from about 10 Å to 14 Å in the “closed” conformation after 200 ns, while it remains around 8 Å in the “open” conformation (Figure 5B).

Figure 5. Evolution of the local structure between H8 and the Gβ subunit. A, Evolution of the angle Gly310(Gβ) -Asp311(H8)-Glu338 (H8) in the “open” and “closed” conformations;


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B, Temporal change of the distance between Gly310(Gβ) -Glu338 (H8) in the “open” and “closed” conformations. C and D are the free-energy landscapes for the “open” and “closed” conformations. Free energy landscapes help us to understand further the evolution of the local structure between H8 and the Gβ subunit in the two conformations. In the “open” conformation, the system evolves from region a to region b, as indicated by the decrease of the angle and distance values, and it eventually remains in region b (Figure 5C). For the “closed” conformation, the system evolves from region a to region b at the beginning, as indicated by the dramatic decrease in the angle and the distance, and then to region c, as reflected mainly by the decrease of the angle. Finally, the system remains in region d, which corresponds to the longer distance and the larger angle (Figure 5D). It is surprising that no direct interaction has been observed between H8 and Gs in the crystal structure6, though H8 has been suggested to facilitate G protein pre-coupling in many GPCRs.3, 33 We note that there exist direct interactions between H8 and Gβ in region b in both the “open” and the “closed” conformations. The simulation of the “open” conformation indicates that the system eventually remains in region b. However, the simulation of the “closed” conformation indicates that the system evolves from region b to region d, via region c, and stays in region d. Thus, the free energy landscapes reflect that in the “open” conformation the complex stays in region b and in the “closed” conformation, the complex stays in region d. This difference likely leads to the different dissociation behaviors of the Gs protein from the β2AR. Community network analysis derived from MD simulations provides us with an effective way to identify and compare signaling pathways in protein complexes.42-44 Through community network analysis, we observed distinct intercommunity flows in both the “open” and the “closed” conformations (Figure 6). In the “open” conformation, the network is split


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into 17 communities (Figure 6A and Figure S3), where communities 16 and 17 formed respectively by the nodes Val57(Gα) and Val367(Gα) are not shown. The ICL3 of β2AR is known to be disordered in the protein complex, which most likely results in weak correlation or anti-correlation values among the residues in ICL3 as well as between the residues in ICL3 and those in the remaining protein complex. We believe that omitting ICL3 would have little effect on the splitting of the communities shown in Figure 6. Of the 17 communities, five communities (2, 7, 8A, 8B, 9) are composed exclusively of the residues in the β2AR (Figure S3). Community 3 covers the AH domain of Gαs, communities 1, 4A, 4B and 10 consist of GαsRas, and communities 5, 6, 11, 12, and 13 form the Gβγ subunits. We can see strong intercommunity communication in both the β2AR and the Gβγ subunits. On the other hand, the communication within the Gαs protein is relatively weak. Although there is a large information flow between communities 2 and 4B resulting from their close spatial positions, we did not see other strong intercommunity flows among the β2AR, Gαs, and Gβγ subunits.


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Figure 6. Community networks in the “open” and “closed” conformations. The width of the line connecting two communities is proportional to the strength of the information flow between the two communities. For the “closed” conformation, we obtained 13 communities (Figure 6 and Figure S3), with communities 2, 7, 8, and 9 in the β2AR, communities 1, 3, 4, and 10 in the Gαs subunit, and communities 5, 6, 11, 12, and 13 in the Gβγ subunits. It is interesting to see that the information flow between the β2AR and the Gαs subunit and that between the Gαs and Gβγ subunits are much larger in this conformation than in the “open” conformation, as reflected by the thick line connecting communities 2 and 4 as well as that connecting communities 4 and 11. Such high intercommunity information flows are most likely necessary for allosteric signaling required for the dissociation of the Gs protein. Conclusion: In conclusion, through combining microsecond MD simulations and community network analysis, we have studied the allosteric communication in the β2AR-Gs protein complex in both the “open” and the “closed” conformations. We found that the agonist in the binding pocket of the β2AR adopts four and two conformational clusters in the “open” and “closed” conformations, respectively. In the “closed” conformation, we observed the significant change of the angle Gly310(Gβ)-Asp311(H8)-Glu338(H8) and the distance between the backbone carbon atoms of Glu338(H8) and Gly310(Gβ). The community network analysis reveals that there are only weak information transfers among the β2AR, Gαs, and Gβγ in the “open” conformation. However, our analysis indicates that there indeed exists strong allosteric communication from the β2AR to the Gβγ subunits in the “closed” conformation. We suggest that this allosteric modulation most likely leads to the uncoupling of the Gβγ subunits from the β2AR.


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ASSOCIATED CONTENT Supporting Information. Figure S1-S4 are included in the supporting information. This material is available free of charge via the internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Dr. Yaoquan Tu *Email: [email protected] Notes: The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by a grant from the Swedish National Infrastructure for Computing (SNIC) for the project “Multiphysics Modeling of Molecular Materials”, SNIC025/12-38. REFERENCES (1) Venkatakrishnan, A. J.; Deupi, X.; Lebon, G.; Tate, C. G.; Schertler, G. F.; Babu, M. M. Molecular Signatures of G-Protein-Coupled Receptors. Nature 2013, 494, 185-194. (2) Kobilka, B. The Structural Basis of G-Protein-Coupled Receptor Signaling (Nobel Lecture). Angew. Chem. Int. Ed. 2013, 52, 6380-6388. (3) Lefkowitz, R. J. A Brief History of G-Protein Coupled Receptors (Nobel Lecture). Angew. Chem. Int. Ed. 2013, 52, 6366-6378. (4) Wacker, D.; Fenalti, G.; Brown, M. A.; Katritch, V.; Abagyan, R.; Cherezov, V.; Stevens, R. C. Conserved Binding Mode of Human Β2 Adrenergic Receptor Inverse Agonists and Antagonist Revealed by X-Ray Crystallography. J. Am. Chem. Soc. 2010, 132, 11443-11445. (5) Chung, K. Y.; Rasmussen, S. G. F.; Liu, T.; Li, S.; DeVree, B. T.; Chae, P. S.; Calinski, D.; Kobilka, B. K.; Woods, V. L.; Sunahara, R. K. Conformational Changes in the G Protein Gs Induced by the [Bgr]2 Adrenergic Receptor. Nature 2011, 477, 611-615. (6) Rasmussen, S. G. F.; DeVree, B. T.; Zou, Y.; Kruse, A. C.; Chung, K. Y.; Kobilka, T. S.; Thian, F. S.; Chae, P. S.; Pardon, E.; Calinski, D.; Mathiesen, J. M.; Shah, S. T. A.; Lyons, J. A.; Caffrey, M.; Gellman, S. H.; Steyaert, J.; Skiniotis, G.; Weis, W. I.; Sunahara, R. K.; Kobilka, B. K. Crystal Structure of the Β2 Adrenergic Receptor–Gs Protein Complex. Nature 2011, 477, 549-555. (7) Van Eps, N.; Preininger, A. M.; Alexander, N.; Kaya, A. I.; Meier, S.; Meiler, J.; Hamm, H. E.; Hubbell, W. L. Interaction of a G Protein with an Activated Receptor Opens the


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Microsecond Molecular Dynamics Simulations Provide Insight into the

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