Interplay between Two Allosteric Sites and Their Influence on Agonist

Article pubs.acs.org/jcim

Interplay between Two Allosteric Sites and Their Influence on Agonist Binding in Human μ Opioid Receptor Damian Bartuzi,*,† Agnieszka A. Kaczor,†,‡ and Dariusz Matosiuk† †

Department of Synthesis and Chemical Technology of Pharmaceutical Substances with Computer Modelling Lab, Medical University of Lublin, 4A Chodźki Str., PL20093 Lublin, Poland ‡ University of Eastern Finland, School of Pharmacy, Yliopistonranta 1, P.O. Box 1627, FI-70211 Kuopio, Finland S Supporting Information *

ABSTRACT: Allostery is a widespread mechanism that allows for precise protein tuning. Its underlying mechanisms are elusive, particularly when there are multiple allosteric sites at the protein. This concerns also G-protein-coupled receptors (GPCRs), which are targets for a vast part of currently used drugs. To address this issue, we performed molecular dynamics simulations of a GPCR−human μ opioid receptor (MOR) in a native-like environment, with full agonist (R)-methadone, Na+ ions, and a positive modulator BMS986122 in various configurations. We found that MOR’s seventh transmembrane helix (TM VII) is central for allosteric signal transmission, and modulators affect its bending and rotation. The PAM stabilizes favorable agonist interactions, while Na+ tends to disrupt agonist binding. We identified two residues involved in allosteric signal transmission: Trp 7.35 at the top and Tyr 7.53 at the bottom of TM VII.

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INTRODUCTION Allostery contributes to great complexity of function of biological macromolecules. In general, any complex macromolecule can be affected by a third-party factor, like a small molecule, ion, etc.1 It has tremendous implications, e.g., for drug design, since it enables the possibility of firm tuning of protein function instead of rough activation or inhibition. The benefits would include preservation of physiological patterns of protein action, limited risk of overdosage due to the so-called “ceiling effect”, improved receptor subtype selectivity, and possibly functional selectivity.2 Unfortunately, investigation of the underlying mechanisms of allostery is difficult, in particular, when multiple modulators act on a protein.3,4 Such a situation is also a concern for G-protein-coupled receptors (GPCRs) many family A (rhodopsin-like) GPCRs possess at least two allosteric sites.5 GPCRs are responsible for a vast part of signaling in vertebrates. They are involved in many diseases and are targeted by about 50% of the currently used drugs. There is a growing number of known allosteric modulators affecting the function of GPCRs, with some already approved as drugs (Cinacalcet, Maraviroc). Moreover, recent studies indicate that the Na+ ion acts as an allosteric modulator of these proteins, binding in a specific site inside the receptor bundle at a conserved Asp 2.50 residue6 (Ballesteros−Weinstein notation7), and that other modulators can indirectly affect the sodium-binding site.8 BMS986122 (BMS) is a recently discovered positive allosteric modulator (PAM) of the human μ opioid receptor (MOR).9 It is characterized by pronounced probe dependence as it affects the binding and action of various ligands in different ways. Its effect on full agonist affinity seems to result from its © XXXX American Chemical Society

remote interplay with the allosteric binding site for sodium ions.8 The Na+ ions themselves have a complicated allosteric effect on GPCRs. They both decrease the agonists’ affinities and facilitate G-protein activation.10 Moreover, clues derived from X-ray structures of GPCRs are intriguing. While many structures of agonist-bound receptors contain Na+ situated at Asp 2.50, they usually lack many hallmarks of activation. In contrast, X-ray structures of agonist-bound GPCRs stabilized in their active-state by nanobodies, G protein, its fragments, or mimetics (i.e., PDB IDs: 3SN6, 3P0G, 4MQT, 4MQS, 3DQB, 3PQR, 2X72, 4A4M, 4BEY, 4PXF, 5C1M) show rearrangements typical for GPCR activation but contain no Na+ at the aspartate. This implies that Na+ has a complicated influence on GPCRs at different stages of activation. In this work, we address the questions regarding the allosteric signal propagation pathway in GPCRs and mechanisms of interplay between distant allosteric sites and the orthosteric binding site with computational methods, i.e., molecular dynamics simulations and principal component analysis (PCA). This tandem has proven itself to be useful in investigation of GPCR allostery.11 We found that the allosteric signal and interplay between two allosteric sites at MOR is mediated by the MOR’s seventh transmembrane helix (TM VII). Results indicate that BMS interacts with Trp 7.35 and remotely affects Tyr 7.53, diminishing the effects of Na+ binding. Received: November 24, 2015

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DOI: 10.1021/acs.jcim.5b00705 J. Chem. Inf. Model. XXXX, XXX, XXX−XXX

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Journal of Chemical Information and Modeling

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MATERIALS AND METHODS The native-like membrane was constructed with the CHARMM-GUI Membrane Builder,12 using a preprocessed 3SN613 crystal structure from the OPM (Orientations of Proteins in Membranes) database14 as a template of protein− membrane orientation. Membrane composition (90 molecules of sphingomyelin, 120 cholesterol, 114 POPC, 114 POPE) was designed basing on the literature.15 The native-like composition of the membrane was chosen for simulations since there is evidence that the composition plays important role in GPCR signaling.16 Maintaining a native-like environment in molecular dynamics (MD) simulations is also important for obtaining accurate results.17 The simulation box contained 40280 TIP3P water molecules and 0.15 M NaCl. The homology model of the active-state MOR in complex with the Gs protein was prepared with Modeller 9.1018 on the templates: mouse MOR (PDB ID: 4DKL)19 deprived of the intracellular part of TM VI, human β2 adrenergic receptor (PDB IDs: 3SN6, 3P0G20). The extensive homology modeling study was described in detail in a previous paper.21 We decided to create a homology model of the activestate MOR in complex with the Gs protein since there is evidence for MOR-Gs protein coupling,22 and availability of the 3SN6 X-ray structure of the β2 receptor−Gs protein complex13 allowed for a more reliable reconstruction of the protein− protein interface. (R)-methadone was chosen for an orthosteric ligand on the basis of the paper by Livingston and Traynor.8 It is a relatively rigid, small-molecule full agonist, which is sensitive to the influence of sodium ions and BMS986122. Molecular docking was performed with the Surflex module of SybylX 1.323 and with Glide.24 Ligands were prepared with Spartan 1025 and optimized by the DFT B3LYP, 6-31G* basis set. ESP charges were obtained by REDServer.26 Ligand topologies were built with ACPYPE.27 An Amber03 force field was used for the protein, GAFF for the ligands, and Stockholm lipids28,29 for the membrane. All of these force fields are compatible.30−32 The random seeds for velocity generation were different between replicas but identical within a series, to make simulations of different complexes within a series directly comparable. A Gromacs 5.033 engine was used for simulations. Results were analyzed with Gromacs tools. VMD34 and PyMOL35 were used for visualization.

The protein was immersed in a raft-imitating membrane since there is evidence for migration of activated MOR to raft regions.16 Moreover, a proper membrane environment was shown to be important for the accurate reproduction of protein behavior11,17 Model Validation. Very recently, an X-ray structure of active-state murine MOR has been published (PDB ID: 5C1M).36 The comparison of the model with the crystal structure has proven its excellent quality: Cα RMSD of whole structures is 2.60 Å, and removal of the most disordered regions (N-terminus, C-terminus, ICL 3) decreases RMSD to 1.91 Å, below the crystal resolution (2.10 Å). Most differences in the truncated structures are located at the TM I−TM VII crystal packing interface, which can be affected by packing forces. Excluding this region from comparison improves RMSD further to 1.81. (Figure S1, Table S1) The result is even more satisfying since the 5C1M structure is bound to a camelid antibody mimicking the G protein instead of the G protein itself, which can be partially responsible for the differences. Similar substitution in the case of the β2 adrenergic receptor X-ray structures causes RMSD of 0.61 Å.13 Allosteric Site Prediction. The hypothetical allosteric binding pocket for BMS was determined by molecular docking. The five most promising poses were simulated for 20 ns. The four most stable poses were simulated further to 50 ns (Figure 1). As two of these poses (poses 3 and 4) converged to very similar orientations and presented favorable interaction energy, one of them (the one with lower protein−BMS interaction energy) was picked for further MD. As during the main simulations, BMS tended to drift toward the fourth best pose; an additional simulation with BMS in this position (pose 5) was performed. The most important BMS interactions involved Trp 7.35, His 7.36, Ile 7.39, Asn 2.63, and Tyr 2.64. Main Simulations. Simulations were run in triplicate. Unrestrained production runs lasted 0.4 μs each. Every replica in a series used the same random seed for velocity generation, so that simulations within a replica set are directly comparable. Surprisingly, the effect of BMS and Na+ ion on RME binding was very pronounced in simulations. Nonmodulated RME remained in the binding pocket. It developed a stable interaction with Asp 3.32 (Figure 2), which is essential for classical agonist binding in MOR as well as in many aminergic GPCRs. One exception was the second replica (RME 2), where initially the interaction was unstable. In all simulations where Na+ was placed at Asp 2.50 and BMS was absent (RME-Na), the RME−Asp 3.32 interaction was disturbed; RME lost its contact with the Asp 3.32 and slightly drifted toward the receptor entrance (in RME-Na-3 it eventually returned). However, introduction of BMS (RME-Na-BMS) completely reversed this effect. Moreover, we observed disruption of the allosteric sodium ion position (Figure S2). The ion left the receptor interior permanently in RME-Na-BMS-2. It also left in RME-Na-BMS-1, but it remained at the receptor entrance and eventually returned to Asp 2.50 after 30 ns. However, in RMENa-BMS-3 the interaction between Na+ and Asp 2.50 is less disturbed than in RME-Na-1, so this is not a universal rule. Interestingly, in RME-Na-BMS-p5 where the modulator was located farther from TM7 and closer to RME, the sodium ion was very stable in its pocket. Since the two binding sitesorthosteric and allostericare located quite closely in the cavity inside MOR (Figure 3) and direct interactions between the agonist and the modulator can occur, the question on the mechanism of modulation arises.

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RESULTS System Preparation. To investigate how BMS affects the binding of full agonists and Na+ ions, we performed all-atom MD simulations of MOR with full agonist (R)-methadone (RME), BMS (Chart 1), and the Na+ ion in various configurations (Table 1) in a native-like environment. We used an active-state homology model of human MOR in complex with the G-protein21 to minimize activation-related events and facilitate identification of modulator-related ones. Chart 1. Structures of Agonist ((R)-methadone) and Modulator (BMS986122) Investigated in the Study

B


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Journal of Chemical Information and Modeling Table 1. Agonist−Modulator−Receptor Configurations Investigated and Number of Replicas complex modulator n replicas t [μs] per replica a

apo-MOR −/Na 1 0.4

+ a

MOR-RME

MOR-RME-Na+

− 3 0.4

MOR-RME- Na+-BMS

+

Na+, BMS986122 3 + 1b 0.4

Na 3 0.4

Na+ ion migrated into MOR during simulation. bOne additional simulation of BMS986122 in a different docking position (pose 5) was performed

Figure 1. Best docking poses of BMS986122, chosen for initial 50 ns simulations: pose 1, blue; pose 3, lime; pose 4 (chosen for main simulations), yellow; pose 5 (additionally simulated), red. The modulator drifted during simulations and eventually assumed poses presented in Figure 3.

Figure 3. Relative location of ligands in the receptor cavity presented on the final snapshot of RME-Na-BMS-1. Additionally, final coordinates of BMS in the other two replicas as well as in RME− Na−BMS-p5 are presented with color coding: yellow, RME-Na-BMS1; red, RME-Na-BMS-2; tan, RME-Na-BMS-3; dark gray, RME-NaBMS-p5.

apparent common denominator,8 the interplay between allosteric sites seems to be more probable than simple direct stabilization of the agonist. BMS binds at the top of TM2 and TM7, while there are highly conserved residues, Asp 2.50 and Ser 7.46, belonging to these helices located at the height of the allosteric Na+. Therefore, it can be hypothesized that BMS diminishes Na+ influence on these TMs mediated by these two residues. The hypothesis is supported by the fact that the solvent-accessible surface area (SASA) of Asp 2.50 is changed upon BMS binding, while it is similar in agonist-bound systems with and without the allosteric sodium at Asp 2.50 (Table 2). Table 2. Average Values of Solvent-Accessible Surface Area (SASA) of Asp 2.50 during Simulations

Figure 2. Distance between the proton at the RME amine and closest oxygen at Asp 3.32 throughout all simulations in all replica series. The plot was split into three parts for clarity.

However, since BMS has a similar effect on structurally different compounds for which the full agonism is the most C

simulation

average SASA (nm2)

standard deviation (nm2)

apo-MOR RME-1 RME-2 RME-3 RME-Na-1 RME-Na-2 RME-Na-3 RME-Na-BMS-1 RME-Na-BMS-2 RME-Na-BMS-3 RME-Na-BMS-p5

0.2274 0.2077 0.1530 0.2207 0.1537 0.2196 0.2043 0.2336 0.2543 0.2236 0.0752

0.0966 0.0687 0.0576 0.0753 0.0733 0.0708 0.0516 0.0740 0.1197 0.,0554 0.0335 DOI: 10.1021/acs.jcim.5b00705 J. Chem. Inf. Model. XXXX, XXX, XXX−XXX

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Figure 4. PCA of MOR fragments reveals rearrangement of TM VII with rotation of Tyr 7.53. The figure presents data for all simulations from all replicas. (A) Conformational space explored through last 200 ns of MD runs. The systems form clusters corresponding to particular rearrangements: cluster 1, rotation of Tyr 7.53 and bending of TM VII at Cys 7.47; cluster 2, Tyr 7.53 protruding inside the receptor bundle, TM VII straightened; cluster 3, Tyr 7.53 displaced by rigid-body movement of entire TM VII. The apo-MOR was assigned to cluster 3 on the basis of the PC1 value and the number of similar rearrangements presented in Figure 5. (B) Extreme projections along PC2 show difference between cluster 1 and clusters 2 and 3. The blue and red cartoon correspond to the PC2 values included in the corresponding legend. (C) Extreme projections along PC1 show difference between cluster 2 and cluster 3, while cluster 1 represents intermediate conformation. The yellow and red cartoon corresponds to the PC1 values included in the corresponding legend.

Rearrangements of TM VII. The relationships pointed out by all PCAs were next analyzed in the original MD trajectories. Evaluation of the TM VII motions revealed that they correspond well to PCA results (Figure 5). There was a significant difference in the bending of the helix at the NPxxY motif. The motif is well conserved in the family A receptors and therefore is suspected to play a crucial role in activation.38 The angle was measured between vectors designated by helix fragments below and above the kink at Ser 7.46−Asn 7.49. In apo-MOR, RME-2, and all RME-Na systems, the TM VII was apparently straightened, while in others it was visibly bent. Also, there was a difference in cumulative rotation of TM VII at its intracellular side, below Asn 7.49. The rotation draws an accurate distinction between these systems. When the influence of sodium ions was diminished or absent, the intracellular part of TM VII rotated clockwise (looking from the extracellular side) with only RME-Na-3 not fitting into the scheme. Bending and rotation of TM VII are apparently connected to the rotameric transition of the Tyr 7.53 side chain and its distance to Phe 345 in helix VIII. These two residues are a part of the tyrosine toggle switch.38 In RME-1, RME-3, and all RME-Na-BMS systems, Tyr 7.53 rotates and points toward Asp 2.50 (Figures 4 and 6). Also, its distance to Phe 345 is decreased. In turn, configuration of the tyrosine toggle switch seems to be in relation to the rotameric position of Trp 7.35 in the binding pocket (Figure 7). In the structures that were negatively modulated by the sodium ion at Asp 2.50, when the Tyr 7.53−Phe 345 distance is high, the side chain of Trp 7.35 tends to point toward the protein interior. In RME1, RME3, and the positively modulated structures where the Tyr 7.53− Phe 345 distance is low, the Trp 7.35 side chain points toward the entrance to the binding pocket. In RME-Na-BMS-p5, BMS is docked in the position more distant to Trp 7.35 (Figure 1) and fails to affect its rotameric position for entire simulation. Therefore, MOR in RME-Na-BMS-p5 explores a similar conformational space as other Na+-modulated systems (Figure 4).

Principal Component Analysis. To shed more light on the intimate rules of BMS-induced modulation, we employed PCA, which is a statistical procedure helpful in finding intricate relationships.37 In all PCAs, all trajectories from all replicas were concatenated so that they were analyzed in a common subspace. The most disordered parts of MOR, i.e., N-terminus and the third intracellular loop were excluded from analyses. PCAs involved the 7TM main chain and side chains protruding inside the receptor bundle to remove noise from the protein− membrane interactions. A series of PCAs was performed. They suggested that modulators have considerable effect on TM VII (Figure 4). In particular, based on separate PCAs performed for particular TMs, we picked up domains presenting favorable descriptions of modulator-specific events and additionally the most stable domains for spatial reference. Therefore, TM II, III, IV, and VII were picked for a more detailed analysis. The analysis presented apparent separation of RME and RME-Na-BMS complexes from RME-Na systems in terms of both PC1 and PC2. This allowed for identification of modulator-specific events (Figures 4−6). This analysis also has revealed that RME-2 is an outlying result. The comparison of the conformational space explored in the intervals from 200 to 300 ns and from 300 to 400 ns shows that the major changes occurred in the first half of the simulations (Figure S3). The possible reason for the unexpected behavior of RME-2 was found after visual analysis of the trajectory. The RME adopted the orientation that hindered the flow of water molecules into the receptor. It approached Tyr 7.43, decreasing the space available for water exchange. In an additional PCA, the central part of TM VI with the conserved CWxP motif was added to the previous selection since it is believed to be a part of a “rotamer toggle switch” in GPCRs.38 This made the division between RME-Na and all other systems blurred, yet it revealed significant motions of this part of TM VI relative to TM II−IV (Figure S4). D


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trajectories revealed that although the water appeared at the COG of HB during these two runs, it did not form a continuous chain and the barrier was rearranged but intact. In particular, in RME-Na-BMS-p5, the overall amount of water inside the receptor was very low (approximately six molecules). Discussion. The overall results are in line with the literature. In particular, simulations show that agonist binding is disturbed in the presence of the sodium ion at conserved Asp 2.50, which was suggested experimentally10,40,41 and computationally,6,42,43 while it is stable in the absence of the ion. Moreover, introduction of BMS into the hypothetical binding pocket stabilizes the MOR−RME interaction (Figure 2) and disrupts the allosteric Na+ binding (Figure S2), which corresponds to the experimentally investigated mechanism of a BMS-induced increase in affinity of the MOR full agonists.8 The hypothetical allosteric site is located near Trp 7.35, which is not conserved among human opioid receptor subtypes; it is replaced by Tyr and Leu in κ- and δ-opioid receptors (KOR and DOR), respectively. Interaction with the 7.35 residue seems to be important for the modulation mechanism since in the RME-Na-BMS-p5 simulation, where BMS was located some distance from Trp 7.35, different MOR behavior was observed (Figures 4−7, Figure S2, Table 2). This seems to be connected to BMS selectivity toward MOR.9 The 7.35 residue was also suggested to be important in the binding and selectivity of salvinorin A and its analogues in opioid receptors.11,44 This is particularly interesting since salvinorin A is a negative modulator of MOR,45 while being a potent selective agonist of KOR.46 The hypothesized MOR allosteric site location is similar to the location of a LY2119620 modulator bound in a human M2 muscarinic receptor (M2M) revealed by X-ray crystallography (PDB ID: 4MQT).47 Similarly to MOR, the M2M possesses Trp at the 7.35 position, and it is one of the main contacts of the allosteric modulator at M2M. This position is also part of a binding pocket for an allosteric modulator maraviroc bound to a CCR5 chemokine receptor, as revealed in an X-ray structure (PDB ID: 4MBS).48 Although the modulator is located deeper in the receptor in this case, the clue is important since chemokine receptors are more closely related to opioid receptors than to muscarinic ones.49 The agreement with the literature allowed us to assume that the system was constructed correctly. Obviously, all approaches aiming to resolve protein structure are condemned to some degree of inaccuracy. Even X-ray structures have limited resolution, and the crystallographic approach often requires introduction of some modifications improving the stability of the transmembrane protein that can affect the protein structure. Moreover, the structure also can be affected by the crystal packing forces. Therefore, validation is very important in all modeling approaches. In our case, the comparison of the observed phenomena with those reported in the literature suggests good quality of the obtained system. There were noticeable differences between replicas, especially in the way the modulators and the agonist affected water and the hydrophobic lock inside the receptor. This is probably a consequence of the presence of three allosteric factors: G protein, sodium ion, and BMS986122. Also, the agonist was not as rigid as, for instance, morphine or etorphine. The conflict of these factors results in a greater variance than in a simple receptor−ligand complex. On the other hand, performing three 400 ns simulations probably allowed for sampling far greater conformational space than would be possible within one 1200

Figure 5. Rearrangements of Tyr 7.53 and TM VII throughout simulations. The values for particular simulations (including all replicas of all systems) are presented as subsequent columns labeled in the first row by analogy with Table 1. Numbers at the end of the simulation name indicate the replica series. Different ligand configurations are shaded for clarity.

Water inside MOR. The motions of TM VII, and particularly of Tyr 7.53, should affect water molecules at the receptor interior. Therefore, we calculated the number of water molecules within 2 Å from the center of geometry (COG) of the hydrophobic barrier (HB) residues (2.43, 2.46, 3.43, 3.46 6.40, and additionally, 7.49, and 7.53). The barrier is another switch, suggested to play a crucial role in GPCR activation.39 Also, we counted water molecules within 1 nm from conserved Asp 2.50, which covers the space between the agonist binding pocket and HB. These analyses revealed that in all systems where Tyr 7.53 was close to Phe 345, either the hydrophobic barrier was broken or the number of water molecules inside the receptor was increased (Figure 6). Unexpectedly, this also applied to some extent to RME-Na-3. In this particular case, Na+ was unable to permanently destabilize RME (Figure 2). Also, similarly to RME-Na-BMS-p5, the distance between Tyr 7.53−Phe 345 was small, but the value of the side chain χ1 dihedral was low. Deeper insight into PCA (Figure 4C) revealed that in these systems the rigid-body tilt of the entire TM VII occurred, which was confirmed by measurement of the angle between TM II and TM VII (Figure S5). This is reflected in the value of PC1 in Figure 4. Visual assessment of the E


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Figure 6. Water inside MOR. (A) Number of water molecules within 2 Å from the center of geometry of the hydrophobic barrier. (B) Number of water molecules within 1 nm from Asp 2.50 (area marked as a yellow spot in the legend) in the second and third replica series. (C) Snapshots of RME-1 (top) and RME-Na-1 (bottom) simulations show how rotameric transition of Tyr 7.53 helps in the hydrophobic barrier (HB) breakdown.

Figure 7. Plot of the χ2 dihedral of Trp 7.35 throughout all simulations from all replicas.

(SME) has a significantly lesser affinity to MOR and promotes lower maximal activation. Therefore, we hypothesize that in RME-2 the ligand adopted the position that is possible for methadone and mostly preferred by the (S)-enantiomer. Moreover, while RME-2 resembles Na+-affected simulations, RME-Na-BMS-2 behaves like other RME or RME-Na-BMS systems. Since, as mentioned, RME-2 and RME-Na-BMS-2 are directly comparable, it is a premise that BMS shifts equilibrium to methadone binding in the full agonist-specific position. Indeed, BMS increases maximal MOR activation by SME.8

ns simulation. Keeping all the differences in mind, we managed to identify patterns related to particular ligand configurations that were common to all replicas, and the conclusions were drawn on these common features. Considering the Kb of BMS of 5 μM,9 the allosteric effect was unexpectedly pronounced in simulations. This indicates that the prediction of the BMS-binding allosteric pocket can be considered successful. The satisfactory in silico reproduction of the experimental data provides information about the location of the allosteric site at MOR, which is important, e.g., for further structure-based drug design efforts. The results indicate that rearrangements of TM VII affect the NPxxY motif, which is very conserved among the family A GPCRs. This suggests that targeting TM VII might be a good strategy in search for novel GPCR allosteric modulators. The successful reproduction of the system allows also for deeper insight into allosteric mechanisms. The unexpected behavior of RME-2 resulted from the RME orientation that hindered the flow of water molecules into the receptor. Its interaction with Tyr 7.43 decreased the space available for water exchange. A similar effect has been described recently.50 There is a suitable explanation for such behavior of methadone. While RME is a potent full agonist, (S)-methadone

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CONCLUSIONS Detailed analysis of the results suggests that a stable interaction of RME at Asp 3.32 promotes rearrangement of TM VII. This includes stabilization of a particular rotameric position of Trp 7.35 in the binding pocket, rotation of TM VII, its bending at the conserved NPxxY motif, and rotation of Tyr 7.53 at the tyrosine toggle switch. Also, the agonist promotes an increase in the TM V−TM VI distance at the height of the conserved CWxP motif (Figure S4). These rearrangements result in the breakdown of HB or increase in the number of water molecules inside the receptor. The presence of the sodium ion at its allosteric site disrupts the stability of the RME−Asp 3.32 F



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interaction, which results in distortion of the motions mentioned above. The stable RME binding is restored in the presence of BMS. The restoration is probably connected to the influence on the Trp 7.35 residue since BMS interacts directly with its side chain. This assumption is supported by the fact that BMS is a MOR-selective PAM, and Trp does not occur at the 7.35 position in any other OR subtype. Moreover, in the RME-Na-BMS-p5 simulation, where BMS did not affect Trp 7.35 (Figures 1 and 7), the amount of water inside the receptor was very low.

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REFERENCES

(1) Gunasekaran, K.; Ma, B.; Nussinov, R. Is Allostery an Intrinsic Property of All Dynamic Proteins? Proteins: Struct., Funct., Genet. 2004, 57, 433−443. (2) Christopoulos, A. Advances in G Protein-Coupled Receptor Allostery: From Function to Structure. Mol. Pharmacol. 2014, 86, 463−478. (3) Hohl, M.; Hürlimann, L. M.; Böhm, S.; Schöppe, J.; Grütter, M. G.; Bordignon, E.; Seeger, M. A. Structural Basis for Allosteric CrossTalk between the Asymmetric Nucleotide Binding Sites of a Heterodimeric ABC Exporter. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 11025−11030. (4) Nussinov, R.; Tsai, C.-J. Allostery without a Conformational Change? Revisiting the Paradigm. Curr. Opin. Struct. Biol. 2015, 30, 17−24. (5) Lazareno, S.; Popham, A.; Birdsall, N. J. M. Analogs of WIN 62,577 Define a Second Allosteric Site on Muscarinic Receptors. Mol. Pharmacol. 2002, 62, 1492−1505. (6) Yuan, S.; Vogel, H.; Filipek, S. The Role of Water and Sodium Ions in the Activation of the μ-Opioid Receptor. Angew. Chem., Int. Ed. 2013, 52, 10112−10115. (7) Ballesteros, J. A.; Weinstein, H. Integrated Methods for the Construction of Three-Dimensional Models and Computational Probing of Structure-Function Relations in G Protein-Coupled Receptors. In Methods in Neurosciences; Sealfon, S. C., Ed.; Receptor Molecular Biology; Academic Press: San Diego, CA, 1995; Vol. 25, pp 366−428. (8) Livingston, K. E.; Traynor, J. R. Disruption of the Na+ Ion Binding Site as a Mechanism for Positive Allosteric Modulation of the Mu-Opioid Receptor. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 18369− 18374. (9) Burford, N. T.; Clark, M. J.; Wehrman, T. S.; Gerritz, S. W.; Banks, M.; O’Connell, J.; Traynor, J. R.; Alt, A. Discovery of Positive Allosteric Modulators and Silent Allosteric Modulators of the μOpioid Receptor. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 10830− 10835. (10) Yabaluri, N.; Medzihradsky, F. Regulation of Mu-Opioid Receptor in Neural Cells by Extracellular Sodium. J. Neurochem. 1997, 68, 1053−1061. (11) Bartuzi, D.; Kaczor, A. A.; Matosiuk, D. Activation and Allosteric Modulation of Human μ Opioid Receptor in Molecular Dynamics. J. Chem. Inf. Model. 2015, 55, 2421−2434. (12) Wu, E. L.; Cheng, X.; Jo, S.; Rui, H.; Song, K. C.; DávilaContreras, E. M.; Qi, Y.; Lee, J.; Monje-Galvan, V.; Venable, R. M.; Klauda, J. B.; Im, W. CHARMM-GUI Membrane Builder toward Realistic Biological Membrane Simulations. J. Comput. Chem. 2014, 35, 1997−2004. (13) 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. (14) Lomize, M. A.; Lomize, A. L.; Pogozheva, I. D.; Mosberg, H. I. OPM: Orientations of Proteins in Membranes Database. Bioinformatics 2006, 22, 623−625. (15) Pike, L. J.; Han, X.; Chung, K.-N.; Gross, R. W. Lipid Rafts Are Enriched in Arachidonic Acid and Plasmenylethanolamine and Their Composition Is Independent of Caveolin-1 Expression: A Quantitative Electrospray Ionization/mass Spectrometric Analysis. Biochemistry 2002, 41, 2075−2088. (16) Saulière-Nzeh Ndong, A.; Saulière-Nzeh, A. N.; Millot, C.; Corbani, M.; Mazères, S.; Lopez, A.; Salomé, L. Agonist-Selective Dynamic Compartmentalization of Human Mu Opioid Receptor as Revealed by Resolutive FRAP Analysis. J. Biol. Chem. 2010, 285, 14514−14520. (17) Mahmood, I.; Liu, X.; Neya, S.; Hoshino, T. Influence of Lipid Composition on the Structural Stability of G-Protein Coupled Receptor. Chem. Pharm. Bull. 2013, 61, 426−437.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jcim.5b00705. Superposition of the 5C1M X-ray structure and the homology model. Distance between the allosteric sodium ion and Asp 2.50 in simulations. Comparison between conformational space explored by the receptor during all simulations at the intervals 200−300 ns and 300−400 ns. Distance between the TM V and the conserved CWxP motif at the TM VI throughout simulations. The angle between TM II and TM VII throughout all simulations. Values of RMSD between X-ray structure (PDB ID: 5C1M) and the homology model in various configurations. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel. +48 81448 7270, +48 81448 7272. E-mail: damian. [email protected], [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The study benefited from the project “The equipment of innovative laboratories doing research on new medicines used in the therapy of civilization and neoplastic diseases”, Operational Program Development of Eastern Poland 20072013, Priority Axis I modern Economy, operations I.3 Innovation promotion. Calculations were partially performed under a grant from the Interdisciplinary Center for Mathematical and Computational Modeling, Grant G30-18, under resources from CSC, Finland, and under the PRACE-3IP project FP7 RI-312763, resource Archer, based in the United Kingdom at the University of Edinburgh. The work was supported by the Foundation for Polish Science (TEAM 20094/5 Program).

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ABBREVIATIONS GPCRs, G protein-coupled receptors; MOR, μ opioid receptor; PAM, positive allosteric modulator; TM, transmembrane helix; BMS, BMS986122; PCA, principal component analysis; OPM, Orientations of Proteins in Membranes; MD, molecular dynamics; GAFF, general Amber force field; RME, (R)methadone; SASA, solvent accessible surface area; COG, center of geometry; HB, hydrophobic barrier; M2M, M2 muscarinic receptor; SME, (S)-methadone G


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(39) Yuan, S.; Filipek, S.; Palczewski, K.; Vogel, H. Activation of GProtein-Coupled Receptors Correlates with the Formation of a Continuous Internal Water Pathway. Nat. Commun. 2014, 5, 4733. (40) Liu, W.; Chun, E.; Thompson, A. A.; Chubukov, P.; Xu, F.; Katritch, V.; Han, G. W.; Roth, C. B.; Heitman, L. H.; IJzerman, A. P.; Cherezov, V.; Stevens, R. C. Structural Basis for Allosteric Regulation of GPCRs by Sodium Ions. Science 2012, 337, 232−236. (41) Katritch, V.; Fenalti, G.; Abola, E. E.; Roth, B. L.; Cherezov, V.; Stevens, R. C. Allosteric Sodium in Class A GPCR Signaling. Trends Biochem. Sci. 2014, 39, 233−244. (42) Miao, Y.; Caliman, A. D.; McCammon, J. A. Allosteric Effects of Sodium Ion Binding on Activation of the m3Muscarinic G-ProteinCoupled Receptor. Biophys. J. 2015, 108, 1796−1806. (43) Shang, Y.; LeRouzic, V.; Schneider, S.; Bisignano, P.; Pasternak, G. W.; Filizola, M. Mechanistic Insights into the Allosteric Modulation of Opioid Receptors by Sodium Ions. Biochemistry 2014, 53, 5140− 5149. (44) Kane, B. E.; McCurdy, C. R.; Ferguson, D. M. Toward a Structure-Based Model of Salvinorin A Recognition of the KappaOpioid Receptor. J. Med. Chem. 2008, 51, 1824−1830. (45) Rothman, R. B.; Murphy, D. L.; Xu, H.; Godin, J. A.; Dersch, C. M.; Partilla, J. S.; Tidgewell, K.; Schmidt, M.; Prisinzano, T. E. Salvinorin A: Allosteric Interactions at the Mu-Opioid Receptor. J. Pharmacol. Exp. Ther. 2007, 320, 801−810. (46) Roth, B. L.; Baner, K.; Westkaemper, R.; Siebert, D.; Rice, K. C.; Steinberg, S.; Ernsberger, P.; Rothman, R. B. Salvinorin A: A Potent Naturally Occurring Nonnitrogenous Kappa Opioid Selective Agonist. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 11934−11939. (47) Kruse, A. C.; Ring, A. M.; Manglik, A.; Hu, J.; Hu, K.; Eitel, K.; Hübner, H.; Pardon, E.; Valant, C.; Sexton, P. M.; Christopoulos, A.; Felder, C. C.; Gmeiner, P.; Steyaert, J.; Weis, W. I.; Garcia, K. C.; Wess, J.; Kobilka, B. K. Activation and Allosteric Modulation of a Muscarinic Acetylcholine Receptor. Nature 2013, 504, 101−106. (48) Tan, Q.; Zhu, Y.; Li, J.; Chen, Z.; Han, G. W.; Kufareva, I.; Li, T.; Ma, L.; Fenalti, G.; Li, J.; Zhang, W.; Xie, X.; Yang, H.; Jiang, H.; Cherezov, V.; Liu, H.; Stevens, R. C.; Zhao, Q.; Wu, B. Structure of the CCR5 Chemokine Receptor-HIV Entry Inhibitor Maraviroc Complex. Science 2013, 341, 1387−1390. (49) Pelé, J.; Abdi, H.; Moreau, M.; Thybert, D.; Chabbert, M. Multidimensional Scaling Reveals the Main Evolutionary Pathways of Class A G-Protein-Coupled Receptors. PLoS One 2011, 6, e19094. (50) Yuan, S.; Palczewski, K.; Peng, Q.; Kolinski, M.; Vogel, H.; Filipek, S. The Mechanism of Ligand-Induced Activation or Inhibition of μ- and κ-Opioid Receptors. Angew. Chem., Int. Ed. 2015, 54, 7560− 7563.

(18) Webb, B.; Sali, A. Comparative Protein Structure Modeling Using MODELLER. Curr. Protoc. Bioinform. 2014, 47, 5.6.1−5.6.32. (19) Manglik, A.; Kruse, A. C.; Kobilka, T. S.; Thian, F. S.; Mathiesen, J. M.; Sunahara, R. K.; Pardo, L.; Weis, W. I.; Kobilka, B. K.; Granier, S. Crystal Structure of the μ-Opioid Receptor Bound to a Morphinan Antagonist. Nature 2012, 485, 321−326. (20) Rasmussen, S. G. F.; Choi, H.-J.; Fung, J. J.; Pardon, E.; Casarosa, P.; Chae, P. S.; Devree, B. T.; Rosenbaum, D. M.; Thian, F. S.; Kobilka, T. S.; Schnapp, A.; Konetzki, I.; Sunahara, R. K.; Gellman, S. H.; Pautsch, A.; Steyaert, J.; Weis, W. I.; Kobilka, B. K. Structure of a Nanobody-Stabilized Active State of the β(2) Adrenoceptor. Nature 2011, 469, 175−180. (21) Kaczor, A.; Bartuzi, D.; Matosiuk, D. Modeling the Active Conformation of Human μ Opioid Receptor. Lett. Drug Des. Discovery 2014, 11, 1053−1061. (22) Chakrabarti, S.; Chang, A.; Gintzler, A. R. Subcellular Localization of Mu-Opioid Receptor G(s) Signaling. J. Pharmacol. Exp. Ther. 2010, 333, 193−200. (23) SYBYL-X 1.3; Tripos International: St. Louis, MO, 2009. (24) Small-Molecule Drug Discovery Suite 2015−3: Glide; Schrödinger, LLC: New York, 2015. (25) Spartan ’10; Wavefunction, Inc.: Irvine, CA, 2010. (26) Vanquelef, E.; Simon, S.; Marquant, G.; Garcia, E.; Klimerak, G.; Delepine, J. C.; Cieplak, P.; Dupradeau, F.-Y. R.E.D. Server: A Web Service for Deriving RESP and ESP Charges and Building Force Field Libraries for New Molecules and Molecular Fragments. Nucleic Acids Res. 2011, 39, W511−W517. (27) Sousa da Silva, A. W.; Vranken, W. F. ACPYPE - AnteChamber PYthon Parser interfacE. BMC Res. Notes 2012, 5, 367. (28) Jämbeck, J. P. M.; Lyubartsev, A. P. Derivation and Systematic Validation of a Refined All-Atom Force Field for Phosphatidylcholine Lipids. J. Phys. Chem. B 2012, 116, 3164−3179. (29) Jämbeck, J. P. M.; Lyubartsev, A. P. Another Piece of the Membrane Puzzle: Extending Slipids Further. J. Chem. Theory Comput. 2013, 9, 774−784. (30) Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A. Development and Testing of a General Amber Force Field. J. Comput. Chem. 2004, 25 (9), 1157−1174. (31) Jämbeck, J. P. M.; Lyubartsev, A. P. An Extension and Further Validation of an All-Atomistic Force Field for Biological Membranes. J. Chem. Theory Comput. 2012, 8, 2938−2948. (32) Paloncýová, M.; Fabre, G.; DeVane, R. H.; Trouillas, P.; Berka, K.; Otyepka, M. Benchmarking of Force Fields for MoleculeMembrane Interactions. J. Chem. Theory Comput. 2014, 10, 4143− 4151. (33) Pronk, S.; Páll, S.; Schulz, R.; Larsson, P.; Bjelkmar, P.; Apostolov, R.; Shirts, M. R.; Smith, J. C.; Kasson, P. M.; van der Spoel, D.; Hess, B.; Lindahl, E. GROMACS 4.5: A High-Throughput and Highly Parallel Open Source Molecular Simulation Toolkit. Bioinformatics 2013, 29, 845−854. (34) Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual Molecular Dynamics. J. Mol. Graphics 1996, 14, 33−38 27−28. (35) The PyMOL Molecular Graphics System; Schrödinger, LLC., 2012. (36) Huang, W.; Manglik, A.; Venkatakrishnan, A. J.; Laeremans, T.; Feinberg, E. N.; Sanborn, A. L.; Kato, H. E.; Livingston, K. E.; Thorsen, T. S.; Kling, R. C.; Granier, S.; Gmeiner, P.; Husbands, S. M.; Traynor, J. R.; Weis, W. I.; Steyaert, J.; Dror, R. O.; Kobilka, B. K. Structural Insights into μ-Opioid Receptor Activation. Nature 2015, 524, 315−321. (37) Kaczor, A. A.; Rutkowska, E.; Bartuzi, D.; Targowska-Duda, K. M.; Matosiuk, D.; Selent, J. Computational Methods for Studying GPCRs. Methods Cell Biol. 2016, DOI: 10.1016/bs.mcb.2015.11.002. (38) Trzaskowski, B.; Latek, D.; Yuan, S.; Ghoshdastider, U.; Debinski, A.; Filipek, S. Action of Molecular Switches in GPCRs– Theoretical and Experimental Studies. Curr. Med. Chem. 2012, 19, 1090−1109. H


Interplay between Two Allosteric Sites and Their Influence on Agonist

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