In Silico Observation of the Conformational Opening of the Glutathione

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Computational Biochemistry

In Silico Observation of the Conformational Opening of the Glutathione-binding Site of Microsomal Prostaglandin E2 Synthase-1 Shuo Zhou, ziyuan zhou, Kai Ding, Yaxia Yuan, Fang Zheng, and Chang-Guo Zhan J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.9b00289 • Publication Date (Web): 20 Aug 2019 Downloaded from pubs.acs.org on August 21, 2019

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Journal of Chemical Information and Modeling

In Silico Observation of the Conformational Opening of the Glutathionebinding Site of Microsomal Prostaglandin E2 Synthase-1 Shuo Zhou,1,2 Ziyuan Zhou,1,2 Kai Ding,1,2 Yaxia Yuan,1,2 Fang Zheng,1,2,* and Chang-Guo Zhan1,2,* 1Molecular

Modeling and Biopharmaceutical Center, College of Pharmacy, University of

Kentucky, 789 South Limestone Street, Lexington, KY 40536. 2Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536

Correspondence: Chang-Guo Zhan, Ph.D. Director, Molecular Modeling and Biopharmaceutical Center (MMBC) Director, Chemoinformatics and Drug Design Core of CPRI University Research Professor Endowed College of Pharmacy Professor in Pharmaceutical Sciences Professor, Department of Pharmaceutical Sciences College of Pharmacy University of Kentucky 789 South Limestone Street Lexington, KY 40536 Phone: 859-323-3943 FAX: 859-257-7585

E-mail: [email protected]

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Abstract Microsomal prostaglandin E2 synthase-1 (mPGES-1) is known as an ideal target for nextgeneration anti-inflammatory drugs to effectively and safely treat a variety of inflammation-related diseases. High-resolution X-ray crystal structures are available for human mPGES-1, but all in a closed conformation for glutathione (GSH)-binding site. Here we report in silico observation of the desirable open conformation of mPGES-1 using a simple computational strategy with fully relaxed molecular dynamics simulations starting a high-resolution X-ray crystal structure in the closed conformation. The open conformation mainly exists in the apo-form. Once GSH enters the binding site, the binding site is closed and, thus, mPGES-1 becomes the closed conformation. According to the determined free energy profile, both the open and closed conformations can coexist in solution with a thermodynamic equilibrium, and the conformational distribution is dependent on the GSH concentration. In addition, the cap domain responsible for the conformational transition is located right on the crystal packing interface, showing that only closed conformation is suitable for the crystal packing. All of the computational insights are consistent with reported experimental observations. The computationally simulated open conformation of mPGES-1 may serve as a new target state for rational design of novel inhibitors of mPGES-1. We anticipate that the similar computational strategy used in this study may also be used to explore open conformation starting from a crystal structure of the corresponding closed conformation with a ligand bound for other proteins. 1. Introduction As well known, prostaglandin E2 (PGE2) is the principal pro-inflammatory agent serving as a mediator of pain and fever in inflammatory reactions in a number of inflammation-related diseases,1 such as various types of pain, cardiovascular diseases, neurodegenerative diseases, and cancers.2-4 The PGE2 biosynthesis5 starts from arachidonic acid (AA) which is converted to prostaglandin H2 (PGH2) by cyclooxygenase (COX)-1 and COX-25 and then to PGE2 by prostaglandin E synthase (PGES).6 The first generation of nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin and ibuprofen used to treat pain and reduce fever or inflammation, weakly and non-selectively inhibit both COX-1 and COX-2. The second generation of NSAIDs, including celecoxib (Celebrex), rofecoxib (Vioxx) and valdecoxib (Bextra), potently and selectively inhibit COX-2. The COX-2-selective inhibitors still have a number of serious side

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effects, such as increasing the risk of fatal heart attack or stroke and causing stomach or intestinal bleeding. The serious side effects led to withdrawal of rofecoxib and valdecoxib, although celecoxib still remains in clinical use. The serious side effects are due to the fact that the synthesis of all physiologically needed prostaglandins downstream of PGH2 are inhibited by the action of the COX-1/2 inhibitors. For example, blocking the production of prostaglandin-I2 (PGI2) will cause significant cardiovascular problems.7 Microsomal PGES-1 (mPGES-1), an inducible enzyme, is recognized as a more promising, ideal target for the next generation of antiinflammatory drugs, because the mPGES-1 inhibition will only block the PGE2 production without affecting the production of PGI2 and other physiologically needed prostaglandins, as confirmed by reported knock-out studies.8-16 Thus, mPGES-1 inhibitors are expected to retain the antiinflammatory effect of COX-1/2 inhibitors, but without the side effects caused by the COX-1/2 inhibition. For development of a next generation of anti-inflammatory drugs, various mPGES-1 inhibitors have been reported in the literature.17-38 Unfortunately, almost all of the previously reported human mPGES-1 inhibitors are inactive against mouse or rat mPGES-1, which prevents using wellestablished mouse/rat models of inflammation, pain, and other diseases for preclinical studies.39 To design improved mPGES-1 inhibitors, one will first need to have a reliable three-dimensional (3D) structure of the protein in a suitable conformational state. As well known, it is usually difficult to obtain a crystal structure of a membrane protein like mPGES-1. Since 2013, Sjogren et. al.40 and Weinert et al.41 reported high-resolution X-ray crystal structures of mPGES-1, demonstrating significant structural differences with the earlier CryoEM structure reported in 2008.42 Since there are major differences between the CryoEM and X-ray crystal structures, it was suggested that mPGES-1 might adopt a different conformation to function in solution.40 In particular, all available X-ray crystal structures exist in a closed conformation with glutathione (GSH) completely trapped inside the active site cavity; according to this conformation, GSH cannot enter or leave the active site cavity without a major conformational change of the enzyme. Here, the open conformation of mPGES-1 refers to the one in which both PGH2 and GSH can freely enter or leave the active site cavity without an energy barrier. Notably, the role of GSH in mPGES-1 is two folds: GSH mainly serves as a co-factor for mPGES-1-catalyzed reaction of PGH2 to produce PGE2. Meanwhile, GSH itself serves as a substrate43 for another type of physiologically irrelevant reaction (which can occur only in certain in vitro conditions including a second reactant).

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Previous computational modeling44 of the mPGES-1 conformation started from the earlier CryoEM structure with a low resolution – 3.5 Å in-plane (x-y plane) resolution and 10 Å z-axis resolution (PDB: 3DWW),45 and only examined the PGH2-binding site without examining whether the GSH-binding site opens or not. To the best of our knowledge, there has been no report of an X-ray crystal structure of mPGES-1 with the open conformation in terms of the GSH-binding site or a computational modeling of such open conformation of mPGES-1 starting from a highresolution X-ray crystal structure. Here we report an in silico observation of the desirable open conformation of mPGES-1 in solution through a fully relaxed molecular dynamics (MD) simulation starting from a highresolution X-ray crystal structure – a simple computational strategy which may be used to simulate open conformations of other proteins with the similar structural questions. According to our computational simulations, in the absence of GSH, both the open and closed conformations can co-exist in solution with the open conformation having a relatively lower free energy, and there is a very low free energy barrier for the conformational transition between the open and closed states. However, in the presence of GSH, mPGES-1 predominantly exists in the closed conformation with a much lower free energy. GSH helps to considerably stabilize the closed conformation, explaining why only the closed conformation has been observed in the reported X-ray crystal structures with GSH. 2. Methods Our computational modeling and simulations started from the X-ray crystal structure40 of human mPGES-1 (PDB 4AL0 with a remarkably high resolution at 1.2 Å). The mPGES-1 protein (trimer) structure was simulated in the presence of a phospholipid bilayer of 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) and a water box in all of the MD simulations. The CHARMMGUI membrane builder46 was used to build the initial 3D structural model and prepare input files for the MD simulations. Only the protein part with/without GSH of the X-ray crystal structure was used in building the initial model. A total of 153 DOPC molecules were used to build the membrane model, and a total of 11365 water molecules were added to fill the water box. In addition, 25 sodium and 46 chloride ions are added to neutralize the system and adjust the salt concentration to 0.15 M.

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The NAMD 2.10 program47 was used to run the MD simulation for 100 ns. The input files were generated by the CHARMM-GUI builder, and the CHARMM force field was used in the MD simulation. The simulation temperature was set at 303.15 K and the time step used was 2 fs. The Nose-Hoover Langevin piston pressure control was used to keep the temperature and pressure constant. The AMBER12 software48 was used for the targeted MD (TMD), potential of mean force (PMF), and Molecular Mechanics (MM)-Poisson-Boltzmann Surface Area (MM-PBSA) simulations/calculations. Input models were generated by using the NAMD simulation results. AMBER ff03 force field and lipid11 force field were used for the simulation. In the TMD simulation, the closed conformation was used as the starting conformation, and the open conformation state was used as the final conformation. Along the reaction coordinate, a conformation in every 5 degrees was selected as a window. For each window, the structure was energy-minimized and equilibrated, followed by an MD simulation (1 ns) for the conformational sampling. The obtained energetic data were then submitted to the WHAM script49 for the PMF calculations. 3. Results and Discussion 3.1. In silico observation of the conformational opening Conformation of mPGES-1 with GSH bound. Starting from the high-resolution X-ray crystal structure (4AL0 with a high resolution at 1.2 Å), we first modeled and simulated the protein structure of mPGES-1 in the presence of co-factor GSH. Throughout the 100-ns MD simulation, the average root-mean-square deviation (RMSD) was only at 1.9 Å (Figure 1A), the closed conformation of the protein structure was very stable. There was no conformational change of mPGES-1 during the MD simulation. Spontaneous conformational opening of mPGES-1 without GSH. To examine whether GSH may induce a significant conformational change, we performed another MD simulation on mPGES-1 in the absence of GSH (but mPGES-1 was still in the phospholipid bilayer and water box). In this simulation, the protein structure appeared to have an average RMSD of 2.8 Å (Figure 1A). In particular, a small portion (F44 to D62) of the protein had a major conformational change, while the remaining protein structure was still very stable (Figure 1B). The flexible residues (from

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F44 to D62, Figure 1C) are located right on the GSH binding site. In the presence of GSH, this domain acts like a cap of the GSH-binding site, preventing GSH from contacting the solvent. When this cap domain adopts this completely different “open” conformation (which may be defined as ϕ < 120˚; see Figure 1D and caption for the definition of ϕ), the catalytic site will be able to gain direct access to cytoplasm, allowing GSH to freely enter or leave the active site. To test the reproducibility of computationally observed computational change from the closed conformation to the open conformation, we also carried out additional 10 parallel MD simulations with the same settings. It turned out that all the additional 10 parallel MD simulations also led to in silico observation of the conformational opening, but with different times when the conformation started to open during the MD simulations. Specifically, four out of the 10 additional MD simulations led to the conformational opening within the first 100 ns of the simulation. The remaining six MD simulations led to the conformational opening at a later time (138.5, 147.1, 149.1, 192.6, 225.2, or 351.8 ns); see Figure S1 and Table S1 in the Supporting Information.

Figure 1. (A) Stability of mPGES-1 in 100 ns MD simulation of mPGES-1 with and without GSH. (B) RMSD of each residue in the MD-simulated mPGES-1 structure without co-factor GSH. (C) The cap domain in its “closed” (red) and “open” (green) conformation during the MD simulation. The other part of mPGES-1 is showed in yellow cartoon. (D) The angle ϕ was defined using the 6


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centers of mass (COM) of P63-E66 (blue), P47-A50, and L51-G54 (green). (E) The change of angle ϕ (red) versus the simulation time compared with the angle between the alpha helix (colored green in 1D) and the x-y plane (black). (F) The free energy profile along the reaction coordinate when GSH is present (black) and absent (red). The major structural difference between the open and closed conformations in the cap domain are shown in Figure 2 and summarized in Table 1. In the simulated open conformation, despite the conformational change and loss of a few polar interactions, new polar interactions are formed at the same time to compensate the loss. Thus, the open conformation is expected to be at least as stable as, or more stable than, the closed conformation in solution.

Figure 2. Detailed interactions between residues of the cap domain of mPGES-1 in the closed conformation observed from different orientations (A, B, and C panels) and the open conformation observed from the corresponding orientations (D, E, and F panels, respectively). Protein is showed in yellow cartoon and the cap domain is showed in red (open conformation) or green (open conformation). Key residues are shown in stick model and polar interactions are showed in black dashed lines.

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Table 1. Comparison between the open and closed conformations in the main interactions of amino-acid residues in the cap domain. Residue Closed conformation Open conformation E48

Salt bridge with R67

Side chain exposed in water

D49

Salt bridge with R126

Hydrogen bond (HB) with N46

L51


Side chain buried

R52


HB with Side chain of H53

H53 Q57

Side chain exposed in water Stab into water

HB with Side chain of R52 HB with S61

R60 E66

HB with backbone oxygen of K41 Side chain exposed in water

Salt bridge with E66 Salt bridge with R60

To further test and validate the computational approach used to simulate the open conformation of mPGES-1, the same computational approach was also used to simulate the outward-open conformation of leucine transporter (LeuT, a transmembrane protein with X-ray crystal structures available for both the outward-open and outward-occluded conformations50-51) starting from an X-ray crystal structure of the outward-occluded conformation (PDB ID: 2A65).51 The 100-ns MD simulation indeed led to the outward-open conformation (see Figures S2 and S3 in the Supporting Information) which is very close to the X-ray crystal structure of the outwardopen conformation (PDB ID: 3TT1) with an RMSD of only 1.30 Å for the α-C atoms. The agreement between the MD-simulated outward-open conformation and the corresponding X-ray crystal structure suggests that the computational approach is reasonable. 3.2. Conformational dynamics and energetics for the conformational transition In order to know the energetics of this significant conformational change, we performed a PMF simulation on the conformational transition of mPGES-1 using the umbrella sampling method. To perform this PMF simulation, a “reaction coordinate” must be defined for the conformational transition. Since the main part of this cap domain is an -helix, we decided to use the angle between the vector of this -helix and the x-y plane as the reaction coordinate. However, due to the technical restriction of the MD simulation software, the x-y plane cannot be chosen as an anchor point, we instead selected the COM (center of mass) of P63-E66 as the anchor point because it was stable during the MD simulation (Figure 1D). The COM(P63-E66)–COM(P47A50)–COM(L51-G54) angle highly correlated with the above-mentioned angle between the 8


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helix and the x-y plane (Figure 1E), indicating that it is reasonable to use this angle as the reaction coordinate. The WHAM program49 was then used to analyze relative free energy changes along the reaction coordinate. The results are shown in Figure 1F, revealing two local minima: one associated with the closed conformation and the other with the open conformation. The free energy difference between these two local minima is about 0.5 kcal/mol and the free energy barrier is only 2.5 kcal/mol, which means that these two conformations can easily change from one to another in solution. Further, we also performed a targeted MD (TMD) simulation, followed by the similar PMF simulation on the mPGES-1 in the presence of GSH. The obtained energetic data are also depicted in Figure 1F, showing that the closed conformation has a much lower free energy than the open conformation in the presence of GSH. Based on the energetic data in Figure 1F, the free mPGES-1 protein (without GSH) should mainly, but not exclusively, exist in the open conformation, whereas the protein with GSH bound should exclusively exist in the closed conformation. These computational results are consistent with the fact that only the closed conformation has been observed in the reported X-ray crystal structures with GSH bound. In addition, the above computational data also predict that, in the absence of GSH, both the open and closed conformations of mPGES-1 co-exist in solution, with a higher probability of being the open conformation. In other words, the computational simulations predict that the GSH-free mPGES-1 structure (apo-form) in solution mainly exists in the open conformation observed in silico. Overall, whether mPGES-1 mainly takes the open or closed conformation is dependent on the actual concentration of GSH (denoted as [GSH]) in solution. It has been known that the catalytic activity of mPGES-1 is dependent on [GSH], with KM = 0.71 mM for GSH.52 Assuming that Kd  KM under the widely used rapid-equilibration assumption,43 when [GSH] = 0.71 mM, we may expect to have [mPGES-1-GSH] = [mPGES-1], where [mPGES-1-GSH] represents the concentration of the enzyme-GSH complex and [mPGES-1] refers to the concentration of the free enzyme without GSH. When [GSH] > 0.71 mM, we should have [mPGES-1-GSH] > [mPGES-1]. Otherwise, when [GSH] < 0.71 mM, we should expect [mPGES-1-GSH] < [mPGES-1]. So, at the usually used GSH concentration of 1 or 2 mM for mPGES-1 crystallization studies,40, 53 both the open and closed conformations are expected to co-exist in solution, but majority of the mPGES-1 molecules should stay in the closed conformation when [GSH] > 0.71 mM.

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3.3. Computational insights into mPGES-1 crystallization and X-ray crystal structures To validate our newly found open conformation, we examined all X-ray crystal structures available. In fact, mPGES-1 has not yet been crystallized in the apo-form. As discussed above, the free energy barrier between the open and closed conformations is rather low (2.5 kcal/mol) in the absence of GSH, which means the conformational transition between the open and closed states is rather fast. Thus, the open and closed states can keep the thermodynamic equilibrium under this condition. With this new computational insight, we re-examined the crystal structure 4AL0 again and found that the cap domain was located right on the crystal packing interface (Figure 3A). The interface is a perfect match between the neighboring mPGES-1 molecules when they all exist in the closed conformation, as seen in Figure 3A. When the cap opens (in the open conformation), the crystal packing could be interrupted in the lack of favorable intermolecular interactions (Figure 3B) and, thus, the crystal will not be able to grow. So, only the closed conformation can be recruited to keep the crystal growth, which explains why only the closed conformation has been observed in the reported X-ray crystal structures. Based on this computational insight, it would be very difficult to obtain an X-ray crystal structure of mPGES-1 in the open conformation. Further, with a given mPGES-1 concentration, the higher the GSH concentration, the more percent of mPGES-1 molecules in the closed conformation available for the crystal growth and, thus, the faster the crystal grows. The crystal is not expected to grow without a sufficiently high concentration of the closed conformation available. This computational insight is consistent with the previous experimental observation by Li et al.53 that the mPGES-1 protein was unable to be crystallized under a low GSH concentration. According to Li et al.,53 in the presence of 1 mM GSH, the crystal started to grow initially, but disappeared between 3 and 10 days post-setup due to the susceptibility of GSH to oxidization. It is well-known that GSH can be oxidized spontaneously to its dimer GSSG (i.e. GSH + GSH  GSSG), with a reaction velocity of 6.6 nM min-1 when [GSH] = 56 µM.54 According to the velocity of 6.6 nM min-1 when [GSH] = 56 µM at the room temperature, the second-order reaction rate constant (k2) should be 2.1 × 10-9 nM min-1. When k2 = 2.1 × 10-9 nM min-1, GSH should have a half-life of 8 hr (at the room temperature) when [GSH] = 1 mM or 16 hr when [GSH] = 0.5 mM,

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or 32 hr when [GSH] = 0.25 mM, …. Clearly, starting at 1 mM, the GSH concentration became too low after 3 days post-setup, according to our kinetic analysis. Realizing the crystal instability due to the spontaneous reduction in the GSH concentration, Li et al.53 increased the GSH concentration in the protein solution to 2 mM and further supplemented the precipitant solution with GSH to the same concentration (2 mM). Under such a high concentration of GSH, Li et al. was able to obtain the qualified single crystal.53 Experimental observation reported by Li et al.53 is consistent with our computational insight that only the closed conformation of mPGES-1 can be recruited to grow the crystal and that GSH can considerably stabilize the closed conformation. Our computational insight helps to better understand the experimental observation.

Figure 3. (A) Crystal packing interface of mPGES-1 in the closed conformation. The protein is showed in yellow cartoon and the cap domain is in red color. The interface shows a perfect match between the two neighboring molecules as shown in the van der Waals surface (grey). (B) Crystal packing interface of mPGES-1 in the open conformation. The protein is showed in yellow cartoon and the cap domain is in green color. Most of van der Waals contacts (grey) have been lost in the open conformation. Conclusion Through fully relaxed molecular dynamics simulations starting from a high-resolution X-ray crystal structure in the closed conformation, we have obtained the open conformation of human mPGES-1 in solution, which is remarkably different from the closed conformation shown in all X-ray crystal structures reported so far. A cap domain has been identified responsible for the conformational transition between the open and closed conformations. The open conformation mainly exists in solution in the absence of GSH. Once GSH enters the binding site, the GSH-

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binding site is closed and, thus, mPGES-1 becomes the closed conformation. The GSH-bound mPGES-1 predominantly exist in the closed conformation. According to the determined free energy profile, both the open and closed conformations can co-exist in solution and can rapidly reach a thermodynamic equilibrium, and the distribution of the two conformations is dependent on the concentration of GSH. The higher the GSH concentration, the more percent of closed conformation of mPGES-1 existing in solution. In addition, the cap domain responsible for the conformational transition is located right on the crystal packing interface. The interface represents a perfect match between the neighboring mPGES-1 molecules when they all exist in the closed conformation. But the favorable interface does not exist in the open conformation. Thus, the crystal is not expected to grow when mPGES1 mainly exists in the open conformation. So, only the closed conformation can be recruited to keep the crystal growth, which explains why only the closed conformation has been observed in the reported X-ray crystal structures and why the mPGES-1 crystal can grow only in the presence of a sufficiently high concentration of GSH. The computationally simulated open conformation of mPGES-1 may serve as a new target state for rational design of novel inhibitors of mPGES-1. In general, this study has used a simple computational strategy of performing the fully relaxed MD simulation on the open conformation of a protein starting from a high-resolution X-ray crystal structure of the corresponding closed conformation with a ligand bound. This simple strategy will allow the protein to spontaneously open the binding site (without any biasing force in the simulation) after the ligand is removed from the binding site. We have demonstrated that such a simple computational strategy can be used to reliably simulate the open conformation of a protein starting from a high-resolution X-ray crystal structure of the corresponding closed conformation with a ligand bound. Acknowledgements This work was supported in part by the funding of the Molecular Modeling and Biopharmaceutical Center at the University of Kentucky College of Pharmacy and the National Science Foundation (NSF grant CHE-1111761). The authors also acknowledge the Computer Center at the University of Kentucky for supercomputing time on a Dell Supercomputer Cluster consisting of 388 nodes or 4,816 processors.

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In Silico Observation of the Conformational Opening of the Glutathione

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