Research Article Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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Flipped Phenyl Ring Orientations of Dopamine Binding with Human and Drosophila Dopamine Transporters: Remarkable Role of Three Nonconserved Residues Yaxia Yuan,† Jun Zhu,‡ and Chang-Guo Zhan*,† †
Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, Kentucky 40536, United States ‡ Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, 715 Sumter Street, Columbia, South Carolina 29208, United States S Supporting Information *
ABSTRACT: Molecular modeling and molecular dynamics simulations were performed in the present study to examine the modes of dopamine binding with human and Drosophila dopamine transporters (hDAT and dDAT). The computational data revealed flipped binding orientations of dopamine in hDAT and dDAT due to the major differences in three key residues (S149, G153, and A423 of hDAT vs A117, D121, and S422 of dDAT) in the binding pocket. These three residues dictate the binding orientation of dopamine in the binding pocket, as the aromatic ring of dopamine tends to take an orientation with both the para- and meta-hydroxyl groups being close to polar residues and away from nonpolar residues of the protein. The flipped binding orientations of dopamine in hDAT and dDAT clearly demonstrate a generally valuable insight concerning how the species difference could drastically affect the protein− ligand binding modes, demonstrating that the species difference, which is a factor rarely considered in early drug design stage, must be accounted for throughout the ligand/drug design and discovery processes in general. KEYWORDS: Protein−ligand binding, transporter, dopamine system, species difference, molecular modeling, molecular dynamics
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INTRODUCTION Human dopamine transporter (hDAT) is a membranespanning protein regulating the uptake and efflux of dopamine (DA). DA releasing from the synaptic cleft can be transported across the cell membrane into presynaptic neurons by hDAT1−3 with the driven force from ion concentration gradient.4 Thus, hDAT plays a critical role in regulating the spatial and temporal extra neuronal DA concentration.4,5 As DA is a vital neurotransmitter for the reward-motivated systems, hDAT is of particular clinical relevance because it is involved in many DA-related diseases such as attention deficit hyperactivity disorder, bipolar disorder, clinical depression, schizophrenia, Parkinson’s disease, and drug dependence.6−9 Similar to other members of neurotransmitter sodium symporters (NSS) family, the transporting process of DA via hDAT is Na+/Cl− dependent,10,11 which may be roughly modeled as involving three typical conformational states of hDAT: the outward-open state (i.e., the extracellular side of binding site for the transmitter is open, while the intracellular side is blocked), the outward-occluded state (i.e., both the extracellular and intracellular sides of binding site are blocked such that the binding site is occluded and no longer accessible for substrate), and the inward-open state (i.e., the intracellular side of binding site for the transmitter is open, while the extracellular side is blocked).12−19 Knowledge about the © XXXX American Chemical Society
detailed binding mode of dopamine with hDAT is essential for understanding the molecular mechanisms concerning how hDAT transports the substrate, and also for providing clues concerning how hDAT interacts with other important ligands. In our previous studies,20−22 molecular modeling was performed on hDAT based on X-ray crystal structure (PDB code, 4M48; resolution, 2.95 Å; complexed with Nortriptyline)23 of Drosophila DAT (dDAT, its sequence similarity and identity with hDAT are 59% and 46%, respectively) released in 2013. The binding conformation of dopamine was determined by molecular docking and molecular dynamics (MD) simulations. More recently, further X-ray structure of dDAT complexed with dopamine was reported (PDB code, 4XP1; resolution, 2.89 Å),24 providing the straightforward information concerning how dopamine interacts with a DAT. However, we noticed that the binding orientation of dopamine in this newly reported X-ray crystal structure of dDAT is ∼180° rotated along the long axis of dopamine compared to the orientation of dopamine in our modeled hDAT-DA binding structure. Generally speaking, according to conventional wisdom, a substrate of homologous proteins with the same function is Received: January 19, 2018 Accepted: March 1, 2018 Published: March 1, 2018 A
DOI: 10.1021/acschemneuro.8b00030 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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ACS Chemical Neuroscience
Figure 1. Sequence alignment of human and Drosophila dopamine transporter (hDAT and dDAT). Residue 79 is colored in red, and residues 149, 153, and 429 are colored in green.
expected to adopt similar binding mode. However, the modeled hDAT-DA binding mode is drastically different from the dDAT-DA binding mode shown in the X-ray crystal structure. On the other hand, the MD simulations demonstrated that dopamine with a flipped orientation in the modeled hDAT structure did match the binding site of hDAT very well. To understand the remarkable difference between the modeled hDAT-DA binding mode and the dDAT-DA binding mode in the X-ray crystal structure, we carried out further computational modeling and MD simulations on dopamine binding with the wild-type transporters (dDAT and hDAT) and their mutants. The computational data reveal that dopamine indeed should have flipped binding orientations in hDAT and dDAT due to the major differences in three key residues in the binding pocket. The flipped binding orientations of DA in hDAT and dDAT clearly demonstrate how the species difference could drastically affect the protein−ligand binding modes.
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RESULTS AND DISCUSSION Modeled Structures of Dopamine Binding with WildType hDAT and dDAT. The amino acid sequence alignment between hDAT and dDAT (Figure 1) shows that 12 regions with high homology can be assigned to 12 transmembrane (TM) helices. The similarity between hDAT and dDAT in amino acid sequence for these 12 TMs is as high as 60%, suggesting that homology modeling of the hDAT structure using the dDAT template is reasonable. Depicted in Figure 2A is a typical snapshot of the MD-simulated 3D structure of the outward-open hDAT-DA binding complex in a biological environment built through homology modeling by using the Xray crystal structure of dDAT (complexed with nortriptyline) reported in 201335 as a template, as used in our previous studies.21,22 The continued MD simulation in this study further confirmed that our previously modeled hDAT-DA binding complex was very stable, without any noticeable structural changes during the MD simulation. However, the modeled hDAT-DA binding mode obtained from the molecular docking and MD simulation is remarkably different from the dDAT-DA binding mode revealed in a more recently reported X-ray crystal structure24 of dDAT complexed with DA (Figure 2B). According to our molecular modeling and MD simulation on the dDAT-DA complex starting from the X-ray crystal
Figure 2. Structure of (A) human dopamine transporter (hDAT)− dopamine (DA) complex and (B) Drosophila dopamine transporter (dDAT)−DA complex (PDB code: 4XP1) in cartoon style. The dopamine-binding site is showed in the dashed box. Key residues around the binding site in (C) hDAT-DA complex and (D) dDAT-DA complex are showed in stick style. Dopamine is colored in purple. The negatively charged D46 and D79 are colored in orange. Key different residues between dDAT and hDAT in the binding site are colored in green. Hydrogen bonds are showed in dashed lines with label distances. (E) Key residues around dopamine-binding site in the superimposed structures of the dDAT (green) and hDAT (cyan).
structure, the simulated dDAT-DA complex structure starting from the X-ray crystal structure was also very stable. According to conventional wisdom, a substrate of homologous proteins with a same function is usually expected to adopt similar protein−substrate binding mode. As noted above, hDAT and dDAT are highly homologous, implying that the binding conformations of DA in hDAT and dDAT should be similar. However, according to our molecular modeling and MD simulations, DA in the binding pocket adopted a very different orientation in the X-ray crystal structure of dDAT-DA complex (Figure 2B) compared to that in the modeled hDATDA complex structure (Figure 2A). Why are they remarkably B
DOI: 10.1021/acschemneuro.8b00030 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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ACS Chemical Neuroscience
Figure 3. (A) Key residues around dopamine in the wt-hDAT-DA complex are showed in stick-ball style. Dopamine is colored in purple. Three nonconserved residues are colored in green. The conserved aspartic acid is colored in orange. (B) Tracked changes of the RMSD for the Cα atoms and the hydrogen bonds between dopamine and wt-hDAT along the MD simulation. (C) Key residues around dopamine in the tm-dDAT-DA complex. (D) Tracked changes of the RMSD for the Cα atoms and the hydrogen bonds between dopamine and tm-dDAT along the MD simulation.
Figure 4. (A) Key residues around dopamine in the wt-dDAT-DA complex are showed in stick-ball style. Dopamine is colored in purple. Three nonconserved residues are colored in green. The conserved aspartic acid is colored in orange. (B) Tracked changes of the RMSD for the Cα atoms and the hydrogen bonds between dopamine and wt-dDAT along the MD simulation. (C) Key residues around dopamine in the tm-hDAT-DA complex. (D) Tracked changes of the RMSD for the Cα atoms and the hydrogen bonds between dopamine and tm-hDAT along the MD simulation.
different? According to the sequence alignment of hDAT and dDAT (Figure 1), there are only three nonconserved residues in the DA-binding pocket, i.e. S149, G153, and A423 of hDAT compared to A117, D121, and S422 of dDAT. Local view of the DA-binding region of hDAT and dDAT (Figure 2C and D) indicates that the three nonconserved residues are involved in the binding with DA. Hence, the differences between hDAT and dDAT in these three residues might be responsible for the difference in the DA binding orientation. According to the local view of the superimposed DA binding region depicted in Figure 2E, the overall shapes of DA in hDAT and dDAT are similar, except for the ∼180° flipping of the aromatic ring of DA. As an asymmetric molecule, the meta-hydroxyl of the aromatic ring uneven hydrophobic and hydrophilic property distribution to the structure of DA, which drives DA to bind to its receptor with a specific orientation. The aromatic ring of DA always took an orientation with both the para- and meta-hydroxyl groups being close to polar residues and away from nonpolar residues of the protein, as shown in Figure 2C and D. For wild-type hDAT, the two hydroxyl groups of DA were orientated to be close to the S149 and away from A423 and G153. For wild-type
dDAT, the two hydroxyl groups of DA were orientated to be close to D121 and S422 and away from A117. Effects of the Three Nonconserved Residues on the DA Orientation in hDAT and dDAT. As only three residues are nonconserved in the DA-binding site of hDAT and dDAT, we hypothesized that these three residues are the decisive factor for the different binding orientations of DA in hDAT and dDAT. Our further modeling and MD simulations were carried out to test this hypothesis. An intuitive way of inspecting this hypothesis is to check whether mutations associated with these three residues would alter the favorable binding orientation of DA. Computational mutations A117S, D121G, and S422A were performed on the wild-type dDAT (wt-dDAT) to mimic the binding site of wild-type hDAT (wt-hDAT), and computational mutations S149A, G153D, and A423S were performed on the wt-hDAT to mimic the binding site of wt-dDAT. The triplemutated hDAT and dDAT were denoted as tm-hDAT and tmdDAT, respectively, for convenience. For each complex, the MD simulation was performed to examine the binding mode. The binding structures of DA with wt-hDAT and tm-dDAT from the equilibrated MD trajectories are showed in Figure 3A and C, respectively. Obviously, DA stayed in wt-hDAT and tmC
DOI: 10.1021/acschemneuro.8b00030 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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the flipped binding orientations of dopamine in hDAT and dDAT are due to the major differences in three key residues (S149, G153, and A423 of hDAT vs A117, D121, and S422 of dDAT) in the binding pocket. These three nonconserved residues dictate the binding orientation of dopamine in the binding pocket, as the aromatic ring of dopamine tends to take an orientation with both the para- and meta-hydroxyl groups being close to polar residues and away from nonpolar residues of the protein. The novel structural insights obtained from this computational study may be valuable in guiding future rational drug design in general. In particular, the species difference between human and an animal model for a drug target was usually ignored in early drug design stage for drug discovery effort. The flipped binding orientations of dopamine in hDAT and dDAT may serve as a convincing example to demonstrate how the species difference could drastically affect a ligand binding with the target proteins from different species and why the species difference must be accounted for throughout the ligand/drug design and discovery processes.
dDAT with an almost identical binding mode. Depicted in Figure 3B and D are the tracked changes of positional rootmean-square deviations (RMSD, from their starting structures) for the Cα atoms of the wt-hDAT and tm-dDAT binding structures (black curve), together with the calculated RMSD values for the atomic positions of DA in the binding pocket (red curve). After a period of ∼20 ns MD run, all of the RMSD curves became flat, indicating that the MD-relaxations were equilibrated very well. The atomic details of intermolecular interactions between DA and the protein as tracked along the MD simulation are also showed in Figure 3B and D. The protonated amino group of DA was hydrogen-bonded with the Oδ atom of wt-hDAT-D79 or tm-dDAT-D46 side chain (blue curve). The para-hydroxyl group of DA also formed a hydrogen bond with Oγ atom of residue wt-hDAT-S149 or tm-dDATS117 (orange curve). The conformation of DA and all of these hydrogen bonds were stable during the MD simulations, suggesting that the simulated binding structures are reasonable. The binding conformations of DA with wt-dDAT and tmhDAT from the equilibrated MD trajectories are depicted in Figure 4A and C, respectively. Similarly, DA stayed in wt-dDAT and tm-hDAT with an almost identical binding mode, which was an ∼180° flipped conformation compared to that in wthDAT or tm-dDAT. The flat curves of RMSD for the protein and ligand in 20 ns production MD simulations depicted in Figure 4B and D suggest that the two complex structures were equilibrated very well. The protonated amino group of DA was hydrogen-bonded with the Oδ atom of wt-dDAT-D46 or tmhDAT-D79 side chain (blue curve). Both the meta- and parahydroxyl groups of DA formed hydrogen bonds with Oδ atom of residue wt-dDAT-D121 (orange curve) or tm-hDAT-D153 (green curve). The conformation of DA and all of these hydrogen bonds were stable during the MD simulations, suggesting that the simulated binding structures are reasonable. Interestingly, Wang et al.24 made D121G/S426M mutations on dDAT in order to mimic the dopamine binding site of hDAT. However, the dopamine transporting activity of dDAT was totally extinguished by the D121G/S426M mutations. D121 is close to dopamine, and S426 is away from the binding site, as seen in Figure S1 (Supporting Information). According to the binding mode depicted in Figure 4, D121 forms hydrogen bonds with dopamine, serving as one of the most important residues for dopamine binding with dDAT. The D121G mutation would destroy the key hydrogen bonds between dopamine and dDAT. So, our modeled proteindopamine binding structures help to understand why the D121G/S426M mutant is inactive. As revealed by all of the computational data discussed above, only exchanging the three nonconserved residues in hDAT and dDAT can lead to the exchange of the favorable binding orientations of DA with hDAT and dDAT. These three residues determine the pattern of the polar and nonpolar interactions of DA with hDAT or dDAT, which is likely the major factor to cause the remarkable difference between wthDAT and wt-dDAT in the dopamine-binding mode.
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METHODS
Molecular dynamics (MD) simulations in the presented work were carried out using the PMEMD module of the AMBER 12 program package.25 Modeling of Wild-Type hDAT and dDAT. Simulation System Preparation. The equilibrated simulation system of wild-type hDAT was obtained from our previous studies,21,22 with the previous MD simulation on the hDAT-DA complex system extended further so that all of the protein−ligand systems in the present study were simulated in a same way. The simulation system for wild-type dDAT was prepared in the same way. The initial X-ray crystal structure of the dDAT-DA complex (outward-open state of dDAT) was downloaded from the RCSB database (PDB entry: 4XP1).24 In order to optimize the structure of dDAT complex in the physiological environment, the initial structure of dDAT complex was inserted into a POPC (1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipid bilayer and then solvated by two layers (upside and downside of lipid bilayer) of water (TIP3P) by using the Membrane-Builder module of Charmm-GUI (http://www.charmm-gui.org/?doc=input/membrane). The orientation data of dDAT in the OPM database (http://opm.phar.umich. edu/) were used to determine the initial orientation of the dDAT complex in the lipid membrane. Standard protonation states were also set to for ionizable residues of the protein at physiological pH (pH = 7.4). Then, the protein was solvated with the TIP3P water and neutralized with Na+ or Cl− ions. Additional Na+/Cl− ion pairs were added to the system in order to reach the 0.15 M concentration of NaCl, which mimicked the physiological environment at pH = 7.4. The atomic force field parameters for residues and lipid were adopted from the ff12SB force field and Lipid 1126 of the AMBER 12,25 respectively. The atomic charges for the cholesterol cofactor and dopamine in the dDAT complex were the restrained electrostatic potential (RESP)-fitted charges based on the first-principles electronic structure calculations at the B3LYP/6-31G* level by using the Gaussian03 program.27 Next, the constructed system was subjected to multiple steps of energy minimization by using the AMBER 12 program package. Energy Minimization. The energy minimization was performed first for 100 000 steps by using the steepest descent method and then 100 000 steps by using the conjugated gradient method implemented in the PMEMD module of the AMBER 12 program package.25 In order to effectively avoid the steric hindrance between the protein atoms and the atoms of the protein environment, additional force with a force constant of 50 kcal/mol were applied as constraints first to all atoms of protein while the rest solvent environment molecules were energy minimized. Here, for convenience, the protein environment was defined as all solvent water molecules, Na+ ions, Cl− ions, and
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CONCLUSION All of the molecular modeling and MD simulations have consistently demonstrated that the binding mode of dopamine in hDAT is remarkably different from that in dDAT concerning the orientation of dopamine in the binding pocket. Specifically, according to the computational data, dopamine took an ∼180° flipped orientation in hDAT compared to that in dDAT, and D
DOI: 10.1021/acschemneuro.8b00030 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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ACS Chemical Neuroscience lipid molecules. The constraints were then applied only to the atoms of protein, while the atomic positions of the protein environment were energy-minimized. In the third step, the constraints were applied to the atoms of protein backbone, while the atoms of protein side chains and the atoms of the protein environment were energy-minimized. Finally, the whole system was energy-minimized without any constraints. Molecular Dynamics Simulation. Each simulation system was gradually heated to a temperature (T) of 310 K by applying Langevin dynamics, and was equilibrated for 50 ns. As long-time simulation was needed for the equilibration of lipids, the backbone atoms of protein were fixed in the first 30 ns of the equilibration stage. Then all of the restraints were removed in the remaining 20 ns of equilibration. During the MD simulations, a 10.0 Å nonbonded interaction cutoff and 2.0 Å nonbonded list updating cutoff were used. The motion for the mass center of the system was removed every 1,000 steps. The particle-mesh Ewald (PME) method28,29 was used to treat long-range electrostatic interactions. The lengths of covalent bonds involving a hydrogen atom were fixed with the SHAKE algorithm,30 enabling the use of a 2 fs time step to numerically integrate the equations of motion. The production MD simulation was kept running for 20 ns with a periodic boundary condition in the NTP (constant temperature and pressure) ensemble at T = 310 K. Modeling of Mutated hDAT and dDAT. Residue Mutation. The mutagenesis function of the PyMol software31 was used to perform mutations on the structure of wild-type hDAT and dDAT based on their equilibrated MD models. To avoid the possible collision between solvent molecules and mutated residues, the solvent molecules in the equilibrated model were removed before the operation of residue mutations. As a negatively charged residue (aspartic acid) was involved in the mutation which would affect the electric neutrality of the system, all ions were also removed in this step. Dopamine in the binding pocket of dDAT or hDAT was also removed to make room for the mutations. That is, only the protein, cholesterol cofactor, and lipid molecules were retained in the structure prepared for the mutation operation. The initial conformation of mutated residues was selected as the most relaxed one among all candidate sidechain conformations based on the backbone-dependent rotamers library.32 Simulation System Preparation. Dopamine molecule was docked into the binding site of mutated hDAT or dDAT (see below for the molecular docking procedure). Then the triple-mutated hDAT or dDAT protein−ligand-lipid system was solvated with the TIP3P water and neutralized with counterions. Additional Na+/Cl− ion pairs were added to the system in order to reach the 0.15 M concentration of NaCl, which mimicked the physiological environment at pH = 7.4. Energy Minimization and Molecular Dynamics Simulation. The energy minimization and MD simulation steps performed for the triple-mutated dDAT and hDAT proteins were similar to those carried out for wild-type systems mentioned above. A slight variation was that 30 ns equilibration for lipids molecules was skipped in the equilibration stage, as lipids molecules in the triple-mutated protein (hDAT or dDAT) were transferred from an equilibrated system with the wild-type protein (hDAT or dDAT). Molecular Docking of Dopamine. In order to explore the binding mode of hDAT with its substrate dopamine (DA), molecular docking was performed by using the AutoDock 4.2 program33 based on the MD-simulated hDAT structure. As observed from the X-ray crystal structures of the NSS members and indicated in our previous reports,21−24,34 the amino-group of DA should have hydrogen bonding with the carboxyl group of residue D79 of hDAT (corresponding to D46 of dDAT), and the aromatic ring of DA should stay with hydrophobic residues in the binding site. Since most of the residues around the DA-binding site were conserved in hDAT or dDAT, the binding conformation of dopamine in hDAT was expected to be similar to the conformation of dopamine in the X-ray crystal structure of DA-dDAT complex.24 During the process of molecular docking, Lamarckian genetic algorithm was applied to treatment of the intermolecular interactions between hDAT and DA. The number of the docking runs was set to 200, and the number of individuals for
each run was set to 300. The maximum number of energy evaluations was set to 25 000 000, and 300 000 was chosen as the maximum number of generations. The size of grid box was 80 × 80 × 80 points along each axis, and the grid spacing was 0.2 Å. All of the conformational candidates generated from docking operations were evaluated and ranked in terms of binding free energies by using the standard scoring function implemented in the AutoDock 4.2 program. The best conformation/orientation of DA binding was selected from the largest docking clusters based on the lowest binding free energy (i.e., the best energy score from the AutoDock 4.2 scoring function) along with good geometric matching and ability of hydrogen bonding with the surrounding residues in the binding site of hDAT. The docked binding structure was used as the initial structure for further energy minimization and MD simulations as noted above.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.8b00030. Wild-type dDAT-dopamine binding structure, showing S426 and other key residues (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Phone: 859-323-3943. Fax: 859-257-7585. E-mail: zhan@uky. edu. ORCID
Chang-Guo Zhan: 0000-0002-4128-7269 Author Contributions
Y.Y. performed the modeling studies and drafted the manuscript. C.-G.Z. designed the research project with contribution from J.Z., and C.-G.Z. finalized the manuscript. Funding
This work was supported in part by the NIH (Grants R01 DA035714, R01 DA035552, R01 DA032910, and R01 DA025100) and the NSF (Grant CHE-1111761). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge the Computer Center at the University of Kentucky for supercomputing time on a Dell Supercomputer Cluster consisting of 388 nodes or 4816 processors.
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DOI: 10.1021/acschemneuro.8b00030 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
