How dopamine transporter interacts with dopamine: insights from molecular modeling and simulation

Abstract

By performing homology modeling, molecular docking, and molecular dynamics simulations, we have developed three-dimensional (3D) structural models of both dopamine transporter and dopamine transporter-dopamine complex in the environment of lipid bilayer and solvent water. According to the simulated structure of dopamine transporter-dopamine complex, dopamine was orientated in a hydrophobic pocket at the midpoint of the membrane. The modeled 3D structures provide some detailed structural and mechanistic insights concerning how dopamine transporter (DAT) interacts with dopamine at atomic level, extending our mechanistic understanding of the dopamine reuptake with the help of Na(+) ions. The general features of the modeled 3D structures are consistent with available experimental data. Based on the modeled structures, our calculated binding free energy (DeltaG(bind) = -6.4 kcal/mol) for dopamine binding with DAT is also reasonably close to the experimentally derived DeltaG(bind) value of -7.4 kcal/mol. Finally, a possible dopamine-entry pathway, which involves formation and breaking of the salt bridge between side chains of Arg(85) and Asp(476), is proposed based on the results obtained from the modeling and molecular dynamics simulation. The new structural and mechanistic insights obtained from this computational study are expected to stimulate future, further biochemical and pharmacological studies on the detailed structures and mechanisms of DAT and other homologous transporters.

FIGURE 1

Sequence alignment of human DAT with the bacterial homolog of Na⁺/Cl⁻-dependent neurotransmitter transporters (LeuT_Aa) by manual adjustment. Twelve helices are labeled above the sequence, and strictly conserved residues among the NSS family are shown in bold.

FIGURE 2

Initial structural model of human DAT in the physiological environment used for MD simulations. (A) DAT protein is represented as ribbon in red, lipid molecules in gray, and water molecules in green. Labeled also are the system sizes along x, y, and z axes. (B) Top view of DAT protein itself shown as ribbon in red, and Na⁺ ions as CPK in magenta. The Na⁺ ions, extracellular loop 2 (EL2), transmembrane helix 12 (TM12), and Nter are labeled. (C) Side view of DAT protein.

FIGURE 3

Plots of the Cα RMSD and key distances in the simulated DAT and DAT-dopamine structures versus the simulation time (nanoseconds) during the MD simulations. (A) The DAT Cα RMSD, the Cα RMSD of the DAT-dopamine complex, and the RMSD of dopamine (DA) in the complex. (B) The minimum-distance changes between the positively charged side-chain atoms (NE, NH1, and NH2) of Arg⁸⁵ and the negatively charged side-chain atoms (OD1 and OD2) of Asp⁴⁷⁶ in both the DAT and DAT-dopamine complex.

FIGURE 4

Representative local structures of DAT surrounding the Na⁺ ions (i.e., Na1 and Na2) captured at 1.50 ns snapshot of the MD simulations on DAT (A for Na1 and B for Na2) and DAT-dopamine complex (C for Na1 and D for Na2). In all cases, Na⁺ ions are shown in CPK style. The coordinating residues are shown in stick, and the protein in ribbon, representation. The coordinating distances are labeled.

FIGURE 5

Typical structure of the DAT-dopamine binding complex, which was the 1.50 ns snapshot of the MD trajectory. (A) Viewing the dopamine molecule (shown as ball-and-stick) in the complex model from the extracellular side. Only part of the DAT is shown as ribbon in red, and Na2 as CPK in magenta. Helices 1, 6, 8, 10, and 12 are labeled to indicate the relative position of dopamine in DAT. (B) Viewing the DAT in the binding pocket in the same orientation as panel A. The binding pocket is represented in molecular surface format, colored with electrostatic potentials in which blue is for positive and red is for negative potentials.

FIGURE 6

(A) Distances from the atoms (the nitrogen and hydroxyl oxygen atoms) of dopamine to the carbonyl oxygen of Phe⁷⁶, side chain CG of Asp⁷⁹, Cα of Phe³²⁰, carbonyl oxygen of Ser⁴²², and carbonyl oxygen of Gly⁴²⁵. (B) Distances from the cationic head of dopamine to the bound Na⁺ ions as observed during the MD simulation of the DAT-dopamine complex.

FIGURE 7

Representative molecular interactions between dopamine and DAT, taken at the 1.50-ns snapshot of the MD-simulated DAT-dopamine complex. Residues from DAT within 5 Å of dopamine are labeled and shown in stick style, while dopamine is shown in ball-and-stick. Critical hydrogen-bonding interactions between dopamine and DAT are represented as dash lines with labeled distances, also labeled the bound Na⁺ ions (Na1 and Na2).

FIGURE 8

Side view of the proposed dopamine-entry pathway in DAT with TM12 parallel to the normal of the membrane. The protein is shown as ribbon with helix 1 in green, helix 3 in cyan, helix 6 in blue, helix 8 in magenta, and other helices in red. With helices 3 and 8 in front, the back wall of the pathway is represented by molecular surface in gray, and residues 220–241 are not shown for clear view of the pathway back wall. The relative position of the membrane bilayer is indicated. The proposed substrate-entry pathway starts from the extracellular side as indicated by the large green arrow, going down inside the tunnel along the dashed green arrow to the binding pocket. The side chains of residues on the way of the tunnel to the binding pocket are shown in stick representation and labeled in blue. These are the salt-bridge pair of Arg⁸⁵ and Asp⁴⁷⁶, aromatic residues as Phe¹⁵⁵, Ty¹⁵⁶, Phe³²⁰, and Phe³²⁶. Dopamine is labeled as DA and the second Na⁺ as Na2 in blue.