Molecular mechanism of HIV-1 Tat interacting with human dopamine transporter

Abstract

Nearly 70% of HIV-1-infected individuals suffer from HIV-associated neurocognitive disorders (HAND). HIV-1 transactivator of transcription (Tat) protein is known to synergize with abused drugs and exacerbate the progression of central nervous system (CNS) pathology. Cumulative evidence suggest that the HIV-1 Tat protein exerts the neurotoxicity through interaction with human dopamine transporter (hDAT) in the CNS. Through computational modeling and molecular dynamics (MD) simulations, we develop a three-dimensional (3D) structural model for HIV-1 Tat binding with hDAT. The model provides novel mechanistic insights concerning how HIV-1 Tat interacts with hDAT and inhibits dopamine uptake by hDAT. In particular, according to the computational modeling, Tat binds most favorably with the outward-open state of hDAT. Residues Y88, K92, and Y470 of hDAT are predicted to be key residues involved in the interaction between hDAT and Tat. The roles of these hDAT residues in the interaction with Tat are validated by experimental tests through site-directed mutagensis and dopamine uptake assays. The agreement between the computational and experimental data suggests that the computationally predicted hDAT-Tat binding mode and mechanistic insights are reasonable and provide a new starting point to design further pharmacological studies on the molecular mechanism of HIV-1-associated neurocognitive disorders.

Keywords: Transactivator of transcription; dopamine uptake; neurotoxicity; protein−protein interaction; viral protein.

Figure 1

The simulated process of binding between HIV-1 Tat and hDAT in the outward-open state. (A) The HIV-1 Tat approaches hDAT through long-range electrostatic attractions, as indicated by the electrostatic potential contour maps for these two proteins. Isopotential surfaces are calculated at +4kBT/e for positively charged surface (shown as blue mesh) and at −3kBT/e for negatively charged surface (shown as red mesh) respectively. Colored arrows indicate the directions of the dipoles of both molecules. (B) The general process of two proteins approaching toward each other and form the initial encounter complex. Both proteins are represented as the surface style, with HIV-1 Tat in gold and hDAT in cyan. The black arrow indicates the HIV-1 Tat aligning to the vestibule near the extracellular side of hDAT. (C) Typical hDAT-Tat binding structure derived from the last snapshot of the MD trajectory #1 followed by the energy minimization. Tat is represented as gold ribbons. hDAT is represented by semi-transparent cyan surface. Substrate dopamine, cholesterol, sodium ions, chloride ion, and zinc ion are represented by the sphere style and colored in red, purple, blue, green, and yellow, respectively. The contact interface of hDAT between Tat and hDAT is colored in pink. It was observed that both TM10 and TM1 are involved in the interaction between Tat and hDAT. (D) Atomic interactions on the binding interface of the typical hDAT-Tat binding structure (as shown in C). HIV-1 Tat protein is shown as ribbon and colored in gold, and hDAT is shown as cyan ribbon. Residues T-M1, T-P18, and T-K19 of HIV-1 Tat are shown in ball-stick style and colored in yellow. Residues D-Y470, D-Y88, and D-K92 are shown in ball-stick style and colored in green. Dashed lines represent intermolecular hydrogen bonds with labeled distances. For D-Y470, the red point indicates the center of its aromatic ring, and the dashed line pointing to the red ball represents the cation-π interaction with labeled distance.

Figure 2

Tracked changes of critical distances and positional RMSD values for the hDAT-Tat binding structure based on the 10 MD trajectories (10 ns for each MD trajectory). Green curve refers to the distance between the positively charged amino group of T-M1 of HIV-1 Tat and the center of the aromatic ring at D-Y470 side chain. Blue curve represents the distance between the hydrogen atom on the positively charged head of T-K19 side chain and the hydroxyl oxygen atom of D-Y88 side chain, and the red curve refers to the distance between the backbone oxygen atom of T-P18 and the hydrogen atom on the positively charged head of D-K92 side chain. The black curve refers to the positional RMSD for the backbone atoms of the hDAT-Tat binding complex.

Figure 3

Interactions between the positively charged amino group of T-M1 and the side chain of residue #470 of hDAT in the modeled complexes of HIV-1 Tat binding with WT-hDAT and its mutants. Structures of the Y470F, Y470H, and Y470A mutants of hDAT were modeled starting from that of WT-hDAT in the hDAT-Tat binding structure shown in Figure 1C and 1D by changing the Y470 side chain into the corresponding one of the mutant and then performing the energy minimization. (A) Residues T-M1 and D-Y470 are represented as sticks and colored by atom types. The red point represents the center of aromatic ring of residue #470 of hDAT, and the dashed line pointing to the red point represents the cation-π interaction with the distance labeled. (B) Y470F mutation retains the cation-π interaction between T-M1 and D-F470. The distance between the center of aromatic side chain of D-F470 and the nitrogen atom of the positively charged amino group of T-M1 is indicated in Å. (C) Residue D-H470 stays far away from the positively charged amino group of T-M1, i.e. no direct cation-π interaction. (D) Residue D-A470 stays further away from the positively charged amino group of T-M1, i.e. no direct cation-π interaction.

Figure 4

Effects of Tat on the kinetic analysis of [³H]DA uptake by WT-hDAT and its mutants. (A) PC12 cells transfected with WT-hDAT (WT) Y470F-hDAT (Y470F), Y470H-hDAT (Y470H), Y470A-hDAT (Y470A), Y88F-hDAT (Y88F) or K92M-hDAT (K92M) were preincubated with or without recombinant Tat_1-86 (500 nM, final concentration) at room temperature for 20 min followed by the addition of 0.05 μM final concentration of the [³H]DA. *p < 0.05 compared to respective control in the absence of Tat (n=4). (B) The corresponding Tat-induced inhibitory effects on [³H]DA uptake in hDAT mutants were presented as the percentage of the Tat-induced inhibitory effect of [³H]DA uptake in WT-hDAT (100%) at the same concentrations of [³H]DA (0.05 μM) and Tat_1-86 (500 nM).