Binding Mode of Human Norepinephrine Transporter Interacting with HIV-1 Tat

Abstract

The increase of HIV infection in macrophages results in HIV proteins being released, like HIV Tat which impairs the function of monoamine transporters. HIV-infected patients have displayed increased synaptic levels of dopamine (DA) due to reduced binding and function of monoamine transporters such as the norepinephrine transporter (NET) and the dopamine transporter (DAT). Development of a three-dimensional model of the HIV-1 Tat-human NET (hNET) binding complex would help reveal how HIV-1 Tat causes toxicity in the neuron by affecting DA uptake. Here we use computational techniques such as molecular modeling to study microscopic properties and molecular dynamics of the HIV-1 Tat-hNET binding. These modeling techniques allow us to analyze noncovalent interactions and observe residue-residue contacts to verify a model structure. The modeling results studied here show that HIV-1 Tat-hNET binding is highly dynamic and that HIV-1 Tat preferentially binds to hNET in its outward-open state. In particular, HIV-1 Tat forms hydrogen bond interactions with side chains of hNET residues Y84, K88, and T544. The favorable hydrogen bonding interactions of HIV-1 Tat with the hNET side chain residues Y84 and T544 have been validated by our subsequently performed DA uptake activity assays and site-directed mutagenesis, suggesting that the modeled HIV-1 Tat-hNET binding mode is reasonable. These mechanistic and structural insights gained through homology models discussed in this study are expected to encourage the pursuit of pharmacological and biochemical studies on HIV-1 Tat interacting with hNET mechanisms and detailed structures.

Keywords: Molecular docking; dopamine; molecular dynamics; norepinephrine transporter; protein−protein interaction; trans-activator of transcription.

Figure 1.

Modeled structure of HIV-1 Tat bound to hNET in the outward-open state after the molecular docking and energy minimization. (A) HIV-1 Tat–hNET binding structure developed from homology modeling. HIV-1 Tat is (orange) shown here with the cartoon representation, hNET (green) is represented using the cartoon representation. The lipid membrane is shown using semitransparent surface and stick representation colored according to heavy atoms. (B) HIV-1 Tat (orange) and hNET (green) are shown here with the surface representation showing the shape complementarity. (C) Highlighting HIV-1 Tat bound to hNET with critical residues important for maintaining the formed complex. HIV-1 Tat is (orange) shown here with the cartoon representation. Dopamine (red), residues Y84, Y88, and T544 of hNET (purple) are all shown in surface representation. (D) Internuclear distances are shown between the binding interface of HIV-1 Tat–hNET binding structure. HIV-1 Tat (orange) and hNET (green) are represented in the cartoon representation. Residues Y84, Y88, and T544 of hNET (cyan) as well as P18, K19, and K40 of Tat (purple) shown as sticks which are important for maintaining the binding between the two proteins. Intermolecular HBs are indicated as dashed lines, and distances are labeled next to the respected lines.

Figure 2.

RMSD and critical distances for the HIV-1 Tat–hNET binding structure based on the MD production simulations. (A) RMSD values (black) for the Tat–hNET binding structures backbone heavy atoms (Cɑ, C, O, N) in the 2.0 μs MD trajectories. (B) Critical distance (red) of the interaction between the nitrogen of hNET K88 side chain and the backbone oxygen of HIV-1 Tat P18. (C) Critical distance (blue) of the interaction between the side chain oxygen of hNET Y84 and side-chain nitrogen of HIV-1 Tat K19. (D) Critical distance (green) of the interaction between the oxygen of hNET T544 side chain and the nitrogen of HIV-1 Tat K40 side chain.

Figure 3.

Critical interactions shown between important residues and mutated residues. Mutants of hNET were modeled from the WT-hNET obtained from the HIV-1 Tat–hNET binding structure followed by energy minimizations. (A) Distances are shown between the side chain oxygen of hNET T544 (cyan) and side chain nitrogen of HIV-1 Tat K40 (purple) as well as the side chain oxygen of hNET Y84 (cyan) and side chain nitrogen of HIV-1 Tat K19 (purple). (B) Y84F mutation shows the disrupted interaction between HIV-1 Tat residue K19 (purple) and hNET residue F84 (cyan) due to a loss of a hydroxyl group from hNET residue Y84. (C) T544A mutation shows the disrupted interaction between HIV-1 Tat residue K40 (purple) and hNET residue A544 (cyan) due to a loss of a hydroxyl group from hNET residue T544.