Molecular Mechanism of HIV-1 Tat Interacting with Human Dopamine Transporter

Abstract

Nearly 70% of HIV-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 evidences 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 in 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 DA 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-associated neurocognitive disorders.

Molecular mechanism: the human dopamine transporter histidine 547 regulates basal and HIV-1 Tat protein-inhibited dopamine transport

“…The hydrogen bond with the H547 side chain is expected to be broken with the H547A mutation, which is consistent with decreased inhibitory activity of Tat on hDAT-H547A. Based on the computational model proposed in our previous work36, the Y548-Y470-Y551 interaction motif (denoted as the YYY motif) (Fig. 1C) could stabilize the first part of transmembrane helix 10 (TM10a) by tying down residue Y470 with extracellular loop 6.…”

“…Based on the constructed hDAT-Tat binding model in our previous work36, both the side chain and backbone of H547 forms a hydrogen bond with residue R49 of HIV-Tat (Fig. 1B).…”

“…1C) could stabilize the first part of transmembrane helix 10 (TM10a) by tying down residue Y470 with extracellular loop 6. Instability of TM10a could promote the formation of a salt bridge between D476 (in TM10a) and R85 (in TM1b); however, the D476-R85 salt bridge would close the “entrance gate” of DA353644454647. As a result, instability of TM10a is expected to block the DA entrance in the outward-open state, which may decrease the DA uptake efficiency of hDAT.…”

“…The H547P, H547A, H547D, and H547R single mutations were tested because proline and alanine mutations are expected to be the most significant change in backbone conformation, while aspartic acid or arginine mutation is expected to introduce significant side-chain conformational disturbances due to their non-neutral electrostatic potentials. According to the molecular dynamics (MD) simulation and the potential of main force (PMF) energy calculation performed in our previous work36, we found that the H547A mutation could enhance the strength of the YYY motif. On the contrary, the H547P mutation could weaken the strength of the YYY motif, which is consistent with the increased DA uptake efficiency for the H547A mutant and decreased DA uptake efficiency for the H547P mutant.…”

“…Through integrated computational modeling prediction and experimental validation, we have identified key residues in hDAT with which Tat interacts, which are critical for Tat-induced inhibition of DAT and transporter conformational transitions353637. Our studies provide mechanistic insights into identifying residues on DAT for Tat binding, which allows the exploration of the molecular targets on DAT for therapeutic interventions, improving neurocognitive function of HAND.…”