Abstract
The entire substrate translocation pathway in the human GABA transporter (GAT-1) was explored for the endogenous substrate GABA and the anti-convulsive drug tiagabine. Following a steered molecular dynamics (SMD) approach, in which a harmonic restraining potential is applied to the ligand, dissociation and re-association of ligands were simulated revealing events leading to substrate (GABA) translocation and inhibitor (tiagabine) mechanism of action. We succeeded in turning the transporter from the outward facing occluded to the open-to-out conformation, and also to reorient the transporter to the open-to-in conformation. The simulations are validated by literature data and provide a substrate pathway fingerprint in terms of which, how, and in which sequence specific residues are interacted with. They reveal the essential functional roles of specific residues, e.g. the role of charged residues in the extracellular vestibule including two lysines (K76 (TM1) and K448 (TM10)) and a TM6-triad (D281, E283, and D287) in attracting and relocating substrates towards the secondary/interim substrate-binding site (S2). Likewise, E101 is highlighted as essential for the relocation of the substrate from the primary substrate-binding site (S1) towards the cytoplasm.
Highlights
The anti-convulsive agent tiagabine is the only approved drug that works by inhibiting the gamma-aminobutyric acid (GABA) transporters (GATs) [1], namely GABA transporter subtype 1 (GAT-1)
Key details of the simulation are collected in the left-hand side of Figure 2 in terms of I) traces of the center of mass (COM) of GABA, II) evolvements of the biasing potential energy, III) non-bonded interactions between GABA and the protein, water, sodium and chloride ions, IV-VII) non-bonded interactions between GABA and residues interacted with along the translocation pathway
We have presented steered molecular dynamics (SMD) simulations covering the entire substrate translocation pathway in GAT-1, and we have rationalized the association of the drug tiagabine inhibiting GAT1
Summary
The anti-convulsive agent tiagabine is the only approved drug that works by inhibiting the gamma-aminobutyric acid (GABA) transporters (GATs) [1], namely GAT-1. The structure revealed a so-called outward-facing occluded state, with the primary substrate binding site (S1) being occluded, shielded from the extracellular solute by two gating residues, Y108 and F253, and on top of these a water-mediated salt bridge formed by R30 and D404. Released crystal structures of LeuT reported outward-facing occluded conformations in complex with various ligands, and a single crystal structure revealed an open-to-out conformation with a small-molecule inhibitor, L-tryptophan, occupying the S1 binding site [6,7,8,9,10]. Based on these crystal structures, diverse computational simulations were performed to explore the translocation of LeuT [4,11,12,13,14]
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