A steered molecular dynamics study of binding and translocation processes in the GABA transporter.
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.
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Søren
S
Skovstrup
Laurent
L
David
Olivier
O
Taboureau
Flemming Steen
FS
Jørgensen
D000927:Anticonvulsants / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites; D003593:Cytoplasm / Q000378:metabolism; D050485:GABA Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008968:Molecular Conformation; D056004:Molecular Dynamics Simulation; D009557:Nipecotic Acids / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D000078308:Tiagabine; D005680:gamma-Aminobutyric Acid / Q000737:chemistry
D000927:Anticonvulsants; D050485:GABA Plasma Membrane Transport Proteins; D008024:Ligands; D009557:Nipecotic Acids; C030278:nipecotic acid; D005680:gamma-Aminobutyric Acid; D000078308:Tiagabine