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.

本研究针对人类γ-氨基丁酸转运蛋白（GAT-1）的完整底物转运通路，围绕其内源性底物γ-氨基丁酸（GABA）与抗惊厥药物替加宾（tiagabine）展开探索。本研究采用定向分子动力学（steered molecular dynamics, SMD）方法，通过对配体施加谐波约束势，模拟了配体的解离与重新结合过程，揭示了与底物（GABA）转运及抑制剂（替加宾）作用机制相关的事件。我们成功将该转运蛋白从外向闭合构象转换为外向开放构象，并将其重定向至内向开放构象。本模拟通过已有文献数据完成验证，并提供了底物转运通路的特征指纹，可明确特异性残基的相互作用对象、作用方式与作用顺序。本研究揭示了特定残基的核心功能作用：例如，细胞外前庭区域的带电残基，包括两个赖氨酸残基（K76，TM1；K448，TM10）以及跨膜6区三联残基（D281、E283与D287），可将底物吸引并转运至次级/临时底物结合位点（S2）。同样，E101残基被证实对底物从初级底物结合位点（S1）向细胞质的转运发挥关键作用。