Journal ArticleConformational free energy landscape of a glutamate transporter and microscopic details of its transport mechanismThangapandian, Sundar; Fakharzadeh, Ashkan; Moradi, Mahmoud; Tajkhorshid, Emad<p>Removing glutamate from the synaptic cleft is vital for proper function of the brain. Excitatory amino acid transporters mediate this process by uptaking the neurotransmitter from the synaptic cleft back to the cell after its release. The archaeal homolog, Glt_Ph, an aspartate transporter from<italic>Pyrococcus horikoshii</italic>, presents the best structurally characterized model for this family of transporters. In order to transport, Glt_Phundergoes elevator-like conformational changes between inward-facing (IF) and outward-facing (OF) states. Here, we characterize, at an atomic level, the OF⇌IF transition of Glt_Phin different<italic>apo</italic>/bound states using a combination of ensemble-based enhanced sampling techniques, employing more than two thousand of coupled simulation replicas of membrane-embedded Glt_Ph. The resulting free-energy profiles portray the transition of<italic>apo</italic>/bound states as a complex four-stage process, while sodium binding alone locks the structure in one of its states. Along the transition, the transport domain (TD) disengages from the scaffold domain (SD), allowing it to move as a piston sliding vertically with respect to the membrane during the elevator-like motion of TD. Lipid interactions with residues comprising the SD–TD interface directly influence the large-scale conformational changes and, consequently, the energetics of transport. Structural intermediates formed during the transition leak water molecules and may correlate to the uncoupled Cl⁻ion conductance observed experimentally in both prokaryotic and mammalian glutamate transporters. Mechanistic insights obtained from our study provide a structural framework for better development of therapeutic for neurological disorders.</p>National Academy of Sciences2025-03-1110600653Proceedings of the National Academy of Sciences122100027-8424https://doi.org/10.1073/pnas.24163811221945465National Science Foundation