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  • Hazel Teague posted an update 1 year, 4 months ago

    We have performed free energy simulations for the Asp transporter GltPh, a homolog of the mammalian glutamate transporters (EAATs). We have investigated in detail the coordination of all the ligands and calculated their binding free energies. The coordination shells found for Asp and Na1 in the closed state are in good agreement with the crystal structure. A small displacement of the experimental Na2 site is observed, which results in replacement of the S349 carbonyl in the coordination shell with the T308 hydroxyl. The new Na2 site is shown to be more stable and also more consistent with the gate-locking function of Na2. Our simulations show that Asp is firmly bound to the open structure in the presence of Na3 and Na1 but becomes unstable when Na1 is removed, unbinding in <5?ns in all three monomers. A close scrutiny of the unbinding Dorsomorphin process has revealed that Na1 and Asp are coupled via a hydrogen-bond network, and removal of Na1 leads to the unzipping of this network destabilizing Asp. The binding sequence of the ligands is determined buy BAY 73-4506 from their binding free energies as: Na3, Na1, Asp (HP2 gate closes), and Na2. The binding order of the second Na+ ion and Asp has not been resolved from experimental observations and previous MD simulations (17). Our results clearly show that Asp binding is contingent upon the presence of Na1, and this happens after the LY2157299 solubility dmso binding of the second Na+ ion. Finally, our MD simulations with bound Glu and FEP calculations for transforming Asp�� Glu in the open and closed states provide a simple mechanistic explanation for why GltPh transports Asp but not Glu��the binding pocket is too small to accommodate the larger Glu, which remains in a higher energy state compared to Asp both in the open and closed states. The insights provided for Asp/Glu selectivity here will provide valuable guidance in constructing homology models for EAATs based on the GltPh structure. Calculations were performed using the High-Performance Computing facilities at the National Computational Infrastructure (Canberra) and ULAKBIM (Ankara). We thank Rob Vandenberg and Renae Ryan for discussions on the structure and function of GltPh. This work was supported by grants from the Australian Research Council and Turkish Scientific and Technical Research Council. ””NMDA receptors are a subtype of glutamate-activated ion channels that mediate rapid excitatory neurotransmission in the mammalian central nervous system. NMDA receptors are involved in a number of higher order brain functions including learning and memory as well as neurodevelopment (1). Aberrant activity of NMDA receptors can trigger and/or complicate both acute (e.g., stroke and seizure) and chronic (e.g., Parkinson’s and Alzheimer’s diseases) neurological and psychiatric (e.g.