Free energy simulations of ligand binding to the aspartate transporter Glt(Ph).

Glutamate/Aspartate transporters cotransport three Na(+) and one H(+) ions with the substrate and countertransport one K(+) ion. The binding sites for the substrate and two Na(+) ions have been observed in the crystal structure of the archeal homolog Glt(Ph), while the binding site for the third Na(+) ion has been proposed from computational studies and confirmed by experiments. Here we perform detailed free energy simulations of Glt(Ph), giving a comprehensive characterization of the substrate and ion binding sites, and calculating their binding free energies in various configurations. Our results show unequivocally that the substrate binds after the binding of two Na(+) ions. They also shed light into Asp/Glu selectivity of Glt(Ph), which is not observed in eukaryotic glutamate transporters.