Mechanism of Selective Cation Binding to Sodium-Coupled Transporters: Insights from Free Energy Simulations and QM/MM Simulations

Abstract

Ion-coupled transport of neurotransmitter molecules by secondary amino-acids transporters plays pivotal role in the regulation of neuronal signaling. One of the major events in the transport cycle is ion-substrate coupling and formation of the high-affinity occluded state with bound ions and substrate. Molecular mechanisms of ion-substrate coupling, specificity for a particular cation and the corresponding ion-substrate stoichiometry in secondary transporters has yet to be understood. We have studied Li+/K+/Tl+/Na+ binding and/or selectivity to several transporters with available crystal structures such as the bacterial aspartate transporter GltPh, leucing transporter LeuT and maltose transporter vSGLT using free energy simulations and QM/MM minimization to evaluate the role of different factors in the observed selectivity and ion binding to the protein. Two different mechanisms were found to co-exist for crystallographically characterized binding sites Na1 and Na2 in LeuT and Glt. Furthermore, site Na1 appeared to be well conserved amongst members of different families. To evaluate the role of Na+ binding in the transporter function, we have performed free energy simulations to determine actual cation selectivity as well as binding affinity for sites Na1 and Na2 in the protein. QM/MM minimization was used to characterize the role of the electronic effects of the stabilization of non-native cations such as Li+ and Tl+ in the Na+-selective sites of different transporters. In the case of Tl+ binding to Glt transporter, neighboring residues from a second solvation shell provide a necessary stabilization to the larger cation due to polarization and charge transfer effects implying a rather large flexibility of the metal binding sites.