Substrate binding and formation of an occluded state in the leucine transporter

Translocation through the extracellular vestibule and binding of leucine in the leucine transporter (LeuT) have been studied with molecular dynamics simulations. More than 0.1 mu s of all-atom molecular dynamics simulations have been performed on different combinations of LeuT, bound substrate, and bound structural Na+ ions to describe molecular events involved in substrate binding and in the formation of the occluded state and to investigate the dynamics of this state. Three structural features are found to be directly involved in the initial steps of leucine transport: a Na+ ion directly coordinated to leucine (Na-1), two aromatic residues closing the binding site toward the extracellular vestibule (Tyr-108 and Phe-253), and a salt bridge in the extracellular vestibule (Arg-30 and Asp-404). These features account for observed differences between simulations of LeuT with and without bound substrate and for a possible pathway for leucine binding and thereby formation of the occluded LeuT binding site.