Modeling of the Inward-Facing State of LeuT and Dynamics of the Outward-To-Inward Transition

Abstract

Leucine transporter (LeuT) is a bacterial aminoacid transporter belonging to the neurotransmitter:sodium symporter family. An ‘alternating-access model’is proposed for LeuT function, wherein the transporter alternates between outward-facing and inward-facing states. While multiple crystal structures of LeuT bound with substrate and inhibitors have been reported, only the outward-facing state is known. The inward facing state, and the dynamics of the outward-to-inward transition remain undescribed.Several transporters from different families report a structural fold similar to the basic ‘LeuT fold’indicating the significance of this fold in transporter function. These structures include some in the inward-facing state. Exploiting this information, we have generated a model for the inward-facing state of LeuT.Since the inward-facing structure employed for modeling had very low sequence similarity, and moderate structural similarity to LeuT, a combination of several techniques was required for model generation. A detailed modeling approach was adopted, including sequence- and structure-based approaches, combined with molecular dynamics techniques, such as targeted MD. The final model retains the secondary structural features of LeuT and the substrate/ion binding sites, while adopting an inward-facing state.We have also employed this model to study the dynamics of outward-to-inward transition of LeuT. The behavior of bound substrate and ions during this transition was recorded and shows interesting features relevant to the transport mechanism. Water permeation was monitored with the progress in transition. The main structural elements of LeuT involved in this transition are described. This study thus presents a model of the inward-facing state of LeuT and a description of a possibly general transport mechanism in transporters adopting the LeuT fold.