Conformational Stability and Substrate Translocation - A Computational Study of the Leucine Transporter

Abstract

The human monoamine transporters are involved in a variety of disorders and thus important medicinal targets.1 There are no available high-resolution structures of the transporters. The leucine transporter (LeuT) is a bacterial homolog of the human monoamine transporters with a sequence similarity of 20-24 %.2 LeuT has been crystallized with a number of different ligands and in several different conformations. Most recently, an inward facing conformation was solved.3 The crystallization process required the use of four point mutations as well as the association of an antibody. We have applied computational methods to investigate whether the conformation of this manipulated protein is relevant for the wild-type transporter in a membrane environment. We have performed several molecular dynamics simulations of both the mutated and wild-type transporter, and analyzed the stability through measurements of helix tilt angles. The simulations reveal that the mutant and wild-type systems types behave similarly and both maintain a relatively stable inward facing conformation. The crystal structure of LeuT in an inward facing conformation does not contain any ligands or ions. To investigate the release of substrate and ions from the transporter, we have also performed simulations of wild-type LeuT in the inward facing conformation with sodium and either leucine or alanine bound. From this, we can conclude that one of the two sodium binding sites is fully disrupted in this conformation leading to the release of this ion in almost all of the simulations. Furthermore, in one of the simulations we observe release of alanine along with the second sodium ion, and are thus able to describe the translocation process in atomic detail.1. Br J Pharmacol (2012) 167, 256-278.2. Mol Pharmacol (2006) 70, 1630-1642.3. Nature (2012) 481, 469-474