Properties of an Inward-Facing State of LeuT: Conformational Stability and Substrate Release

Abstract

The leucine transporter (LeuT) is a bacterial homolog of the human monoamine transporters, which are important pharmaceutical targets. There are no high-resolution structures of the human transporters available; however, LeuT has been crystallized in several different conformational states. Recently, an inward-facing conformation of LeuT was solved revealing an unexpectedly large movement of transmembrane helix 1a (TM1a). We have performed molecular dynamics simulations of the mutated and wild-type transporter, with and without the cocrystallized Fab antibody fragment, to investigate the properties of this inward-facing conformation in relation to transport by LeuT within the membrane environment. In all of the simulations, local conformational changes with respect to the crystal structure are consistently observed, especially in TM1a. Umbrella sampling revealed a soft potential for TM1a tilting. Furthermore, simulations of inward-facing LeuT with Na(+) ions and substrate bound suggest that one of the Na(+) ion binding sites is fully disrupted. Release of alanine and the second Na(+) ion is also observed, giving insight into the final stage of the translocation process in atomistic detail.

Energy landscape of LeuT from molecular simulations

“…4), whereas that of the crystal structure was ∼46 • . These results are in accord with previous MD studies 28,41 of several hundreds of ns of simulations for the apo and holo (Ala/2Na + -bound) K288A LeuT, showing slightly smaller TM1a openings than the crystal structure. The smaller TM1a opening in our simulations also relates to the reorientation angle of TM1a, which differs from the one observed in the crystal structure; it opens rather toward TM7 which prevents it from lifting up into the membrane (Fig.…”

“…Many computational studies 28,41,42 have focused on the dynamics of the K288A LeuT mutant instead of WT LeuT, mainly because this mutation has been shown to enhance substrate flux in proteoliposomes. 43 K288 is located on TM7 facing the membrane bilayer and distant from the LeuT central binding cavity.…”

Two Na+ Sites Control Conformational Change in a Neurotransmitter Transporter Homolog

“…TM1 is shown in two positions, the occluded state in red (6) and in an inward-open conformation in transparent salmon (47). The separation between TMs 1 and 8 found in the inward-open LeuT structure (PDB code 3TT3) (9) is even larger than shown here for an inward-open model, possibly reflecting the detergent/lipid environment in the crystal (21). H͔leucine concentrations 10% of the measured K D for each mutant.…”

“…Na2 is formed by transmembrane helices 1 and 8 (TM1 and TM8), which are close enough to form a coordination shell for Na ϩ in outward-facing (occluded and open) LeuT structures (6,7,9) but not in the inward-open structure where the two helices have separated to open the cytoplasmic permeation pathway (Fig. 1B) (9,21,22). This observation suggests that Na ϩ binding to Na2 may foster interaction between TMs 1 and 8 that stabilizes LeuT in outward-open conformations (9,16).…”

Conformational dynamics of a neurotransmitter:sodium symporter in a lipid bilayer

“…We therefore hypothesized that interpretation of these data could be aided by comparison of ensembles of structures generated with MD simulations (43,44) starting with X-ray crystallographic structures of different conformations of LeuT. The outward-and inward-open conformational ensembles of LeuT were mimicked in silico using MD simulations of the following, respectively: the substrate-free, sodiumbound, outward-open structure [Protein Data Bank (PDB) ID code 3TT1] after reversing the Y108F mutation (12); and the substratefree, inward-open, structure (PDB ID code 3TT3) after reversing the modifications at the Na2 site (T354A and S355A) and TM7 (K288A) (10), but maintaining the intracellular gate mutant Y268A. In each case, the protein was embedded in a hydrated lipid bilayer.…”