Structure and mechanism of a bacterial homolog of neurotransmitter:sodium symporters

Abstract

Re-uptake of neurotransmitters through membrane-bound transport proteins is the primary mechanism of terminating synaptic transmission in the central nervous system. Biogenic monoamine transporters couple the movement of substrate to the pre-existing Na+ gradient that provides the thermodynamic driving force for translocation. However, the conspicuous absence of high resolution structural details precludes a fundamental understanding of the molecular basis for energy coupling to the conformational motion that underlies the transport process. The crystal structure of LeuT, a bacterial amino acid transporter homolog, provided the first molecular glimpse into the architecture of these transporters. Site-directed spin labeling and EPR spectroscopy was used to characterize the structural features of conformational intermediates sampled by LeuT under biochemical conditions that mimic distinct steps of a transport cycle. Spin labels reported local and global conformational changes on the extracellular side of LeuT, showing that ion binding produces an outward-facing state that exposes the substrate permeation pathway. In contrast, substrate binding reverses this effect and establishes an occluded conformation. These results, combined with molecular dynamics simulations and functional analysis, provide a unique dynamic perspective on an emerging model of Na+-coupled transport.