Structure, Dynamics, and Mechanism of the Leucine Transporter Studied by Double Electron Electron Resonance Spectroscopy

Abstract

The neurotransmitter/sodium symporter (NSS) family of transporters includes the human dopamine, serotonin, and norepinephrine transporters. These transporters harness sodium and chloride gradients to facilitate reuptake of neurotransmitters from the synapse thus terminating neurotransmission. Due to their critical regulatory role, NSSs are the targets of numerous psychiatric therapeutics and drugs of abuse. Progress in understanding and treating diverse conditions including depression and anxiety will require a detailed description of the structure and function of this class of proteins. Structural investigations of NSS have focused on a bacterial homolog of these transporters, LeuT. This work will describe a systematic investigation of the intracellular dynamics of LeuT as well as detail progress in defining the LeuT transport mechanism and open-inward structure. Using double electron-electron resonance (DEER) spectroscopy, a pulsed EPR technique, over 40 double mutants were analyzed for their distance distributions and relative dynamics. Mechanistic descriptions were inferred by tracking shifts in equilibria between multi-component distance distributions and relating these shifts to biochemical conditions across all mutants. Closed-inward distances were largely consistent with the LeuT crystal structures, while primary gating motions were identified at helices 1 and 6 with additional reorientations of helices 5 and 7, resulting in the open-inward structure. Distance data are being implemented into restrained computational modeling to generate preliminary models of the open-inward conformation of LeuT. These results will be compared to the current models of LeuT open-in structure and transport mechanism derived using alternative approaches.