Complete Mapping of Substrate Translocation Highlights the Role of LeuT N-terminal Segment in Regulating Transport Cycle

Neurotransmitter: sodium symporters (NSSs) regulate neuronal signal transmission by clearing excess neurotransmitters from the synapse, assisted by the co-transport of sodium ions. Extensive structural data have been collected in recent years for several members of the NSS family, which opened the way to structure-based studies for a mechanistic understanding of substrate transport. Leucine transporter (LeuT), a bacterial orthologue, has been broadly adopted as a prototype in these studies. This goal has been elusive, however, due to the complex interplay of global and local events as well as missing structural data on LeuT N-terminal segment. We provide here for the first time a comprehensive description of the molecular events leading to substrate/Na+ release to the postsynaptic cell, including the structure and dynamics of the N-terminal segment using a combination of molecular simulations. Substrate and Na+-release follows an influx of water molecules into the substrate/Na+-binding pocket accompanied by concerted rearrangements of transmembrane helices. A redistribution of salt bridges and cation-π interactions at the N-terminal segment prompts substrate release. Significantly, substrate release is followed by the closure of the intracellular gate and a global reconfiguration back to outward-facing state to resume the transport cycle. Two minimally hydrated intermediates, not structurally resolved to date, are identified: one, substrate-bound, stabilized during the passage from outward- to inward-facing state (holo-occluded), and another, substrate-free, along the reverse transition (apo-occluded).