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

Mary Hongying Cheng and Ivet Bahar

PLOS Computational Biology, 2014, vol. 10, issue 10, 1-15

Abstract: 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).Author Summary: Bacterial leucine transporter (LeuT) belongs to neurotransmitter:sodium symporter (NSS) family. Its human orthologs include dopamine transporter and serotonin transporter. Malfunction of NSS members has been implicated in neurological diseases, hence the significance of elucidating their mechanism of function as clinically relevant drug targets. NSSs co-transport substrates (neurotransmitters or amino acids) and sodium ions across the cell membrane via alternating access to extracellular and intracellular media, which enables the uptake of substrate and ions from the extracellular region and their release to the intracellular region. Despite significant progress in elucidating the structure and function of NSS family members, their mechanism of function and the role of their N-terminal segment exposed to the cell interior remain elusive. Here, we provide for the first time a full-atomic time-resolved description of the complete transport cycle of LeuT using multiscale simulations. Two major findings are (i) elucidation of the structure and dynamics of the N-terminal segment which helps in mediating substrate and cation release and resuming the transport cycle, and (ii) determination of the structures of two minimally hydrated intermediates occluded to both extracellular and intracellular environments.

Date: 2014
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Persistent link: https://EconPapers.repec.org/RePEc:plo:pcbi00:1003879

DOI: 10.1371/journal.pcbi.1003879

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