Complete Mapping of Substrate Translocation Implicates the Secondary Binding Site and Highlights the Significance of LeuT N-Terminal Segment in Regulating Transport Cycle

Abstract

Neurotransmitter:sodium symporters (NSSs) regulate neurotransmission by clearing excess neurotransmitters from the synaptic cleft, assisted by co-transport of sodium ions. Crystal structures resolved for a prokaryotic orthologue LeuT opened the way to structure-based studies in search of a mechanistic understanding of substrate transport by NSSs. Yet, this goal has been elusive due to the complex interplay of global and local events as well as missing structural data on LeuT N-terminal segment implicated in intracellular gating. We have extended our recent study,1 to obtain for the first time a comprehensive time-resolved mechanistic description of the complete transport cycle, using a combination of conventional and advanced molecular dynamics simulations. Our simulations suggest that LeuT harbors two substrate-binding sites for alanine. In the outward-facing open state, binding of substrate (and sodium ions) to the primary-site S1 regulates subsequent redistribution of molecular interactions to trigger extracellular gate closure; whereas the secondary-site S2 is only a transient binding site. Substrate-binding affinity at S2 increases in an intermediate close to inward-facing state. Small displacements in the second substrate near S2 are observed to induce concerted small translocations in the substrate bound to S1, although complete release requires collective structural rearrangements that fully expose the intracellular vestibule to the cytoplasm. Redistribution of salt bridges and cation-π interactions involving the N-terminal segment residues plays a pivotal role in mediating substrate release and closure of intracellular gate, and triggering a global reconfiguration to resume the transport cycle.Reference:1. Cheng MH, Bahar I (2013) Coupled Global and Local Changes Direct Substrate Translocation by Neurotransmitter-Sodium Symporter Ortholog LeuT Biophys J105: 630-639.