Significant advances have been made in recent years in characterizing neurotransmitter:sodium symporter (NSS) family structure and function. Yet, many time-resolved events and intermediates that control the various stages of transport cycle remain to be elucidated. Whether NSSs harbor one or two sites for binding their substrates (neurotransmitters or amino acids), and what the role of the secondary site S2 is, if any, are still unresolved. Using molecular modeling and simulations for LeuT, a bacterial NSS, we present a comprehensive account of substrate-binding and -stabilization events, and subsequently triggered interactions leading to substrate (alanine) release. LeuT instantaneous conformation as it reconfigures from substrate-receiving (outward-facing) to -releasing (inward-facing) state appears to be a determinant of its affinity to bind substrate at site S2. In the outward-facing state, S1 robustly binds alanine and regulates subsequent redistribution of interactions to trigger extracellular gate closure; whereas S2 is only a transient binding site. The substrate-binding affinity at S2 increases in an intermediate close to inward-facing state. LeuT harbors the two substrate-binding sites, and small displacements of second substrate near S2 are observed to induce concerted small translocations in the substrate bound to primary site S1, although complete release requires collective structural rearrangements that fully expose the intracellular vestibule to the cytoplasm.
Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.