Abstract
Neurotransmitter:sodium symporters (NSS) are conserved from bacteria to man and serve as targets for drugs, including antidepressants and psychostimulants. Here we report the X-ray structure of the prokaryotic NSS member, LeuT, in a Na+/substrate-bound, inward-facing occluded conformation. To obtain this structure, we were guided by findings from single-molecule fluorescence spectroscopy and molecular dynamics simulations indicating that L-Phe binding and mutation of the conserved N-terminal Trp8 to Ala both promote an inward-facing state. Compared to the outward-facing occluded conformation, our structure reveals a major tilting of the cytoplasmic end of transmembrane segment (TM) 5, which, together with release of the N-terminus but without coupled movement of TM1, opens a wide cavity towards the second Na+ binding site. The structure of this key intermediate in the LeuT transport cycle, in the context of other NSS structures, leads to the proposal of an intracellular release mechanism of substrate and ions in NSS proteins.
Highlights
Neurotransmitter:sodium symporters (NSS) are conserved from bacteria to man and serve as targets for drugs, including antidepressants and psychostimulants
Another bacterial NSS member, MhsT, has been crystallized in two Na+/substrate-bound, inward-facing occluded states (PDB-IDs: 4US3 and 4US4)[13], and structures have been obtained for the Drosophila melanogaster dopamine transporter[14,15] and for the human SERT16,17
It was shown that this intermediate conformation could be converted to the high-FRET, inwardly occluded state in the presence of high Na+ concentrations relative to the EC50
Summary
Neurotransmitter:sodium symporters (NSS) are conserved from bacteria to man and serve as targets for drugs, including antidepressants and psychostimulants. Compared to the outward-facing occluded conformation, our structure reveals a major tilting of the cytoplasmic end of transmembrane segment (TM) 5, which, together with release of the N-terminus but without coupled movement of TM1, opens a wide cavity towards the second Na+ binding site. The structure of this key intermediate in the LeuT transport cycle, in the context of other NSS structures, leads to the proposal of an intracellular release mechanism of substrate and ions in NSS proteins. Results from single-molecule fluorescence resonance energy transfer (smFRET), spin labeling and site-directed fluorescence quenching spectroscopy (SDFQS) studies support the existence of distinct conformations on the trajectory from the outward-facing occluded state to the full apo inward-facing open conformation[25,28,29,35]
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