A conserved leucine occupies the empty substrate site of LeuT in the Na + -free return state

Bacterial members of the neurotransmitter:sodium symporter (NSS) family perform Na + -dependent amino-acid uptake and extrude H + in return. Previous NSS structures represent intermediates of Na + /substrate binding or intracellular release, but not the inward-to-outward return transition. Here we report crystal structures of Aquifex aeolicus LeuT in an outward-oriented, Na + - and substrate-free state likely to be H + -occluded. We find a remarkable rotation of the conserved Leu25 into the empty substrate-binding pocket and rearrangements of the empty Na + sites. Mutational studies of the equivalent Leu99 in the human serotonin transporter show a critical role of this residue on the transport rate. Molecular dynamics simulations show that extracellular Na + is blocked unless Leu25 is rotated out of the substrate-binding pocket. We propose that Leu25 facilitates the inward-to-outward transition by compensating a Na + - and substrate-free state and acts as the gatekeeper for Na + binding that prevents leak in inward-outward return transitions.