A Conserved Leucine Occupies the Empty Substrate Site of LeuT in the Na+-free Return State

Lina Malinauskaite, Saida Said, Caglanur Sahin, Julie Grouleff, Azadeh Shahsavar, Henriette Bjerregaard, Pernille Rimmer Noer, Kasper Severinsen, Thomas Boesen, Birgit Schiøtt, Steffen Sinning, Poul Nissen

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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.
Original languageEnglish
Article number11673
JournalNature Communications
Publication statusPublished - 2016


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