title: A conserved leucine occupies the empty substrate site of LeuT in the Na(+)-free return state.
abstract: 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.
authors.affiliation: Danish Research Institute of Translational Neuroscience-DANDRITE, Nordic-EMBL Partnership for Molecular Medicine, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, Aarhus C DK-8000, Denmark.
authors.forename: Lina
authors.initials: L
authors.lastname: Malinauskaite
authors.affiliation: Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, Skovagervej 2, Risskov DK-8240, Denmark.
authors.forename: Saida
authors.initials: S
authors.lastname: Said
authors.affiliation: Danish Research Institute of Translational Neuroscience-DANDRITE, Nordic-EMBL Partnership for Molecular Medicine, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, Aarhus C DK-8000, Denmark.
authors.forename: Caglanur
authors.initials: C
authors.lastname: Sahin
authors.affiliation: inSPIN and iNANO centers, Department of Chemistry, Aarhus University, Langelandsgade 140, Aarhus C DK-8000, Denmark.
authors.forename: Julie
authors.initials: J
authors.lastname: Grouleff
authors.affiliation: Danish Research Institute of Translational Neuroscience-DANDRITE, Nordic-EMBL Partnership for Molecular Medicine, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, Aarhus C DK-8000, Denmark.
authors.forename: Azadeh
authors.initials: A
authors.lastname: Shahsavar
authors.affiliation: Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, Skovagervej 2, Risskov DK-8240, Denmark.
authors.forename: Henriette
authors.initials: H
authors.lastname: Bjerregaard
authors.affiliation: Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, Skovagervej 2, Risskov DK-8240, Denmark.
authors.forename: Pernille
authors.initials: P
authors.lastname: Noer
authors.affiliation: Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, Skovagervej 2, Risskov DK-8240, Denmark.
authors.forename: Kasper
authors.initials: K
authors.lastname: Severinsen
authors.affiliation: Danish Research Institute of Translational Neuroscience-DANDRITE, Nordic-EMBL Partnership for Molecular Medicine, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, Aarhus C DK-8000, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Boesen
authors.affiliation: inSPIN and iNANO centers, Department of Chemistry, Aarhus University, Langelandsgade 140, Aarhus C DK-8000, Denmark.
authors.forename: Birgit
authors.initials: B
authors.lastname: Schiøtt
authors.affiliation: Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, Skovagervej 2, Risskov DK-8240, Denmark.
authors.forename: Steffen
authors.initials: S
authors.lastname: Sinning
authors.affiliation: Danish Research Institute of Translational Neuroscience-DANDRITE, Nordic-EMBL Partnership for Molecular Medicine, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, Aarhus C DK-8000, Denmark.
authors.forename: Poul
authors.initials: P
authors.lastname: Nissen
mesh_terms: D000595:Amino Acid Sequence; D026905:Amino Acid Transport Systems / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli; D057809:HEK293 Cells; D006801:Humans; D007930:Leucine / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D050480:Plasma Membrane Neurotransmitter Transport Proteins / Q000378:metabolism*; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism
chemical_list: D026905:Amino Acid Transport Systems; D050480:Plasma Membrane Neurotransmitter Transport Proteins; D012964:Sodium; D007930:Leucine