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

doi:10.1038/ncomms11673

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

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avis › Tidsskriftartikel › Forskning › peer review

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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 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.

Originalsprog	Engelsk
Artikelnummer	11673
Tidsskrift	Nature Communications
Vol/bind	7
Nummer	1
ISSN	2041-1723
DOI	https://doi.org/10.1038/ncomms11673 https://doi.org/10.1038/ncomms11673
Status	Udgivet - 1 sep. 2016

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Malinauskaite, L., Said, S., Sahin, C., Grouleff, J., Shahsavar, A., Bjerregaard, H., Noer, P. R., Severinsen, K., Boesen, T., Schiøtt, B., Sinning, S., & Nissen, P. (2016). A Conserved Leucine Occupies the Empty Substrate Site of LeuT in the Na+-free Return State. Nature Communications, 7(1), Artikel 11673. https://doi.org/10.1038/ncomms11673, https://doi.org/10.1038/ncomms11673

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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.",

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AU - Malinauskaite, Lina

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AU - Grouleff, Julie

AU - Shahsavar, Azadeh

AU - Bjerregaard, Henriette

AU - Noer, Pernille Rimmer

AU - Severinsen, Kasper

AU - Boesen, Thomas

AU - Schiøtt, Birgit

AU - Sinning, Steffen

AU - Nissen, Poul

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N2 - 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.

AB - 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.

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

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