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

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

46 Citations (Scopus)

400 Downloads (Pure)

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.

Original language	English
Article number	11673
Journal	Nature Communications
Volume	7
Issue	1
ISSN	2041-1723
DOIs	https://doi.org/10.1038/ncomms11673 https://doi.org/10.1038/ncomms11673
Publication status	Published - 2016

Access to Document

10.1038/ncomms11673
10.1038/ncomms11673Licence: Unspecified

ncomms11673 (2)Final published version, 2.15 MBLicence: Unspecified

http://www.nature.com/ncomms/2016/160525/ncomms11673/full/ncomms11673.html

Cite this

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), Article 11673. https://doi.org/10.1038/ncomms11673, https://doi.org/10.1038/ncomms11673

@article{b801e84943fb4b9ab6cf04c0ccc80718,

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.",

author = "Lina Malinauskaite and Saida Said and Caglanur Sahin and Julie Grouleff and Azadeh Shahsavar and Henriette Bjerregaard and Noer, {Pernille Rimmer} and Kasper Severinsen and Thomas Boesen and Birgit Schi{\o}tt and Steffen Sinning and Poul Nissen",

year = "2016",

doi = "10.1038/ncomms11673",

language = "English",

volume = "7",

journal = "Nature Communications",

issn = "2041-1723",

publisher = "Nature Research",

number = "1",

}

TY - JOUR

T1 - A Conserved Leucine Occupies the Empty Substrate Site of LeuT in the Na+-free Return State

AU - Malinauskaite, Lina

AU - Said, Saida

AU - Sahin, Caglanur

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

PY - 2016

Y1 - 2016

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.

UR - http://www.scopus.com/inward/record.url?scp=84987813686&partnerID=8YFLogxK

U2 - 10.1038/ncomms11673

DO - 10.1038/ncomms11673

M3 - Journal article

C2 - 27221344

SN - 2041-1723

VL - 7

JO - Nature Communications

JF - Nature Communications

IS - 1

M1 - 11673

ER -

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

Abstract

Access to Document

Other files and links

Fingerprint

Cite this