Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR

Simon Erlendsson; Kamil Gotfryd; Flemming Hofmann Larsen; Jonas Sigurd Mortensen; Michel-Andreas Geiger; Barth-Jan van Rossum; Hartmut Oschkinat; Ulrik Gether; Kaare Teilum; Claus Juul Løland

doi:10.7554/eLife.19314

Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR

Simon Erlendsson, Kamil Gotfryd, Flemming Hofmann Larsen, Jonas Sigurd Mortensen, Michel-Andreas Geiger, Barth-Jan van Rossum, Hartmut Oschkinat, Ulrik Gether, Kaare Teilum, Claus Juul Løland

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Abstract

The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT. We were able to produce high-quality NMR spectra of substrate bound to microcrystalline LeuT samples and identify one set of sodium-dependent substrate-specific chemical shifts. Furthermore, our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein. We conclude that under our experimental conditions there is only one detectable leucine molecule bound to LeuT.

Original language	English
Article number	e19314
Journal	eLife
Volume	6
Number of pages	9
ISSN	2050-084X
DOIs	https://doi.org/10.7554/eLife.19314
Publication status	Published - 24 Jan 2017

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Direct assessment of substrate binding to the Neurotransmitter Sodium Symporter LeuT by solid state NMRFinal published version, 1.02 MBLicence: CC BY

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title = "Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR",

abstract = "The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT. We were able to produce high-quality NMR spectra of substrate bound to microcrystalline LeuT samples and identify one set of sodium-dependent substrate-specific chemical shifts. Furthermore, our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein. We conclude that under our experimental conditions there is only one detectable leucine molecule bound to LeuT.",

author = "Simon Erlendsson and Kamil Gotfryd and Larsen, {Flemming Hofmann} and Mortensen, {Jonas Sigurd} and Michel-Andreas Geiger and {van Rossum}, Barth-Jan and Hartmut Oschkinat and Ulrik Gether and Kaare Teilum and L{\o}land, {Claus Juul}",

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doi = "10.7554/eLife.19314",

language = "English",

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T1 - Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR

AU - Erlendsson, Simon

AU - Gotfryd, Kamil

AU - Larsen, Flemming Hofmann

AU - Mortensen, Jonas Sigurd

AU - Geiger, Michel-Andreas

AU - van Rossum, Barth-Jan

AU - Oschkinat, Hartmut

AU - Gether, Ulrik

AU - Teilum, Kaare

AU - Løland, Claus Juul

PY - 2017/1/24

Y1 - 2017/1/24

N2 - The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT. We were able to produce high-quality NMR spectra of substrate bound to microcrystalline LeuT samples and identify one set of sodium-dependent substrate-specific chemical shifts. Furthermore, our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein. We conclude that under our experimental conditions there is only one detectable leucine molecule bound to LeuT.

AB - The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT. We were able to produce high-quality NMR spectra of substrate bound to microcrystalline LeuT samples and identify one set of sodium-dependent substrate-specific chemical shifts. Furthermore, our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein. We conclude that under our experimental conditions there is only one detectable leucine molecule bound to LeuT.

U2 - 10.7554/eLife.19314

DO - 10.7554/eLife.19314

M3 - Journal article

C2 - 28117663

SN - 2050-084X

VL - 6

JO - eLife

JF - eLife

M1 - e19314

ER -

Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR

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