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Title: A partially-open inward-facing intermediate conformation of LeuT is associated with Na + release and substrate transport

Abstract

Here, neurotransmitter:sodium symporters (NSS), targets of antidepressants and psychostimulants, clear neurotransmitters from the synaptic cleft through sodium (Na +)-coupled transport. Substrate and Na + are thought to be transported from the extracellular to intracellular space through an alternating access mechanism by coordinated conformational rearrangements in the symporter that alternately expose the binding sites to each side of the membrane. However, the mechanism by which the binding of ligands coordinates conformational changes occurring on opposite sides of the membrane is not well understood. Here, we report the use of single-molecule fluorescence resonance energy transfer (smFRET) techniques to image transitions between distinct conformational states on both the extracellular and intracellular sides of the prokaryotic NSS LeuT, including partially open intermediates associated with transport activity. The nature and functional context of these hitherto unidentified intermediate states shed new light on the allosteric mechanism that couples substrate and Na + symport by the NSS family through conformational dynamics.

Authors:
ORCiD logo [1];  [2];  [2];  [3];  [3];  [1]; ORCiD logo [3];  [4];  [1]
  1. Weill Cornell Medicine, New York, NY (United States). Dept. of Physiology and Biophysics
  2. Columbia Univ. College of Physicians and Surgeons and New York State Psychiatric Inst, New York, NY (United States). Dept. of Psychiatry
  3. Weill Cornell Medicine, New York, NY (United States). Dept. of Physiology and Biophysics and HRH Prince Alwaleed Bin Talal Bin Abdulazaz Alsaud Inst. for Computational Biomedicine
  4. Columbia Univ. College of Physicians and Surgeons and New York State Psychiatric Inst, New York, NY (United States). Dept. of Psychiatry and Dept. of Pharmacology
Publication Date:
Research Org.:
Oak Ridge National Laboratory, Oak Ridge Leadership Computing Facility (OLCF); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). National Energy Research Scientific Computing Center
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1489406
Grant/Contract Number:  
AC05-00OR22725; AC02-05CH11231
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Nature Communications
Additional Journal Information:
Journal Volume: 9; Journal Issue: 1; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES

Citation Formats

Terry, Daniel S., Kolster, Rachel A., Quick, Matthias, LeVine, Michael V., Khelashvili, George, Zhou, Zhou, Weinstein, Harel, Javitch, Jonathan A., and Blanchard, Scott C. A partially-open inward-facing intermediate conformation of LeuT is associated with Na+ release and substrate transport. United States: N. p., 2018. Web. doi:10.1038/s41467-017-02202-y.
Terry, Daniel S., Kolster, Rachel A., Quick, Matthias, LeVine, Michael V., Khelashvili, George, Zhou, Zhou, Weinstein, Harel, Javitch, Jonathan A., & Blanchard, Scott C. A partially-open inward-facing intermediate conformation of LeuT is associated with Na+ release and substrate transport. United States. doi:10.1038/s41467-017-02202-y.
Terry, Daniel S., Kolster, Rachel A., Quick, Matthias, LeVine, Michael V., Khelashvili, George, Zhou, Zhou, Weinstein, Harel, Javitch, Jonathan A., and Blanchard, Scott C. Mon . "A partially-open inward-facing intermediate conformation of LeuT is associated with Na+ release and substrate transport". United States. doi:10.1038/s41467-017-02202-y. https://www.osti.gov/servlets/purl/1489406.
@article{osti_1489406,
title = {A partially-open inward-facing intermediate conformation of LeuT is associated with Na+ release and substrate transport},
author = {Terry, Daniel S. and Kolster, Rachel A. and Quick, Matthias and LeVine, Michael V. and Khelashvili, George and Zhou, Zhou and Weinstein, Harel and Javitch, Jonathan A. and Blanchard, Scott C.},
abstractNote = {Here, neurotransmitter:sodium symporters (NSS), targets of antidepressants and psychostimulants, clear neurotransmitters from the synaptic cleft through sodium (Na+)-coupled transport. Substrate and Na+ are thought to be transported from the extracellular to intracellular space through an alternating access mechanism by coordinated conformational rearrangements in the symporter that alternately expose the binding sites to each side of the membrane. However, the mechanism by which the binding of ligands coordinates conformational changes occurring on opposite sides of the membrane is not well understood. Here, we report the use of single-molecule fluorescence resonance energy transfer (smFRET) techniques to image transitions between distinct conformational states on both the extracellular and intracellular sides of the prokaryotic NSS LeuT, including partially open intermediates associated with transport activity. The nature and functional context of these hitherto unidentified intermediate states shed new light on the allosteric mechanism that couples substrate and Na+ symport by the NSS family through conformational dynamics.},
doi = {10.1038/s41467-017-02202-y},
journal = {Nature Communications},
issn = {2041-1723},
number = 1,
volume = 9,
place = {United States},
year = {2018},
month = {1}
}

Journal Article:
Free Publicly Available Full Text
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Cited by: 12 works
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Figures / Tables:

Fig. 1 Fig. 1 : Detection of intermediate-FRET states at extracellular and intracellular sides of LeuT. a Cartoon representation of LeuT showing the extracellularlabeling sites K239C and H480C as orange stars with TM11 and EL3 hidden for clarity. The arrow highlights the axis of motion expected in FRET experiments from relative positionsmore » of TM6a in crystal structures with closed (red, 3TT3), intermediate (yellow, 2A65), and open (green, 3TT1) extracellular sides. b Representative raw (blue) and filtered (cyan) smFRET trace from the extracellular side (K239C/H480C) in the absence of Na+ and substrate. c Histogram of the FRET values in b. d Histogram of all FRET values in all traces (gray bars), fitted with a Gaussian distribution for each FRET state (colored lines), along with the summed model distribution (black line). e–h As in a–d, but monitoring FRET from the intracellular perspective (H7C/ R86C) and the movements of the N terminus« less

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