Article | Published:

Ion/substrate-dependent conformational dynamics of a bacterial homolog of neurotransmitter:sodium symporters

Nature Structural & Molecular Biology volume 17, pages 822829 (2010) | Download Citation

Abstract

Crystallographic, computational and functional analyses of LeuT have revealed details of the molecular architecture of Na+-coupled transporters and the mechanistic nature of ion/substrate coupling, but the conformational changes that support a functional transport cycle have yet to be described fully. We have used site-directed spin labeling and electron paramagnetic resonance (EPR) analysis to capture the dynamics of LeuT in the region of the extracellular vestibule associated with the binding of Na+ and leucine. The results outline the Na+-dependent formation of a dynamic outward-facing intermediate that exposes the primary substrate binding site and the conformational changes that occlude this binding site upon subsequent binding of the leucine substrate. Furthermore, the binding of the transport inhibitors tryptophan, clomipramine and octyl-glucoside is shown to induce structural changes that distinguish the resulting inhibited conformation from the Na+/leucine-bound state.

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Acknowledgements

This work was financially supported by US National Institutes of Health grants DA022413 and DA17293 to J.A.J., DA023694 to L.S. and DA012408 to H.W., unrestricted funds from Vanderbilt University to H.S.M. and a predoctoral US National Research Service Award (F31NS063699) from the US National Institute of Neurological Disorders and Stroke to D.P.C. Computations were performed on the Ranger at the Texas Advanced Computing Center (TG-MCB090022) and the Cofrin Center for Bioinformation of the Institute for Computational Biomedicine at Weill Cornell Medical College.

Author information

Affiliations

  1. Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

    • Derek P Claxton
    •  & Hassane S Mchaourab
  2. Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, New York, New York, USA.

    • Matthias Quick
    • , Fernanda Delmondes de Carvalho
    •  & Jonathan A Javitch
  3. Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York, USA.

    • Matthias Quick
    •  & Jonathan A Javitch
  4. New York State Psychiatric Institute, Division of Molecular Therapeutics, New York, New York, USA.

    • Matthias Quick
    •  & Jonathan A Javitch
  5. Department of Physiology and Biophysics and the HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, New York, USA.

    • Lei Shi
    •  & Harel Weinstein
  6. Department of Pharmacology, Columbia University, New York, New York, USA.

    • Jonathan A Javitch

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Contributions

D.P.C. designed and performed the EPR experiments and analyzed the results; M.Q. designed, carried out and analyzed the binding and transport experiments with the help of F.D.d.C.; L.S. designed, carried out and analyzed the computational studies; F.D.d.C. helped construct, express and purify membranes harboring LeuT mutations; H.W., J.A.J. and H.S.M. helped to design experiments and analyze data related to the computational, biochemical and EPR studies, respectively; D.P.C., M.Q., L.S., H.W., J.A.J. and H.S.M. contributed to writing and editing the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Hassane S Mchaourab.

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DOI

https://doi.org/10.1038/nsmb.1854

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