Article | Published:

Experimental conditions can obscure the second high-affinity site in LeuT

Nature Structural & Molecular Biology volume 19, pages 207211 (2012) | Download Citation

Abstract

Neurotransmitter:Na+ symporters (NSSs), the targets of antidepressants and psychostimulants, recapture neurotransmitters from the synapse in a Na+-dependent symport mechanism. The crystal structure of the NSS homolog LeuT from Aquifex aeolicus revealed one leucine substrate in an occluded, centrally located (S1) binding site next to two Na+ ions. Computational studies combined with binding and flux experiments identified a second substrate (S2) site and a molecular mechanism of Na+-substrate symport that depends upon the allosteric interaction of substrate molecules in the two high-affinity sites. Here we show that the S2 site, which has not yet been identified by crystallographic approaches, can be blocked during preparation of detergent-solubilized LeuT, thereby obscuring its crucial role in Na+-coupled symport. This finding points to the need for caution in selecting experimental environments in which the properties and mechanistic features of membrane proteins can be delineated.

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Acknowledgements

We thank M. Miller-Cruz for the preparation of membranes, S. Stolzenberg for help with the FEP-MD simulations, and the Keck Biotechnology Resource Laboratory at Yale University for the quantitative amino acid analysis. This work was supported in part by US National Institutes of Health grants DA17293 and DA022413 (J.A.J.), U54GM087519 (H.W. and J.A.J.), DA12408 (H.W.) and DA023694 (L.S.).

Author information

Affiliations

  1. Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, New York, New York, USA.

    • Matthias Quick
    • , Britta Zehnpfennig
    •  & Jonathan A Javitch
  2. Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York, USA.

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

    • Matthias Quick
    •  & Jonathan A Javitch
  4. Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York, USA.

    • Lei Shi
    •  & Harel Weinstein
  5. HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College of Cornell University, New York, New York, USA.

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

    • Jonathan A Javitch

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Contributions

M.Q. designed, carried out and analyzed the functional characterization of LeuT. B.Z. expressed, purified and helped with the preparation of LeuT. J.A.J. helped design the functional characterization and, with L.S. and H.W., helped to interpret the data. All the authors participated in writing and editing the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jonathan A Javitch.

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DOI

https://doi.org/10.1038/nsmb.2197

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