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

Nature Structural & Molecular Biology volume 17, pages 822–829 (2010)Cite this article

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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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Figure 1: Na+ binding establishes an outward-facing conformation, and leucine binding closes access to the extracellular vestibule.
Figure 2: Changes in the EPR spectrum are dependent on Na+ binding.
Figure 3: Substrate binding induces dehydration of the S1 site.
Figure 4: Distance measurements reveal Na+- and leucine-dependent spatial rearrangements.
Figure 5: Leucine binding to the S1 site is the primary determinant of leucine-dependent conformational changes.
Figure 6: Binding of inhibitors leads to structural changes distinct from the Na+/leucine-bound conformation.
Figure 7: Configuration changes of the TM3-TM6 aromatic cluster associated with the conformational transition simulated in the 660 ns molecular dynamics trajectory.

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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.

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Authors and 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. Division of Molecular Therapeutics, New York State Psychiatric Institute, 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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  1. Derek P Claxton
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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.

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Correspondence to Hassane S Mchaourab.

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Claxton, D., Quick, M., Shi, L. et al. Ion/substrate-dependent conformational dynamics of a bacterial homolog of neurotransmitter:sodium symporters. Nat Struct Mol Biol 17, 822–829 (2010). https://doi.org/10.1038/nsmb.1854

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