Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structures of LeuT in bicelles define conformation and substrate binding in a membrane-like context

Nature Structural & Molecular Biology volume 19, pages 212–219 (2012)Cite this article

Subjects

Abstract

Neurotransmitter sodium symporters (NSSs) catalyze the uptake of neurotransmitters into cells, terminating neurotransmission at chemical synapses. Consistent with the role of NSSs in the central nervous system, they are implicated in multiple diseases and disorders. LeuT, from Aquifex aeolicus, is a prokaryotic ortholog of the NSS family and has contributed to our understanding of the structure, mechanism and pharmacology of NSSs. At present, however, the functional state of LeuT in crystals grown in the presence of n-octyl-β-D-glucopyranoside (β-OG) and the number of substrate binding sites are controversial issues. Here we present crystal structures of LeuT grown in DMPC-CHAPSO bicelles and demonstrate that the conformations of LeuT–substrate complexes in lipid bicelles and in β-OG detergent micelles are nearly identical. Furthermore, using crystals grown in bicelles and the substrate leucine or the substrate analog selenomethionine, we find only a single substrate molecule in the primary binding site.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Experimental flow chart and lattice contacts in bicelle-based crystal forms.
Figure 2: LeuT crystal structures derived from bicelles and β-OG are similar.
Figure 3: Mapping detergent sites in LeuT.
Figure 4: LeuT has a single high-affinity substrate site in bicelles.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Masson, J., Sagne, C., Hamon, M. & Mestikawy, S.E. Neurotransmitter transporters in the central nervous system. Pharmacol. Rev. 51, 439–464 (1999).

    CAS  PubMed  Google Scholar 

  2. Amara, S.G. & Sonders, M.S. Neurotransmitter transporters as molecular targets for addictive drugs. Drug Alcohol Depend. 51, 87–96 (1998).

    Article  CAS  PubMed  Google Scholar 

  3. Gouaux, E. The molecular logic of sodium-coupled neurotransmitter transporters. Phil. Trans. R. Soc. Lond. B 364, 149–154 (2009).

    Article  CAS  Google Scholar 

  4. Hahn, M.K. & Blakely, R.D. Monoamine transporter gene structure and polymorphisms in relation to psychiatric and other complex disorders. Pharmacogenomics J. 2, 217–235 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Richerson, G.B. & Wu, Y. Role of the GABA transporter in epilepsy. Adv. Exp. Med. Biol. 548, 76–91 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. Shannon, J.R. et al. Orthostatic intolerance and tachycardia associated with norepinephrine-transporter deficiency. N. Engl. J. Med. 342, 541–549 (2000).

    Article  CAS  PubMed  Google Scholar 

  7. White, K.J., Walline, C. & Barker, E. Serotonin transporters: implications for antidepressant drug development. AAPS J. 7, E421–E433 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Dalby, N.O. Inhibition of γ-aminobutyric acid uptake: anatomy, physiology and effects against epileptic seizures. Eur. J. Pharmacol. 479, 127–137 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. Krogsgaard-Larsen, P., Frolund, B. & Frydenvang, K. GABA uptake inhibitors. Design, molecular pharmacology and therapeutic aspects. Curr. Pharm. Des. 6, 1193–1209 (2000).

    Article  CAS  PubMed  Google Scholar 

  10. Yamashita, A., Singh, S.K., Kawate, T., Jin, Y. & Gouaux, E. Crystal structure of a bacterial homologue of Na+/Cl−-dependent neurotransmitter transporters. Nature 437, 215–223 (2005).

    Article  CAS  PubMed  Google Scholar 

  11. Singh, S.K., Piscitelli, C.L., Yamashita, A. & Gouaux, E. A competitive inhibitor traps LeuT in an open-to-out conformation. Science 322, 1655–1661 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Singh, S.K., Yamashita, A. & Gouaux, E. Antidepressant binding site in a bacterial homologue of neurotransmitter transporters. Nature 448, 952–956 (2007).

    Article  CAS  PubMed  Google Scholar 

  13. Zhou, Z. et al. LeuT-desipramine structure reveals how antidepressants block neurotransmitter reuptake. Science 317, 1390–1393 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Claxton, D.P. et al. Ion/substrate-dependent conformational dynamics of a bacterial homolog of neurotransmitter:sodium symporters. Nat. Struct. Mol. Biol. 17, 822–829 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Zhao, Y. et al. Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue. Nature 474, 109–113 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zhao, Y. et al. Single-molecule dynamics of gating in a neurotransmitter transporter homologue. Nature 465, 188–193 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Field, J.R., Henry, L.K. & Blakely, R.D. Transmembrane domain 6 of the human serotonin transporter contributes to an aqueously accessible binding pocket for serotonin and the psychostimulant 3,4-methylene dioxymethamphetamine. J. Biol. Chem. 285, 11270–11280 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Forrest, L.R. et al. Mechanism for alternating access in neurotransmitter transporters. Proc. Natl. Acad. Sci. USA 105, 10338–10343 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sinning, S. et al. Binding and orientation of tricyclic antidepressants within the central substrate site of the human serotonin transporter. J. Biol. Chem. 285, 8363–8374 (2010).

    Article  CAS  PubMed  Google Scholar 

  20. Beuming, T. et al. The binding sites for cocaine and dopamine in the dopamine transporter overlap. Nat. Neurosci. 11, 780–789 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kniazeff, J. et al. An intracellular interaction network regulates conformational transitions in the dopamine transporter. J. Biol. Chem. 283, 17691–17701 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kanner, B.I. & Zomot, E. Sodium-coupled neurotransmitter transporters. Chem. Rev. 108, 1654–1668 (2008).

    Article  CAS  PubMed  Google Scholar 

  23. Quick, M. et al. Binding of an octylglucoside detergent molecule in the second substrate (S2) site of LeuT establishes an inhibitor-bound conformation. Proc. Natl. Acad. Sci. USA 106, 5563–5568 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Shi, L., Quick, M., Zhao, Y., Weinstein, H. & Javitch, J.A. The mechanism of a neurotransmitter: sodium symporter–inward release of Na+ and substrate is triggered by substrate in a second binding site. Mol. Cell 30, 667–677 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Piscitelli, C.L., Krishnamurthy, H. & Gouaux, E. Neurotransmitter/sodium symporter orthologue LeuT has a single high-affinity substrate site. Nature 468, 1129–1132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Faham, S. & Bowie, J.U. Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure. J. Mol. Biol. 316, 1–6 (2002).

    Article  CAS  PubMed  Google Scholar 

  27. Faham, S., Ujwal, R., Abramson, J. & Bowie, J.U. Chapter 5 Practical aspects of membrane proteins crystallization in bicelles. Curr. Top. Membr 63, 109–125 (2009).

    Article  CAS  Google Scholar 

  28. Ostermeier, C. & Michel, H. Crystallization of membrane proteins. Curr. Opin. Struct. Biol. 7, 697–701 (1997).

    Article  CAS  PubMed  Google Scholar 

  29. Matthews, B.W. Solvent content of protein crystals. J. Mol. Biol. 33, 491–497 (1968).

    Article  CAS  PubMed  Google Scholar 

  30. Boudker, O. & Verdon, G. Structural perspectives on secondary active transporters. Trends Pharmacol. Sci. 31, 418–426 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Smicun, Y., Campbell, S.D., Chen, M.A., Gu, H. & Rudnick, G. The role of external loop regions in serotonin transport. J. Biol. Chem. 274, 36058–36064 (1999).

    Article  CAS  PubMed  Google Scholar 

  32. Stephan, M.M., Chen, M.A., Penado, K.M. & Rudnick, G. An extracellular loop region of the serotonin transporter may be involved in the translocation mechanism. Biochemistry 36, 1322–1328 (1997).

    Article  CAS  PubMed  Google Scholar 

  33. Mitchell, S.M., Lee, E., Garcia, M.L. & Stephan, M.M. Structure and function of extracellular loop 4 of the serotonin transporter as revealed by cysteine-scanning mutagenesis. J. Biol. Chem. 279, 24089–24099 (2004).

    Article  CAS  PubMed  Google Scholar 

  34. Huber, R.E. & Criddle, R.S. The isolation and properties of β-galactosidase from Escherichia coli grown on sodium selenate. Biochim. Biophys. Acta 141, 587–599 (1967).

    Article  CAS  PubMed  Google Scholar 

  35. Hendrickson, W.A., Horton, J.R. & LeMaster, D.M. Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure. EMBO J. 9, 1665–1672 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Mudd, S.H. & Cantoni, G.L. Selenomethionine in enzymatic transmethylations. Nature 180, 1052 (1957).

    Article  CAS  PubMed  Google Scholar 

  37. Reyes, N. & Tavoulari, S. To be, or not to be two sites: that is the question about LeuT substrate binding. J. Gen. Physiol. 138, 467–471 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Celik, L., Schiøtt, B. & Tajkhorshid, E. Substrate binding and formation of an occluded state in the leucine transporter. Biophys. J. 94, 1600–1612 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  PubMed  Google Scholar 

  40. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Adams, P.D. et al. Phenix: a comprehensive python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  43. Chen, V.B. et al. Molprobity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  44. Collaborative Computing Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

Download references

Acknowledgements

We thank P. Shaffer for crystallization and measurement of diffraction data for the LeuT–β-SeHG complex; K. Wang and A. Penmatsa for assistance with LeuT expression and purifications; R. Hibbs for suggestions on structure refinement; M. Kavanaugh and D. Claxton for comments; and L. Vaskalis for assistance with illustrations. We also thank the staff at beamlines 24-ID-E and 24-ID-C of the Advanced Photon Source, and the staff at 8.2.1 and 5.0.2 of the Advanced Light Source. This work was supported by the US National Institutes of Health. E.G. is an investigator with the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

  1. Vollum Institute, Oregon Health & Science University, Portland, Oregon, USA

    Hui Wang & Eric Gouaux

  2. Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon, USA

    Johannes Elferich

  3. Howard Hughes Medical Institute, Oregon Health & Science University, Portland, Oregon, USA

    Eric Gouaux

Authors
  1. Hui Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Johannes Elferich
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eric Gouaux
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.W. and E.G. designed the research; H.W., J.E. and E.G. carried out the research and analyzed the data; and H.W. and E.G. wrote the paper.

Corresponding author

Correspondence to Eric Gouaux.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 5204 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, H., Elferich, J. & Gouaux, E. Structures of LeuT in bicelles define conformation and substrate binding in a membrane-like context. Nat Struct Mol Biol 19, 212–219 (2012). https://doi.org/10.1038/nsmb.2215

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2215

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing