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Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters

Nature volume 437, pages 215–223 (2005)Cite this article

Abstract

Na+/Cl--dependent transporters terminate synaptic transmission by using electrochemical gradients to drive the uptake of neurotransmitters, including the biogenic amines, from the synapse to the cytoplasm of neurons and glia. These transporters are the targets of therapeutic and illicit compounds, and their dysfunction has been implicated in multiple diseases of the nervous system. Here we present the crystal structure of a bacterial homologue of these transporters from Aquifex aeolicus, in complex with its substrate, leucine, and two sodium ions. The protein core consists of the first ten of twelve transmembrane segments, with segments 1–5 related to 6–10 by a pseudo-two-fold axis in the membrane plane. Leucine and the sodium ions are bound within the protein core, halfway across the membrane bilayer, in an occluded site devoid of water. The leucine and ion binding sites are defined by partially unwound transmembrane helices, with main-chain atoms and helix dipoles having key roles in substrate and ion binding. The structure reveals the architecture of this important class of transporter, illuminates the determinants of substrate binding and ion selectivity, and defines the external and internal gates.

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Figure 1: Amino acid sequence alignment and secondary structure of LeuTAa.
Figure 2: LeuT Aa structure.
Figure 3: Leucine binding site.
Figure 4: Sodium ion binding sites.
Figure 5: Extracellular and cytoplasmic gates.
Figure 6: Speculative transport mechanism.

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References

  1. Vanhatalo, S. & Soinila, S. The concept of chemical neurotransmission — variations on the theme. Ann. Med. 19, 151–158 (1998)

    Article  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  3. 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  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. 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  Google Scholar 

  7. Barker, E. L. & Blakely, R. D. in Psychopharmacology—the Fourth Generation of Progress (eds Bloom, F. E. & Kupfer, D. J.) (Raven Press, New York, 2000)

    Google Scholar 

  8. Guastella, J. et al. Cloning and expression of a rat brain GABA transporter. Science 249, 1303–1306 (1990)

    Article  ADS  CAS  Google Scholar 

  9. Nelson, N. The family of Na+/Cl--dependent neurotransmitter transporters. J. Neurochem. 71, 1785–1803 (1998)

    Article  CAS  Google Scholar 

  10. Torres, G. E., Gainetdinov, R. R. & Caron, M. G. Plasma membrane monoamine transporters: structure, regulation, and function. Nature Rev. Neurosci. 4, 13–25 (2003)

    Article  CAS  Google Scholar 

  11. Chen, J. G., Liu-Chen, S. & Rudnick, G. Determination of external loop topology in the serotonin transporter by site-directed chemical labeling. J. Biol. Chem. 273, 12675–12681 (1998)

    Article  CAS  Google Scholar 

  12. Bismuth, Y., Kavanaugh, M. P. & Kanner, B. I. Tyrosine 140 of the γ-aminobutyric acid transporter GAT-1 plays a critical role in neurotransmitter recognition. J. Biol. Chem. 272, 16096–16102 (1997)

    Article  CAS  Google Scholar 

  13. Chen, J. G., Sachpatzidis, A. & Rudnick, G. The third transmembrane domain of the serotonin transporter contains residues associated with substrate and cocaine binding. J. Biol. Chem. 272, 28321–28327 (1997)

    Article  CAS  Google Scholar 

  14. Cao, Y., Li, M., Mager, S. & Lester, H. A. Amino acid residues that control pH modulation of transport-associated current in mammalian serotonin transporters. J. Neurosci. 18, 7739–7749 (1998)

    Article  CAS  Google Scholar 

  15. Rudnick, G. in Neurotransmitter Transporters: Structure, Function, and Regulation (ed. Reith, E. A.) 25–52 (Humana Press, Totowa, New Jersey, 2002)

    Book  Google Scholar 

  16. Kavanaugh, M. P., Arriza, J. L., North, R. A. & Amara, S. G. Electrogenic uptake of γ-aminobutyric acid by a cloned transporter expressed in Xenopus oocytes. J. Biol. Chem. 267, 22007–22009 (1992)

    CAS  PubMed  Google Scholar 

  17. Roux, M. & Supplisson, S. Neuronal and glial glycine transporters have different stoichiometries. Neuron 25, 373–383 (2000)

    Article  CAS  Google Scholar 

  18. Androutsellis-Theotokis, A. et al. Characterization of a functional bacterial homologue of sodium-dependent neurotransmitter transporters. J. Biol. Chem. 278, 12703–12709 (2003)

    Article  CAS  Google Scholar 

  19. Hendrickson, W. A. Determination of macromolecular structures from anomalous diffraction of synchrotron radiation. Science 254, 51–58 (1991)

    Article  ADS  CAS  Google Scholar 

  20. Murata, K. et al. Structural determinants of water permeation through aquaporin-1. Nature 407, 599–605 (2000)

    Article  ADS  CAS  Google Scholar 

  21. Hirai, T. et al. Three-dimensional structure of a bacterial oxalate transporter. Nature Struct. Biol. 9, 597–600 (2002)

    CAS  PubMed  Google Scholar 

  22. Chen, J. G., Liu-Chen, S. & Rudnick, G. External cysteine residues in the serotonin transporter. Biochemistry 36, 1479–1486 (1997)

    Article  CAS  Google Scholar 

  23. Wang, J. B., Moriwaki, A. & Uhl, G. R. Dopamine transporter cysteine mutants: second extracellular loop cysteines are required for transporter expression. J. Neurochem. 64, 1416–1419 (1995)

    Article  CAS  Google Scholar 

  24. Just, H., Sitte, H. H., Schmid, J. A., Freissmuth, M. & Kudlacek, O. Identification of an additional interaction domain in transmembrane domains 11 and 12 that supports oligomer formation in the human serotonin transporter. J. Biol. Chem. 279, 6650–6657 (2004)

    Article  CAS  Google Scholar 

  25. Sitte, H. H., Farhan, H. & Javitch, J. A. Sodium-dependent neurotransmitter transporters: oligomerization as a determinant of transporter function and trafficking. Mol. Interv. 4, 38–47 (2004)

    Article  CAS  Google Scholar 

  26. Ponce, J., Biton, B., Benavides, J., Avenet, P. & Aragon, C. Transmembrane domain III plays an important role in ion binding and permeation in the glycine transporter GLYT2. J. Biol. Chem. 275, 13856–13862 (2000)

    Article  CAS  Google Scholar 

  27. Keshet, G. I. et al. Glutamate-101 is critical for the function of the sodium and chloride-coupled GABA transporter GAT-1. FEBS Lett. 371, 39–42 (1995)

    Article  CAS  Google Scholar 

  28. Nayal, M. & Di Cera, E. Valence screening of water in protein crystals reveals potential Na+ binding sites. J. Mol. Biol. 256, 228–234 (1996)

    Article  CAS  Google Scholar 

  29. Harding, M. M. Metal-ligand geometry relevant to proteins and in proteins: sodium and potassium. Acta Crystallogr. D 58, 872–874 (2002)

    Article  Google Scholar 

  30. Mager, S. et al. Ion binding and permeation at the GABA transporter GAT1. J. Neurosci. 16, 5405–5414 (1996)

    Article  CAS  Google Scholar 

  31. Penado, K. M., Rudnick, G. & Stephan, M. M. Critical amino acid residues in transmembrane span 7 of the serotonin transporter identified by random mutagenesis. J. Biol. Chem. 273, 28098–28106 (1998)

    Article  CAS  Google Scholar 

  32. Chen, N. & Reith, M. E. Na+ and the substrate permeation pathway in dopamine transporters. Eur. J. Pharmacol. 479, 213–221 (2003)

    Article  CAS  Google Scholar 

  33. Mari, S. A. et al. Aspartate 338 contributes to the cationic specificity and to driver-amino acid coupling in the insect cotransporter KAAT1. Cell. Mol. Life Sci. 61, 243–256 (2004)

    Article  CAS  Google Scholar 

  34. Jardetzky, O. Simple allosteric model for membrane pumps. Nature 211, 969–970 (1966)

    Article  ADS  CAS  Google Scholar 

  35. Pantanowitz, S., Bendahan, A. & Kanner, B. I. Only one of the charged amino acids located in the transmembrane alpha-helices of the γ-aminobutyric acid transporter (subtype A) is essential for its activity. J. Biol. Chem. 268, 3222–3225 (1993)

    CAS  PubMed  Google Scholar 

  36. Bennett, E. R., Su, H. & Kanner, B. I. Mutation of arginine 44 of GAT-1, a (Na+ + Cl-)-coupled γ-aminobutyric acid transporter from rat brain, impairs net flux but not exchange. J. Biol. Chem. 275, 34106–34113 (2000)

    Article  CAS  Google Scholar 

  37. Loland, C. J., Granas, C., Javitch, J. A. & Gether, U. Identification of intracellular residues in the dopamine transporter critical for regulation of transporter conformation and cocaine binding. J. Biol. Chem. 279, 3228–3238 (2004)

    Article  CAS  Google Scholar 

  38. Loland, C. J., Norregaard, L., Litman, T. & Gether, U. Generation of an activating Zn2+ switch in the dopamine transporter: mutation of an intracellular tyrosine constitutively alters the conformational equilibrium of the transport cycle. Proc. Natl Acad. Sci. USA 99, 1683–1688 (2002)

    Article  ADS  CAS  Google Scholar 

  39. Toyoshima, C. & Nomura, H. Structural changes in the calcium pump accompanying the dissociation of calcium. Nature 418, 605–611 (2002)

    Article  ADS  CAS  Google Scholar 

  40. Lopez-Corcuera, B., Nunez, E., Martinez-Maza, R., Geerlings, A. & Aragon, C. Substrate-induced conformational changes of extracellular loop 1 in the glycine transporter GLYT2. J. Biol. Chem. 276, 43463–43470 (2001)

    Article  CAS  Google Scholar 

  41. Sato, Y., Zhang, Y. W., Androutsellis-Theotokis, A. & Rudnick, G. Analysis of transmembrane domain 2 of rat serotonin transporter by cysteine scanning mutagenesis. J. Biol. Chem. 279, 22926–22933 (2004)

    Article  CAS  Google Scholar 

  42. 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  Google Scholar 

  43. 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  Google Scholar 

  44. Yernool, D., Boudker, O., Jin, Y. & Gouaux, E. Structure of a glutamate transporter homologue from Pyrococcus horikoshii. Nature 431, 811–818 (2004)

    Article  ADS  CAS  Google Scholar 

  45. Guerrero, S. A., Hecht, H. J., Hofmann, B., Biebl, H. & Singh, M. Production of selenomethionine-labelled proteins using simplified culture conditions and generally applicable host/vector systems. Appl. Microbiol. Biotechnol. 56, 718–723 (2001)

    Article  CAS  Google Scholar 

  46. Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849–861 (1999)

    Article  CAS  Google Scholar 

  47. Collaborative Computational Project, No. 4, The CCP4 suite: program for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

  48. Perrakis, A., Morris, R. & Lamzin, V. S. Automated protein model building combined with iterative structure refinement. Nature Struct. Biol. 6, 458–463 (1999)

    Article  CAS  Google Scholar 

  49. Brünger, A. T. et al. Crystallography & NMR system: a new software suite for maclomolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  Google Scholar 

  50. Yernool, D., Boudker, O., Folta-Stogniew, E. & Gouaux, E. Trimeric subunit stoichiometry of the glutamate transporters from Bacillus caldotenax and Bacillus stearothermophilus. Biochemistry 42, 12981–12988 (2003)

    Article  CAS  Google Scholar 

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Acknowledgements

We appreciate the beamtime and the assistance of the personnel at beamline 8.2.2 of the Advanced Light Source and beamlines X4A, X12B and X29 of the National Synchrotron Light Source; B. Honig and L. Forrest for help with sequence alignment; M. A. Gawinowicz for mass spectrometry and free amino acid analysis; J. Moon for help with bacterial culture and crystallization; H. Furukawa for assistance with vector construction and light scattering experiments; O. Boudker for liposome reconstitution; and D. Yernool, O. Boudker and R. Ryan for comments on the manuscript. A.Y. is on leave from the Laboratory for Structural Biochemistry, RIKEN Harima Institute at SPring-8, Japan. S.K.S. is supported by an NIH NRSA postdoctoral fellowship. The work was supported by the NIH. E.G. is an investigator with the Howard Hughes Medical Institute.

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  1. Department of Biochemistry and Molecular Biophysics,

    Atsuko Yamashita, Satinder K. Singh, Toshimitsu Kawate & Eric Gouaux

  2. Howard Hughes Medical Institute, Columbia University, 650 West 168th Street, New York, 10032, New York, USA

    Yan Jin & Eric Gouaux

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Correspondence to Eric Gouaux.

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The coordinates for the structure have been deposited in the Protein Data Bank under the accession code 2A65. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

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Supplementary Notes

This file contains Supplementary Table S1 (data collection and phasing statistics), Supplementary Methods, Supplementary Figures S1-S5 and accompanying Supplementary Figure Legends. (PDF 2016 kb)

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Yamashita, A., Singh, S., Kawate, T. et al. Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters. Nature 437, 215–223 (2005). https://doi.org/10.1038/nature03978

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