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.

  • Letter
  • Published:

Structure and mechanism of a glutamate–GABA antiporter

Nature volume 483, pages 632–636 (2012)Cite this article

Subjects

Abstract

Food-borne hemorrhagic Escherichia coli, exemplified by the strains O157:H7 and O104:H4 (refs 1, 2), require elaborate acid-resistance systems (ARs)3 to survive the extremely acidic environment such as the stomach (pH ≈ 2). AR2 expels intracellular protons through the decarboxylation of l-glutamate (Glu) in the cytoplasm and exchange of the reaction product γ-aminobutyric acid (GABA) with extracellular Glu. The latter process is mediated by the Glu–GABA antiporter GadC4,5, a representative member of the amino-acid–polyamine–organocation superfamily of membrane transporters. The functional mechanism of GadC remains largely unknown. Here we show, with the use of an in vitro proteoliposome-based assay, that GadC transports GABA/Glu only under acidic conditions, with no detectable activity at pH values higher than 6.5. We determined the crystal structure of E. coli GadC at 3.1 Å resolution under basic conditions. GadC, comprising 12 transmembrane segments (TMs), exists in a closed state, with its carboxy-terminal domain serving as a plug to block an otherwise inward-open conformation. Structural and biochemical analyses reveal the essential transport residues, identify the transport path and suggest a conserved transport mechanism involving the rigid-body rotation of a helical bundle for GadC and other amino acid antiporters.

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: Functional characterization of GadC.
Figure 2: Overall structure of GadC.
Figure 3: The C-plug regulates substrate transport.
Figure 4: The transport path in GadC.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and the structure factor file have been deposited in the Protein Data Bank under accession numbers 4DJK and 4DJI.

References

  1. Riley, L. W. et al. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N. Engl. J. Med. 308, 681–685 (1983)

    Article  CAS  Google Scholar 

  2. Rohde, H. et al. Open-source genomic analysis of Shiga-toxin-producing E. coli O104:H4. N. Engl. J. Med. 365, 718–724 (2011)

    Article  CAS  Google Scholar 

  3. Foster, J. W. Escherichia coli acid resistance: tales of an amateur acidophile. Nature Rev. Microbiol. 2, 898–907 (2004)

    Article  CAS  Google Scholar 

  4. Hersh, B. M., Farooq, F. T., Barstad, D. N., Blankenhorn, D. L. & Slonczewski, J. L. A glutamate-dependent acid resistance gene in Escherichia coli. J. Bacteriol. 178, 3978–3981 (1996)

    Article  CAS  Google Scholar 

  5. Castanie-Cornet, M. P., Penfound, T. A., Smith, D., Elliott, J. F. & Foster, J. W. Control of acid resistance in Escherichia coli. J. Bacteriol. 181, 3525–3535 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Iyer, R., Williams, C. & Miller, C. Arginine–agmatine antiporter in extreme acid resistance in Escherichia coli. J. Bacteriol. 185, 6556–6561 (2003)

    Article  CAS  Google Scholar 

  7. Gong, S., Richard, H. & Foster, J. W. YjdE (AdiC) is the arginine:agmatine antiporter essential for arginine-dependent acid resistance in Escherichia coli. J. Bacteriol. 185, 4402–4409 (2003)

    Article  CAS  Google Scholar 

  8. Gao, X. et al. Structure and mechanism of an amino acid antiporter. Science 324, 1565–1568 (2009)

    Article  ADS  CAS  Google Scholar 

  9. Gao, X. et al. Mechanism of substrate recognition and transport by an amino acid antiporter. Nature 463, 828–832 (2010)

    Article  ADS  CAS  Google Scholar 

  10. Fang, Y. et al. Structure of a prokaryotic virtual proton pump at 3.2 Å resolution. Nature 460, 1040–1043 (2009)

    Article  ADS  CAS  Google Scholar 

  11. Kowalczyk, L. et al. Molecular basis of substrate-induced permeation by an amino acid antiporter. Proc. Natl Acad. Sci. USA 108, 3935–3940 (2011)

    Article  ADS  CAS  Google Scholar 

  12. 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  ADS  CAS  Google Scholar 

  13. Ressl, S., Terwisscha van Scheltinga, A. C., Vonrhein, C., Ott, V. & Ziegler, C. Molecular basis of transport and regulation in the Na+/betaine symporter BetP. Nature 458, 47–52 (2009)

    Article  ADS  CAS  Google Scholar 

  14. Faham, S. et al. The crystal structure of a sodium galactose transporter reveals mechanistic insights into Na+/sugar symport. Science 321, 810–814 (2008)

    Article  ADS  CAS  Google Scholar 

  15. Weyand, S. et al. Structure and molecular mechanism of a nucleobase-cation-symport-1 family transporter. Science 322, 709–713 (2008)

    Article  ADS  CAS  Google Scholar 

  16. Shaffer, P. L., Goehring, A., Shankaranarayanan, A. & Gouaux, E. Structure and mechanism of a Na+-independent amino acid transporter. Science 325, 1010–1014 (2009)

    Article  ADS  CAS  Google Scholar 

  17. Fang, Y., Kolmakova-Partensky, L. & Miller, C. A bacterial arginine–agmatine exchange transporter involved in extreme acid resistance. J. Biol. Chem. 282, 176–182 (2007)

    Article  CAS  Google Scholar 

  18. Sasahara, A., Nanatani, K., Enomoto, M., Kuwahara, S. & Abe, K. Substrate specificity of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus in reconstituted liposomes. J. Biol. Chem. 286, 29044–29052 (2011)

    Article  CAS  Google Scholar 

  19. Robertson, J. L., Kolmakova-Partensky, L. & Miller, C. Design, function and structure of a monomeric CIC transporter. Nature 468, 844–847 (2010)

    Article  ADS  CAS  Google Scholar 

  20. Waterman, S. R. & Small, P. L. Identification of sigma S-dependent genes associated with the stationary-phase acid-resistance phenotype of Shigella flexneri. Mol. Microbiol. 21, 925–940 (1996)

    Article  CAS  Google Scholar 

  21. Kobashi, K. Catalytic oxidation of sulfhydryl groups by o-phenanthroline copper complex. Biochim. Biophys. Acta 158, 239–245 (1968)

    Article  CAS  Google Scholar 

  22. Abramson, J. et al. The lactose permease of Escherichia coli: overall structure, the sugar-binding site and the alternating access model for transport. FEBS Lett. 555, 96–101 (2003)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  24. Shimamura, T. et al. Molecular basis of alternating access membrane transport by the sodium-hydantoin transporter Mhp1. Science 328, 470–473 (2010)

    Article  ADS  CAS  Google Scholar 

  25. Watanabe, A. et al. The mechanism of sodium and substrate release from the binding pocket of vSGLT. Nature 468, 988–991 (2010)

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta Crystallogr. D Biol. Crystallogr. 58, 1772–1779 (2002)

    Article  Google Scholar 

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

    Article  Google Scholar 

  29. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002)

    Article  Google Scholar 

  30. DeLano, W. L. The PyMOL Molecular Graphics System, 〈http://www.pymol.org〉 (2002)

  31. Strong, M. et al. Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis. Proc. Natl Acad. Sci. USA 103, 8060–8065 (2006)

    Article  ADS  CAS  Google Scholar 

  32. Bailey, S. The Ccp4 suite—programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994)

    Article  Google Scholar 

  33. Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta Crystallogr. D Biol. Crystallogr. 58, 1772–1779 (2002)

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  35. Cowtan, K. dm: an automated procedure for phase improvement by density modification. CCP4 ESF-EACBM Newsl. Protein Crystallogr. 31, 34–38 (1994)

    Google Scholar 

  36. Veenhoff, L. M. & Poolman, B. Substrate recognition at the cytoplasmic and extracellular binding site of the lactose transport protein of Streptococcus thermophilus. J. Biol. Chem. 274, 33244–33250 (1999)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Huang and J. He (at SSRF beamline BL17U) and N. Shimizu, T. Kumasaka and S. Baba at the Spring-8 beamline BL41XU for on-site assistance, and N. Yan for discussion and comments on the manuscript. This work was supported by funds from the Ministry of Science and Technology (grant 2009CB918801), National Natural Science Foundation, and Beijing Municipal Commissions of Education and Science and Technology.

Author information

Authors and Affiliations

  1. Ministry of Education Protein Science Laboratory, Center for Structural Biology, School of Life Sciences and School of Medicine, Tsinghua University, Beijing, 100084, China

    Dan Ma, Peilong Lu, Chao Fan & Yigong Shi

  2. State Key Laboratory of Biomembrane and Membrane Biotechnology, Center for Structural Biology, School of Life Sciences and School of Medicine, Tsinghua University, Beijing, 100084, China

    Chuangye Yan, Ping Yin & Jiawei Wang

  3. Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences and School of Medicine, Tsinghua University, Beijing, 100084, China

    Dan Ma, Peilong Lu, Chuangye Yan & Yigong Shi

Authors
  1. Dan Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peilong Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chuangye Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chao Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ping Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jiawei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yigong Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.M., P.L. and Y.S. designed all experiments. D.M., P.L., C.Y., C.F. and P.Y. performed the experiments. D.M., P.L., C.Y., C.F., P.Y., J.W. and Y.S. analysed the data. D.M., P.L., C.Y., J.W. and Y.S. contributed to manuscript preparation. Y.S. wrote the manuscript.

Corresponding author

Correspondence to Yigong Shi.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains supplementary Tables 1-2, Supplementary Figures 1-14, a Supplementary Discussion and additional references. (PDF 1727 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ma, D., Lu, P., Yan, C. et al. Structure and mechanism of a glutamate–GABA antiporter. Nature 483, 632–636 (2012). https://doi.org/10.1038/nature10917

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature10917

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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