Skip to main content

Thank you for visiting 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:

Crystal structure of a nitrate/nitrite exchanger

A Corrigendum to this article was published on 12 March 2014


Mineral nitrogen in nature is often found in the form of nitrate (NO3). Numerous microorganisms evolved to assimilate nitrate and use it as a major source of mineral nitrogen uptake1. Nitrate, which is central in nitrogen metabolism, is first reduced to nitrite (NO2) through a two-electron reduction reaction2,3. The accumulation of cellular nitrite can be harmful because nitrite can be reduced to the cytotoxic nitric oxide. Instead, nitrite is rapidly removed from the cell by channels and transporters, or reduced to ammonium or dinitrogen through the action of assimilatory enzymes3. Despite decades of effort no structure is currently available for any nitrate transport protein and the mechanism by which nitrate is transported remains largely unknown. Here we report the structure of a bacterial nitrate/nitrite transport protein, NarK, from Escherichia coli, with and without substrate. The structures reveal a positively charged substrate-translocation pathway lacking protonatable residues, suggesting that NarK functions as a nitrate/nitrite exchanger and that protons are unlikely to be co-transported. Conserved arginine residues comprise the substrate-binding pocket, which is formed by association of helices from the two halves of NarK. Key residues that are important for substrate recognition and transport are identified and related to extensive mutagenesis and functional studies. We propose that NarK exchanges nitrate for nitrite by a rocker switch mechanism facilitated by inter-domain hydrogen bond networks.

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

Access options

Buy this article

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

Figure 1: The crystal structure of NarK.
Figure 2: The substrate-binding site in NarK.
Figure 3: Protons are probably excluded from the substrate translocation pathway of NarK.
Figure 4: Proposed mechanism of nitrate/nitrite exchange.

Similar content being viewed by others

Accession codes


Protein Data Bank

Data deposits

Structures of substrate-free and nitrite-bound NarK have been deposited inPDB under accession numbers 4JR9 and 4JRE, respectively.


  1. Wood, N. J., Alizadeh, T., Richardson, D. J., Ferguson, S. J. & Moir, J. W. Two domains of a dual-function NarK protein are required for nitrate uptake, the first step of denitrification in Paracoccus pantotrophus. Mol. Microbiol. 44, 157–170 (2002)

    Article  CAS  Google Scholar 

  2. Martínez-Espinosa, R. M., Cole, J. A., Richardson, D. J. & Watmough, N. J. Enzymology and ecology of the nitrogen cycle. Biochem. Soc. Trans. 39, 175–178 (2011)

    Article  Google Scholar 

  3. Einsle, O. & Kroneck, P. M. Structural basis of denitrification. Biol. Chem. 385, 875–883 (2004)

    Article  CAS  Google Scholar 

  4. Saier, M. H., Jr et al. Phylogenetic characterization of novel transport protein families revealed by genome analyses. Biochim. Biophys. Acta 1422, 1–56 (1999)

    Article  CAS  Google Scholar 

  5. Pao, S. S., Paulsen, I. T. & Saier, M. H., Jr Major facilitator superfamily. Microbiol. Mol. Biol. Rev. 62, 1–34 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Jia, W. & Cole, J. A. Nitrate and nitrite transport in Escherichia coli. Biochem. Soc. Trans. 33, 159–161 (2005)

    Article  CAS  Google Scholar 

  7. DeMoss, J. A. & Hsu, P. Y. NarK enhances nitrate uptake and nitrite excretion in Escherichia coli. J. Bacteriol. 173, 3303–3310 (1991)

    Article  CAS  Google Scholar 

  8. Rowe, J. J., Ubbink-Kok, T., Molenaar, D., Konings, W. N. & Driessen, A. J. NarK is a nitrite-extrusion system involved in anaerobic nitrate respiration by Escherichia coli. Mol. Microbiol. 12, 579–586 (1994)

    Article  CAS  Google Scholar 

  9. Moir, J. W. & Wood, N. J. Nitrate and nitrite transport in bacteria. Cell. Mol. Life Sci. 58, 215–224 (2001)

    Article  CAS  Google Scholar 

  10. Jia, W., Tovell, N., Clegg, S., Trimmer, M. & Cole, J. A single channel for nitrate uptake, nitrite export and nitrite uptake by Escherichia coli NarU and a role for NirC in nitrite export and uptake. Biochem. J. 417, 297–304 (2009)

    Article  CAS  Google Scholar 

  11. Wang, Y. Y., Hsu, P. K. & Tsay, Y. F. Uptake, allocation and signaling of nitrate. Trends Plant Sci. 17, 458–467 (2012)

    Article  CAS  Google Scholar 

  12. Law, C. J., Maloney, P. C. & Wang, D. N. Ins and outs of major facilitator superfamily antiporters. Annu. Rev. Microbiol. 62, 289–305 (2008)

    Article  CAS  Google Scholar 

  13. Sun, L. et al. Crystal structure of a bacterial homologue of glucose transporters GLUT1–4. Nature 490, 361–366 (2012)

    Article  ADS  CAS  Google Scholar 

  14. Solcan, N. et al. Alternating access mechanism in the POT family of oligopeptide transporters. EMBO J. 31, 3411–3421 (2012)

    Article  CAS  Google Scholar 

  15. Newstead, S. et al. Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2. EMBO J. 30, 417–426 (2011)

    Article  CAS  Google Scholar 

  16. Dang, S. et al. Structure of a fucose transporter in an outward-open conformation. Nature 467, 734–738 (2010)

    Article  ADS  CAS  Google Scholar 

  17. Yin, Y., He, X., Szewczyk, P., Nguyen, T. & Chang, G. Structure of the multidrug transporter EmrD from Escherichia coli. Science 312, 741–744 (2006)

    Article  ADS  CAS  Google Scholar 

  18. Huang, Y., Lemieux, M. J., Song, J., Auer, M. & Wang, D. N. Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli. Science 301, 616–620 (2003)

    Article  ADS  CAS  Google Scholar 

  19. Abramson, J. et al. Structure and mechanism of the lactose permease of Escherichia coli. Science 301, 610–615 (2003)

    Article  ADS  CAS  Google Scholar 

  20. Trueman, L. J., Richardson, A. & Forde, B. G. Molecular cloning of higher plant homologues of the high-affinity nitrate transporters of Chlamydomonas reinhardtii and Aspergillus nidulans. Gene 175, 223–231 (1996)

    Article  CAS  Google Scholar 

  21. Mirza, O., Guan, L., Verner, G., Iwata, S. & Kaback, H. R. Structural evidence for induced fit and a mechanism for sugar/H+ symport in LacY. EMBO J. 25, 1177–1183 (2006)

    Article  CAS  Google Scholar 

  22. Lü, W. et al. Structural and functional characterization of the nitrite channel NirC from Salmonella typhimurium. Proc. Natl Acad. Sci. USA 109, 18395–18400 (2012)

    Article  ADS  Google Scholar 

  23. Qin, L. et al. Sialin (SLC17A5) functions as a nitrate transporter in the plasma membrane. Proc. Natl Acad. Sci. USA 109, 13434–13439 (2012)

    Article  ADS  CAS  Google Scholar 

  24. Harlow, E. & Lane, D. Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988)

    Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  28. Fokin, A. V. et al. Spatial structure of a Fab-fragment of a monoclonal antibody to human interleukin-2 in two crystalline forms at a resolution of 2.2 and 2.9 angstroms [in Russian with English abstract]. Bioorg. Khim. 26, 571–578 (2000)

    CAS  PubMed  Google Scholar 

  29. Zhang, K. Y., Cowtan, K. & Main, P. Combining constraints for electron-density modification. Methods Enzymol. 277, 53–64 (1997)

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  31. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    Article  CAS  Google Scholar 

  32. Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    Article  CAS  Google Scholar 

  33. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC

  34. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007)

    Article  CAS  Google Scholar 

Download references


We thank E. McCleskey for critically reading this manuscript and for discussions. We thank D. Cawley for development and production of monoclonal antibodies, and staff at the Advanced Light Source, Lawrence Berkeley National Laboratory for assistance with X-ray data collection. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under Contract no. DE-AC02-05CH11231. Research in the Gonen laboratory is funded by the Howard Hughes Medical Institute.

Author information

Authors and Affiliations



H.Z. and T.G. designed the project. H.Z. performed all biochemical experiments including cloning, expression, purification, antibody production and binding assays, crystallization and X-ray data collection for both apo- and nitrite-bound NarK. H.Z. and G.W. built and refined the structures. All authors participated in data analysis and figure preparation. H.Z. and T.G. wrote the manuscript.

Corresponding author

Correspondence to Tamir Gonen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Text, Supplementary Figures 1-5 and Supplementary References. (PDF 882 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zheng, H., Wisedchaisri, G. & Gonen, T. Crystal structure of a nitrate/nitrite exchanger. Nature 497, 647–651 (2013).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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.


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