Abstract
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
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
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)
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)
Einsle, O. & Kroneck, P. M. Structural basis of denitrification. Biol. Chem. 385, 875–883 (2004)
Saier, M. H., Jr et al. Phylogenetic characterization of novel transport protein families revealed by genome analyses. Biochim. Biophys. Acta 1422, 1–56 (1999)
Pao, S. S., Paulsen, I. T. & Saier, M. H., Jr Major facilitator superfamily. Microbiol. Mol. Biol. Rev. 62, 1–34 (1998)
Jia, W. & Cole, J. A. Nitrate and nitrite transport in Escherichia coli. Biochem. Soc. Trans. 33, 159–161 (2005)
DeMoss, J. A. & Hsu, P. Y. NarK enhances nitrate uptake and nitrite excretion in Escherichia coli. J. Bacteriol. 173, 3303–3310 (1991)
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)
Moir, J. W. & Wood, N. J. Nitrate and nitrite transport in bacteria. Cell. Mol. Life Sci. 58, 215–224 (2001)
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)
Wang, Y. Y., Hsu, P. K. & Tsay, Y. F. Uptake, allocation and signaling of nitrate. Trends Plant Sci. 17, 458–467 (2012)
Law, C. J., Maloney, P. C. & Wang, D. N. Ins and outs of major facilitator superfamily antiporters. Annu. Rev. Microbiol. 62, 289–305 (2008)
Sun, L. et al. Crystal structure of a bacterial homologue of glucose transporters GLUT1–4. Nature 490, 361–366 (2012)
Solcan, N. et al. Alternating access mechanism in the POT family of oligopeptide transporters. EMBO J. 31, 3411–3421 (2012)
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)
Dang, S. et al. Structure of a fucose transporter in an outward-open conformation. Nature 467, 734–738 (2010)
Yin, Y., He, X., Szewczyk, P., Nguyen, T. & Chang, G. Structure of the multidrug transporter EmrD from Escherichia coli. Science 312, 741–744 (2006)
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)
Abramson, J. et al. Structure and mechanism of the lactose permease of Escherichia coli. Science 301, 610–615 (2003)
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)
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)
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)
Qin, L. et al. Sialin (SLC17A5) functions as a nitrate transporter in the plasma membrane. Proc. Natl Acad. Sci. USA 109, 13434–13439 (2012)
Harlow, E. & Lane, D. Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988)
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)
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)
Zhang, K. Y., Cowtan, K. & Main, P. Combining constraints for electron-density modification. Methods Enzymol. 277, 53–64 (1997)
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)
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)
Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)
The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC http://www.pymol.org/
Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007)
Acknowledgements
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
Contributions
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
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)
Rights 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). https://doi.org/10.1038/nature12139
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature12139
This article is cited by
-
Structure and mechanism of oxalate transporter OxlT in an oxalate-degrading bacterium in the gut microbiota
Nature Communications (2023)
-
Green roof drained rainwater quality assessment: a physicochemical analysis from a case study in Northeastern Brazil
Sustainable Water Resources Management (2022)
-
Structure of a proton-dependent lipid transporter involved in lipoteichoic acids biosynthesis
Nature Structural & Molecular Biology (2020)
-
Electrochemical reduction of nitrate to ammonia via direct eight-electron transfer using a copper–molecular solid catalyst
Nature Energy (2020)
-
Crystal structure of the plant symporter STP10 illuminates sugar uptake mechanism in monosaccharide transporter superfamily
Nature Communications (2019)
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.