Structure of the proton-gated urea channel from the gastric pathogen Helicobacter pylori

Abstract

Half the world’s population is chronically infected with Helicobacter pylori1, causing gastritis, gastric ulcers and an increased incidence of gastric adenocarcinoma2. Its proton-gated inner-membrane urea channel, HpUreI, is essential for survival in the acidic environment of the stomach3. The channel is closed at neutral pH and opens at acidic pH to allow the rapid access of urea to cytoplasmic urease4. Urease produces NH3 and CO2, neutralizing entering protons and thus buffering the periplasm to a pH of roughly 6.1 even in gastric juice at a pH below 2.0. Here we report the structure of HpUreI, revealing six protomers assembled in a hexameric ring surrounding a central bilayer plug of ordered lipids. Each protomer encloses a channel formed by a twisted bundle of six transmembrane helices. The bundle defines a previously unobserved fold comprising a two-helix hairpin motif repeated three times around the central axis of the channel, without the inverted repeat of mammalian-type urea transporters. Both the channel and the protomer interface contain residues conserved in the AmiS/UreI superfamily, suggesting the preservation of channel architecture and oligomeric state in this superfamily. Predominantly aromatic or aliphatic side chains line the entire channel and define two consecutive constriction sites in the middle of the channel. Mutation of Trp 153 in the cytoplasmic constriction site to Ala or Phe decreases the selectivity for urea in comparison with thiourea, suggesting that solute interaction with Trp 153 contributes specificity. The previously unobserved hexameric channel structure described here provides a new model for the permeation of urea and other small amide solutes in prokaryotes and archaea.

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Figure 1: The Hp UreI urea channel hexamer.
Figure 2: Residues lining the channel.
Figure 3: Structural conservation of a two-helical hairpin motif.
Figure 4: Views of the Hp UreI channel traversed by urea.

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Protein Data Bank

Change history

  • 09 January 2013

    In the Fig. 4c legend, amino acid numbering of Phe 84 was corrected.

References

  1. 1

    Pounder, R. E. & Ng, D. The prevalence of Helicobacter pylori infection in different countries. Aliment. Pharmacol. Ther. 9, 33–39 (1995)

    PubMed  Google Scholar 

  2. 2

    Peek, R. M. & Blaser, M. J. Helicobacter pylori and gastrointestinal tract adenocarcinomas. Nature Rev. Cancer 2, 28–37 (2002)

    CAS  Article  Google Scholar 

  3. 3

    Skouloubris, S., Thiberge, J. M., Labigne, A. & De Reuse, H. The Helicobacter pylori UreI protein is not involved in urease activity but is essential for bacterial survival in vivo. Infect. Immun. 66, 4517–4521 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Weeks, D. L., Eskandari, S., Scott, D. R. & Sachs, G. A. H+-gated urea channel: the link between Helicobacter pylori urease and gastric colonization. Science 287, 482–485 (2000)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Graham, D. Y. & Fischbach, L. Helicobacter pylori treatment in the era of increasing antibiotic resistance. Gut 59, 1143–1153 (2010)

    CAS  Article  Google Scholar 

  6. 6

    Scott, D. R., Marcus, E. A., Weeks, D. L. & Sachs, G. Mechanisms of acid resistance due to the urease system of Helicobacter pylori. Gastroenterology 123, 187–195 (2002)

    CAS  Article  Google Scholar 

  7. 7

    Krulwich, T. A., Sachs, G. & Padan, E. Molecular aspects of bacterial pH sensing and homeostasis. Nature Rev. Microbiol. 9, 330–343 (2011)

    CAS  Article  Google Scholar 

  8. 8

    Meyer-Rosberg, K., Scott, D. R., Rex, D., Melchers, K. & Sachs, G. The effect of environmental pH on the proton motive force of Helicobacter pylori. Gastroenterology 111, 886–900 (1996)

    CAS  Article  Google Scholar 

  9. 9

    Luecke, H. et al. Structure of bacteriorhodopsin at 1.55 Å resolution. J. Mol. Biol. 291, 899–911 (1999)

    CAS  Article  Google Scholar 

  10. 10

    Gray, L. R., Gu, S. X., Quick, M. & Khademi, S. Transport kinetics and selectivity of HpUreI, the urea channel from Helicobacter pylori. Biochemistry 50, 8656–8663 (2011)

    CAS  Article  Google Scholar 

  11. 11

    Huysmans, G. H. et al. A urea channel from Bacillus cereus reveals a novel hexameric structure. Biochem. J. 445, 157–166 (2012)

    CAS  Article  Google Scholar 

  12. 12

    Pebay-Peyroula, E. et al. Structure of mitochondrial ATP/ADP carrier in complex with carboxyatractyloside. Nature 426, 39–44 (2003)

    ADS  CAS  Article  Google Scholar 

  13. 13

    Levin, E. J., Quick, M. & Zhou, M. Crystal structure of a bacterial homologue of the kidney urea transporter. Nature 462, 757–761 (2009)

    ADS  CAS  Article  Google Scholar 

  14. 14

    Levin, E. J. et al. Structure and permeation mechanism of a mammalian urea transporter. Proc. Natl Acad. Sci. USA 109, 11194–11199 (2012)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Weeks, D. L. & Sachs, G. Sites of pH regulation of the urea channel of Helicobacter pylori. Mol. Microbiol. 40, 1249–1259 (2001)

    CAS  Article  Google Scholar 

  16. 16

    Lü, W. et al. pH-dependent gating in a FocA formate channel. Science 332, 352–354 (2011)

    ADS  Article  Google Scholar 

  17. 17

    Mobley, H. L., Island, M. D. & Hausinger, R. P. Molecular biology of microbial ureases. Microbiol. Rev. 59, 451–480 (1995)

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Scott, D. R. et al. Cytoplasmic histidine kinase (HP0244)-regulated assembly of urease with UreI, a channel for urea and its metabolites, CO2, NH3 and NH4+, is necessary for acid survival of Helicobacter pylori. J. Bacteriol. 192, 94–103 (2010)

    CAS  Article  Google Scholar 

  19. 19

    Hong, W. et al. Medium pH-dependent redistribution of the urease of Helicobacter pylori. J. Med. Microbiol. 52, 211–216 (2002)

    Article  Google Scholar 

  20. 20

    Gonen, T., Sliz, P., Kistler, J., Chen, Y. & Walz, T. Aquaporin-0 membrane junctions reveal the structure of a closed water pore. Nature 429, 193–197 (2004)

    ADS  CAS  Article  Google Scholar 

  21. 21

    Callenberg, K. M. et al. APBSmem: a graphical interface for electrostatic calculations at the membrane. PLoS ONE 5, e12722 (2010)

    ADS  Article  Google Scholar 

  22. 22

    Kim, C., Basner, J. & Lee, B. Detecting internally symmetric protein structures. BMC Bioinformatics 11, 303–318 (2010)

    Article  Google Scholar 

  23. 23

    Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010)

    CAS  Article  Google Scholar 

  24. 24

    Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. D Biol. Crystallogr. 64, 112–122 (2008)

    ADS  CAS  Article  Google Scholar 

  25. 25

    Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007)

    CAS  PubMed  Google Scholar 

  26. 26

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010)

    CAS  Article  Google Scholar 

  27. 27

    Cowtan, K. Error estimation and bias correction in phase-improvement calculations. Acta Crystallogr. D Biol. Crystallogr. 55, 1555–1567 (1999)

    CAS  Article  Google Scholar 

  28. 28

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

    CAS  Article  Google Scholar 

  29. 29

    Furnham, N. et al. Knowledge-based real-space explorations for low-resolution structure determination. Structure 14, 1313–1320 (2006)

    CAS  Article  Google Scholar 

  30. 30

    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)

    ADS  CAS  Article  Google Scholar 

  31. 31

    Winn, M. D., Murshudov, G. N. & Papiz, M. Z. Macromolecular TLS refinement in REFMAC at moderate resolutions. Methods Enzymol. 374, 300–321 (2003)

    CAS  Article  Google Scholar 

  32. 32

    Scott, D. R. et al. Expression of the Helicobacter pylori ureI gene is required for acidic pH activation of cytoplasmic urease. Infect. Immun. 68, 470–477 (2000)

    CAS  Article  Google Scholar 

  33. 33

    Orbach, E. & Finkelstein, A. The nonelectrolyte permeability of planar lipid bilayer membranes. J. Gen. Physiol. 75, 427–436 (1980)

    CAS  Article  Google Scholar 

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Acknowledgements

We thank the following for assistance with X-ray data collection at the Stanford Synchrotron Radiation Lightsource (T. Doukov), the Advanced Photon Source (D. Cascio & R. Rajashankar), the Advanced Light Source (J. Nix) and the Swiss Light Source (C. Schulze-Briese). We also thank J. Whitelegge for mass spectrometry characterization of potential heavy atom derivatives; D. R. Scott for the urease assays; B. Hirayama for advice on the oocyte experiments; N. Echols and T. Terwilliger for assistance with the development version of the program Phenix; A. Murzin for fold characterization; T. Silkov for pseudosymmetry analysis; and D. R. Scott, F. Tombola, V. De Souza, K. Luecke, S. Luecke and J. Lanyi for general suggestions. This work was supported by National Institutes of Health (NIH) grants R01AI78000 and P30CA062203, National Cancer Institute institutional training grant 5 T32 CA9054-34, the University of California Irvine Center for Biomembrane Systems (H.L.), NIH grants R01DK53462 and R01DK58333 (G.S.) and the US Veterans Administration (G.S). Portions of this research were performed at the Stanford Synchrotron Radiation Lightsource, a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Stanford University. The Stanford Synchrotron Radiation Lightsource Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the HIN, National Center for Research Resources, Biomedical Technology Program (P41RR001209) and the National Institute of General Medical Sciences.

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G.S. and H.L. contributed to the design of the project. D.S. and K.M. expressed, purified and crystallized HpUreI. D.S. conducted the oocyte uptake measurements. C.-K.C. helped with expression and native gels. R.M. and H.L. performed the crystallographic experiments and analysis of data. S.M.S. assisted with aspects of phasing. G.S. and H.L. were responsible for overall project management and wrote the manuscript with K.M.

Corresponding authors

Correspondence to George Sachs or Hartmut Luecke.

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The authors declare no competing financial interests.

Additional information

The coordinates and structure factors are deposited at the Protein Data Bank under PDB code 3UX4.

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This file contains Supplementary Text and Figures 1-13, Supplementary Tables 1-2 and Supplementary References. (PDF 7240 kb)

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Strugatsky, D., McNulty, R., Munson, K. et al. Structure of the proton-gated urea channel from the gastric pathogen Helicobacter pylori. Nature 493, 255–258 (2013). https://doi.org/10.1038/nature11684

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