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.

Crystal structure of a bacterial homologue of the kidney urea transporter

Abstract

Urea is highly concentrated in the mammalian kidney to produce the osmotic gradient necessary for water re-absorption. Free diffusion of urea across cell membranes is slow owing to its high polarity, and specialized urea transporters have evolved to achieve rapid and selective urea permeation. Here we present the 2.3 Å structure of a functional urea transporter from the bacterium Desulfovibrio vulgaris. The transporter is a homotrimer, and each subunit contains a continuous membrane-spanning pore formed by the two homologous halves of the protein. The pore contains a constricted selectivity filter that can accommodate several dehydrated urea molecules in single file. Backbone and side-chain oxygen atoms provide continuous coordination of urea as it progresses through the filter, and well-placed α-helix dipoles provide further compensation for dehydration energy. These results establish that the urea transporter operates by a channel-like mechanism and reveal the physical and chemical basis of urea selectivity.

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

Access options

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

Figure 1: dvUT mediated urea flux and binding.
Figure 2: Fold and oligomeric structure of dvUT.
Figure 3: Structure of the dvUT pore and DMU-binding sites.
Figure 4: Schematic view of the selectivity filter.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited with the Protein Data Bank under accession 3K3F and 3K3G.

References

  1. Sebbane, F. et al. The Yersinia pseudotuberculosis Yut protein, a new type of urea transporter homologous to eukaryotic channels and functionally interchangeable in vitro with the Helicobacter pylori UreI protein. Mol. Microbiol. 45, 1165–1174 (2002)

    Article  CAS  Google Scholar 

  2. 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)

    Article  ADS  CAS  Google Scholar 

  3. Hediger, M. A. et al. Structure, regulation and physiological roles of urea transporters. Kidney Int. 49, 1615–1623 (1996)

    Article  CAS  Google Scholar 

  4. Sands, J. M. Mammalian urea transporters. Annu. Rev. Physiol. 65, 543–566 (2003)

    Article  CAS  Google Scholar 

  5. Bagnasco, S. M. Role and regulation of urea transporters. Pflugers Arch. 450, 217–226 (2005)

    Article  CAS  Google Scholar 

  6. Finkelstein, A. Water and nonelectrolyte permeability of lipid bilayer membranes. J. Gen. Physiol. 68, 127–135 (1976)

    Article  CAS  Google Scholar 

  7. Valladares, A., Montesinos, M. L., Herrero, A. & Flores, E. An ABC-type, high-affinity urea permease identified in cyanobacteria. Mol. Microbiol. 43, 703–715 (2002)

    Article  CAS  Google Scholar 

  8. Kojima, S., Bohner, A. & von Wiren, N. Molecular mechanisms of urea transport in plants. J. Membr. Biol. 212, 83–91 (2006)

    Article  CAS  Google Scholar 

  9. You, G. et al. Cloning and characterization of the vasopressin-regulated urea transporter. Nature 365, 844–847 (1993)

    Article  ADS  CAS  Google Scholar 

  10. MacIver, B., Smith, C. P., Hill, W. G. & Zeidel, M. L. Functional characterization of mouse urea transporters UT-A2 and UT-A3 expressed in purified Xenopus laevis oocyte plasma membranes. Am. J. Physiol. Renal Physiol. 294, F956–F964 (2008)

    Article  CAS  Google Scholar 

  11. Raunser, S. et al. Oligomeric structure and functional characterization of the urea transporter from Actinobacillus pleuropneumoniae . J. Mol. Biol. 387, 619–627 (2009)

    Article  CAS  Google Scholar 

  12. Zhao, D., Sonawane, N. D., Levin, M. H. & Yang, B. Comparative transport efficiencies of urea analogues through urea transporter UT-B. Biochim. Biophys. Acta 1768, 1815–1821 (2007)

    Article  CAS  Google Scholar 

  13. Mannuzzu, L. M., Moronne, M. M. & Macey, R. I. Estimate of the number of urea transport sites in erythrocyte ghosts using a hydrophobic mercurial. J. Membr. Biol. 133, 85–97 (1993)

    Article  CAS  Google Scholar 

  14. Minocha, R., Studley, K. & Saier, M. H. The urea transporter (UT) family: bioinformatic analyses leading to structural, functional, and evolutionary predictions. Receptors Channels 9, 345–352 (2003)

    Article  CAS  Google Scholar 

  15. Chou, C. L. & Knepper, M. A. Inhibition of urea transport in inner medullary collecting duct by phloretin and urea analogues. Am. J. Physiol. 257, F359–F365 (1989)

    CAS  PubMed  Google Scholar 

  16. Quick, M. & Javitch, J. A. Monitoring the function of membrane transport proteins in detergent-solubilized form. Proc. Natl Acad. Sci. USA 104, 3603–3608 (2007)

    Article  ADS  CAS  Google Scholar 

  17. Shi, L., Quick, M., Zhao, Y., Weinstein, H. & Javitch, J. A. The mechanism of a neurotransmitter:sodium symporter—inward release of Na+ and substrate is triggered by substrate in a second binding site. Mol. Cell 30, 667–677 (2008)

    Article  CAS  Google Scholar 

  18. von Heijne, G. & Gavel, Y. Topogenic signals in integral membrane proteins. Eur. J. Biochem. 174, 671–678 (1988)

    Article  CAS  Google Scholar 

  19. Shayakul, C., Steel, A. & Hediger, M. A. Molecular cloning and characterization of the vasopressin-regulated urea transporter of rat kidney collecting ducts. J. Clin. Invest. 98, 2580–2587 (1996)

    Article  CAS  Google Scholar 

  20. Bradford, A. D. et al. 97- and 117-kDa forms of collecting duct urea transporter UT-A1 are due to different states of glycosylation. Am. J. Physiol. Renal Physiol. 281, F133–F143 (2001)

    Article  CAS  Google Scholar 

  21. Lucien, N. et al. Antigenic and functional properties of the human red blood cell urea transporter hUT-B1. J. Biol. Chem. 277, 34101–34108 (2002)

    Article  CAS  Google Scholar 

  22. Yang, B. & Verkman, A. S. Analysis of double knockout mice lacking aquaporin-1 and urea transporter UT-B. Evidence for UT-B-facilitated water transport in erythrocytes. J. Biol. Chem. 277, 36782–36786 (2002)

    Article  CAS  Google Scholar 

  23. Imai, Y. N., Inoue, Y., Nakanishi, I. & Kitaura, K. Amide–π interactions between formamide and benzene. J. Comput. Chem. 30, 2267–2276 (2009)

    CAS  PubMed  Google Scholar 

  24. Doyle, D. A. et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 69–77 (1998)

    Article  ADS  CAS  Google Scholar 

  25. Dutzler, R., Campbell, E. B., Cadene, M., Chait, B. T. & MacKinnon, R. X-ray structure of a ClC chloride channel at 3.0 Å reveals the molecular basis of anion selectivity. Nature 415, 287–294 (2002)

    Article  ADS  CAS  Google Scholar 

  26. Terris, J. M., Knepper, M. A. & Wade, J. B. UT-A3: localization and characterization of an additional urea transporter isoform in the IMCD. Am. J. Physiol. Renal Physiol. 280, F325–F332 (2001)

    Article  CAS  Google Scholar 

  27. Blount, M. A., Klein, J. D., Martin, C. F., Tchapyjnikov, D. & Sands, J. M. Forskolin stimulates phosphorylation and membrane accumulation of UT-A3. Am. J. Physiol. Renal Physiol. 293, F1308–F1313 (2007)

    Article  CAS  Google Scholar 

  28. Khademi, S. et al. Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 Å. Science 305, 1587–1594 (2004)

    Article  ADS  CAS  Google Scholar 

  29. Zheng, L., Kostrewa, D., Berneche, S., Winkler, F. K. & Li, X. D. The mechanism of ammonia transport based on the crystal structure of AmtB of Escherichia coli . Proc. Natl Acad. Sci. USA 101, 17090–17095 (2004)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  31. Sui, H., Han, B. G., Lee, J. K., Walian, P. & Jap, B. K. Structural basis of water-specific transport through the AQP1 water channel. Nature 414, 872–878 (2001)

    Article  ADS  CAS  Google Scholar 

  32. Fu, D. et al. Structure of a glycerol-conducting channel and the basis for its selectivity. Science 290, 481–486 (2000)

    Article  ADS  CAS  Google Scholar 

  33. Levin, M. H., de la Fuente, R. & Verkman, A. S. Urearetics: a small molecule screen yields nanomolar potency inhibitors of urea transporter UT-B. FASEB J. 21, 551–563 (2007)

    Article  CAS  Google Scholar 

  34. Shayakul, C. & Hediger, M. A. The SLC14 gene family of urea transporters. Pflugers Arch. 447, 603–609 (2004)

    Article  CAS  Google Scholar 

  35. Zhang, C., Sands, J. M. & Klein, J. D. Vasopressin rapidly increases phosphorylation of UT-A1 urea transporter in rat IMCDs through PKA. Am. J. Physiol. Renal Physiol. 282, F85–F90 (2002)

    Article  CAS  Google Scholar 

  36. Punta, M. et al. Structural genomics target selection for the New York Consortium on Membrane Protein Structure. J. Struct. Funct. Genomics 10.1007/s10969-009-9071-1 (in press)

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

    Article  CAS  Google Scholar 

  38. Pape, T. & Schneider, T. R. HKL2MAP: a graphical user interface for macromolecular phasing with SHELX. J. Appl. Crystallogr. 37, 843–844 (2004)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  42. Collaborative Computational Project number 4 The CCP4 suite: programs for protein cystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

  43. Potterton, L. et al. Developments in the CCP4 molecular-graphics project. Acta Crystallogr. D 60, 2288–2294 (2004)

    Article  Google Scholar 

  44. Lebedev, A. A., Vagin, A. A. & Murshudov, G. N. Model preparation in MOLREP and examples of model improvement using X-ray data. Acta Crystallogr. D 64, 33–39 (2008)

    Article  CAS  Google Scholar 

  45. Vagin, A. A. et al. REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr. D 60, 2184–2195 (2004)

    Article  Google Scholar 

  46. Winn, M. D., Isupov, M. N. & Murshudov, G. N. Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr. D 57, 122–133 (2001)

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

Download references

Acknowledgements

We thank R. MacKinnon for advice and support throughout the project, C. Miller and E. Gouaux for comments on the manuscript, J. Love and NYCOMPS for cloning and the initial screen of protein expression levels, Y. Pan for messenger RNA preparation and oocyte injection, and J. Weng and Y. Cao for crystal screening and data collection at the synchrotrons. Data for this study were measured at beamlines X4A, X4C, X25 and X29 of the NSLS and the NE-CAT 24ID-E at the Advanced Photon Source. This work was supported by the US National Institutes of Health (HL086392 to M.Z. and T32HL087745 to E.J.L.), the NYCOMPS that is supported by the NIH Protein Structure Initiatives PSI-II (GM075026 to W. A. Hendrickson), and the American Heart Association (0630148N to M.Z.). M.Z. is a Pew Scholar in Biomedical Sciences.

Author Contributions E.J.L. and M.Z. conceived and designed the experiments. E.J.L. purified and crystallized the protein; M.Q. performed and analysed the radiotracer flux and SPA binding assays; E.J.L. and M.Z. collected and processed the X-ray data, solved the structure, and wrote the paper.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ming Zhou.

Supplementary information

Supplementary Information

This file contains Supplementary Table 1 and Supplementary Figures S1-S6 with Legends. (PDF 9066 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Levin, E., Quick, M. & Zhou, M. Crystal structure of a bacterial homologue of the kidney urea transporter . Nature 462, 757–761 (2009). https://doi.org/10.1038/nature08558

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08558

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

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