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:

Crystal structure of a bacterial homologue of the bile acid sodium symporter ASBT

Nature volume 478, pages 408–411 (2011)Cite this article

Subjects

Abstract

High cholesterol levels greatly increase the risk of cardiovascular disease. About 50 per cent of cholesterol is eliminated from the body by its conversion into bile acids. However, bile acids released from the bile duct are constantly recycled, being reabsorbed in the intestine by the apical sodium-dependent bile acid transporter (ASBT, also known as SLC10A2). It has been shown in animal models that plasma cholesterol levels are considerably lowered by specific inhibitors of ASBT1,2, and ASBT is thus a target for hypercholesterolaemia drugs. Here we report the crystal structure of a bacterial homologue of ASBT from Neisseria meningitidis (ASBTNM) at 2.2 Å. ASBTNM contains two inverted structural repeats of five transmembrane helices. A core domain of six helices harbours two sodium ions, and the remaining four helices pack in a row to form a flat, ‘panel’-like domain. Overall, the architecture of the protein is remarkably similar to the sodium/proton antiporter NhaA3, despite having no detectable sequence homology. The ASBTNM structure was captured with the substrate taurocholate present, bound between the core and panel domains in a large, inward-facing, hydrophobic cavity. Residues near this cavity have been shown to affect the binding of specific inhibitors of human ASBT4. The position of the taurocholate molecule, together with the molecular architecture, suggests the rudiments of a possible transport mechanism.

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: Sodium-dependent transport of bile acid by ASBTNM.
Figure 2: ASBT NM structure.
Figure 3: ASBT NM structure is inward facing and contains bound sodium and bile acid.
Figure 4: Putative mechanism for ASBT NM transport.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates and the structure factors for ASBTNM and ASBTNM_1 have been deposited in the Protein Data Bank under accession numbers 3ZUY and 3ZUX, respectively.

References

  1. Lewis, M. C., Brieaddy, L. E. & Root, C. Effects of 2164U90 on ileal bile acid absorption and serum cholesterol in rats and mice. J. Lipid Res. 36, 1098–1105 (1995)

    CAS  PubMed  Google Scholar 

  2. Bhat, B. G. et al. Inhibition of ileal bile acid transport and reduced atherosclerosis in apoE−/− mice by SC-435. J. Lipid Res. 44, 1614–1621 (2003)

    Article  CAS  Google Scholar 

  3. Hunte, C. et al. Structure of a Na+/H+ antiporter and insights into mechanism of action and regulation by pH. Nature 435, 1197–1202 (2005)

    Article  ADS  CAS  Google Scholar 

  4. Hallén, S., Bjorquist, A., Ostlund-Lindqvist, A. M. & Sachs, G. Identification of a region of the ileal-type sodium/bile acid cotransporter interacting with a competitive bile acid transport inhibitor. Biochemistry 41, 14916–14924 (2002)

    Article  Google Scholar 

  5. Hagenbuch, B. & Dawson, P. The sodium bile salt cotransport family SLC10. Pflugers Arch. 447, 566–570 (2004)

    Article  CAS  Google Scholar 

  6. Wong, M. H., Oelkers, P., Craddock, A. L. & Dawson, P. A. Expression cloning and characterization of the hamster ileal sodium-dependent bile acid transporter. J. Biol. Chem. 269, 1340–1347 (1994)

    CAS  PubMed  Google Scholar 

  7. Weinman, S. A., Carruth, M. W. & Dawson, P. A. Bile acid uptake via the human apical sodium-bile acid cotransporter is electrogenic. J. Biol. Chem. 273, 34691–34695 (1998)

    Article  CAS  Google Scholar 

  8. Oelkers, P., Kirby, L. C., Heubi, J. E. & Dawson, P. A. Primary bile acid malabsorption caused by mutations in the ileal sodium-dependent bile acid transporter gene (SLC10A2). J. Clin. Invest. 99, 1880–1887 (1997)

    Article  CAS  Google Scholar 

  9. Kramer, W. & Wess, G. Bile acid transport systems as pharmaceutical targets. Eur. J. Clin. Invest. 26, 715–732 (1996)

    Article  CAS  Google Scholar 

  10. Drew, D., Lerch, M., Kunji, E., Slotboom, D. J. & de Gier, J. W. Optimization of membrane protein overexpression and purification using GFP fusions. Nature Methods 3, 303–313 (2006)

    Article  CAS  Google Scholar 

  11. Sonoda, Y. et al. Benchmarking membrane protein detergent stability for improving throughput of high-resolution X-ray structures. Structure 19, 17–25 (2011)

    Article  CAS  Google Scholar 

  12. Geyer, J., Wilke, T. & Petzinger, E. The solute carrier family SLC10: more than a family of bile acid transporters regarding function and phylogenetic relationships. Naunyn Schmiedebergs Arch. Pharmacol. 372, 413–431 (2006)

    Article  CAS  Google Scholar 

  13. Sun, A. Q., Balasubramaniyan, N., Chen, H., Shahid, M. & Suchy, F. J. Identification of functionally relevant residues of the rat ileal apical sodium-dependent bile acid cotransporter. J. Biol. Chem. 281, 16410–16418 (2006)

    Article  CAS  Google Scholar 

  14. Chignard, N. et al. Bile acid transport and regulating functions in the human biliary epithelium. Hepatology 33, 496–503 (2001)

    Article  CAS  Google Scholar 

  15. Craddock, A. L. et al. Expression and transport properties of the human ileal and renal sodium-dependent bile acid transporter. Am. J. Physiol. 274, G157–G169 (1998)

    CAS  PubMed  Google Scholar 

  16. Zheng, X., Ekins, S., Raufman, J. P. & Polli, J. E. Computational models for drug inhibition of the human apical sodium-dependent bile acid transporter. Mol. Pharm. 6, 1591–1603 (2009)

    Article  CAS  Google Scholar 

  17. Banerjee, A. & Swaan, P. W. Membrane topology of human ASBT (SLC10A2) determined by dual label epitope insertion scanning mutagenesis. New evidence for seven transmembrane domains. Biochemistry 45, 943–953 (2006)

    Article  CAS  Google Scholar 

  18. Hallén, S., Branden, M., Dawson, P. A. & Sachs, G. Membrane insertion scanning of the human ileal sodium/bile acid co-transporter. Biochemistry 38, 11379–11388 (1999)

    Article  Google Scholar 

  19. Screpanti, E. & Hunte, C. Discontinuous membrane helices in transport proteins and their correlation with function. J. Struct. Biol. 159, 261–267 (2007)

    Article  CAS  Google Scholar 

  20. Boudker, O. & Verdon, G. Structural perspectives on secondary active transporters. Trends Pharmacol. Sci. 31, 418–426 (2010)

    Article  CAS  Google Scholar 

  21. Hagenbuch, B. & Meier, P. J. Sinusoidal (basolateral) bile salt uptake systems of hepatocytes. Semin. Liver Dis. 16, 129–136 (1996)

    Article  CAS  Google Scholar 

  22. Zahner, D., Eckhardt, U. & Petzinger, E. Transport of taurocholate by mutants of negatively charged amino acids, cysteines, and threonines of the rat liver sodium-dependent taurocholate cotransporting polypeptide Ntcp. Eur. J. Biochem. 270, 1117–1127 (2003)

    Article  CAS  Google Scholar 

  23. Olkhova, E., Hunte, C., Screpanti, E., Padan, E. & Michel, H. Multiconformation continuum electrostatics analysis of the NhaA Na+/H+ antiporter of Escherichia coli with functional implications. Proc. Natl Acad. Sci. USA 103, 2629–2634 (2006)

    Article  ADS  CAS  Google Scholar 

  24. Hussainzada, N., Banerjee, A. & Swaan, P. W. Transmembrane domain VII of the human apical sodium-dependent bile acid transporter ASBT (SLC10A2) lines the substrate translocation pathway. Mol. Pharmacol. 70, 1565–1574 (2006)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  26. Padan, E. The enlightening encounter between structure and function in the NhaA Na+-H+ antiporter. Trends Biochem. Sci. 33, 435–443 (2008)

    Article  CAS  Google Scholar 

  27. Appel, M., Hizlan, D., Vinothkumar, K. R., Ziegler, C. & Kuhlbrandt, W. Conformations of NhaA, the Na+/H+ exchanger from Escherichia coli, in the pH-activated and ion-translocating states. J. Mol. Biol. 388, 659–672 (2009)

    Article  CAS  Google Scholar 

  28. Forrest, L. R. et al. Mechanism for alternating access in neurotransmitter transporters. Proc. Natl Acad. Sci. USA 105, 10338–10343 (2008)

    Article  ADS  CAS  Google Scholar 

  29. 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 

  30. Tzubery, T., Rimon, A. & Padan, E. Structure-based functional study reveals multiple roles of transmembrane segment IX and loop VIII–IX in NhaA Na+/H+ antiporter of Escherichia coli at physiological pH. J. Biol. Chem. 283, 15975–15987 (2008)

    Article  CAS  Google Scholar 

  31. Drew, D. E., von Heijne, G., Nordlund, P. & de Gier, J. W. Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli. FEBS Lett. 507, 220–224 (2001)

    Article  CAS  Google Scholar 

  32. Miroux, B. & Walker, J. E. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298 (1996)

    Article  CAS  Google Scholar 

  33. Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006)

    Article  CAS  Google Scholar 

  34. Drew, D. et al. GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae . Nature Protocols 3, 784–798 (2008)

    Article  CAS  Google Scholar 

  35. Alexandrov, A. I., Mileni, M., Chien, E. Y., Hanson, M. A. & Stevens, R. C. Microscale fluorescent thermal stability assay for membrane proteins. Structure 16, 351–359 (2008)

    Article  CAS  Google Scholar 

  36. Winter, G. Xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, 186–190 (2010)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  38. Collaborative Computational Project, Number 4 . The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  39. Knight, S. D. RSPS version 4.0: a semi-interactive vector-search program for solving heavy-atom derivatives. Acta Crystallogr. D 56, 42–47 (2000)

    Article  CAS  Google Scholar 

  40. de La Fortelle, E. & Bricogne, G. Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–494 (1997)

    Article  CAS  Google Scholar 

  41. Abrahams, J. P. & Leslie, A. G. W. Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D 52, 30–42 (1996)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  43. Jones, T. A. & Kjeldgaard, M. Electron-density map interpretation. Methods Enzymol. 277, 173–208 (1997)

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  45. Leslie, A. G. W. Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 ESF-EACBM Newslett. Protein Crystallogr. 26, (1992)

  46. Blanc, E. et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr. D 60, 2210–2221 (2004)

    Article  CAS  Google Scholar 

  47. 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 

  48. Harding, M. M. Metal-ligand geometry relevant to proteins and in proteins: sodium and potassium. Acta Crystallogr. D 58, 872–874 (2002)

    Article  Google Scholar 

  49. Nayal, M. & Di Cera, E. Valence screening of water in protein crystals reveals potential Na+ binding sites. J. Mol. Biol. 256, 228–234 (1996)

    Article  CAS  Google Scholar 

  50. Sanchez-Weatherby, J. et al. Improving diffraction by humidity control: a novel device compatible with X-ray beamlines. Acta Crystallogr. D 65, 1237–1246 (2009)

    Article  CAS  Google Scholar 

  51. Kleywegt, G. J. & Jones, T. A. A super position. ESF/CCP4 Newslett. 31, 9–14 (1994)

    Google Scholar 

  52. Kleywegt, G. J. & Jones, T. A. Detection, delineation, measurement and display of cavities in macromolecular structures. Acta Crystallogr. D 50, 178–185 (1994)

    Article  CAS  Google Scholar 

  53. DeLano, W. L. PyMOL Molecular Viewer 〈http://www.pymol.org〉 (2002)

    Google Scholar 

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

    Article  Google Scholar 

  55. 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 

Download references

Acknowledgements

We are grateful to C. Lee and Y. Sekiguchi for assistance with cloning and expression screening of ASBTNM mutants, and to S. van de Graaf for donating fluorescently labelled bile acid, which was used in the initial functional characterization of ASBTNM. Data were collected at the European Synchrotron Radiation Facility, France, and the Diamond Light Source, UK, with assistance from beamline scientists; in particular, we would like to thank J. Sanchez-Weatherby for help with the HCl. We are also grateful to K. Beis and G. von Heijne for reading the manuscript. This work was funded by the Medical Research Council (MRC_G0900990(91997), to A.D.C. and D.D.), the European Union (EMeP grant LSHG-CT-2004-504601, to S.I.) and the Biotechnology and Biological Sciences Research Council (BB/G023425/1, to S.I.). Part of this work was also supported by a grant from the Targeted Proteins Research Program of MEXT, Japan, and the ERATO Iwata Human Receptor Crystallography Project, Japan Science and Technology Agency. The authors are grateful for the use of the Membrane Protein Laboratory funded by the Wellcome Trust (WT089809) at the Diamond Light Source. D.D. acknowledges personal support from The Royal Society through the University Research Fellow scheme.

Author information

Authors and Affiliations

  1. Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK

    Nien-Jen Hu, So Iwata, Alexander D. Cameron & David Drew

  2. Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Chilton OX11 0DE, UK

    Nien-Jen Hu, So Iwata & Alexander D. Cameron

  3. Research Complex at Harwell Rutherford, Appleton Laboratory, Harwell, Oxford, Didcot OX11 0FA, UK

    Nien-Jen Hu, So Iwata & Alexander D. Cameron

  4. Japan Science and Technology Agency, ERATO, Human Crystallography Project, Yoshida Konoe, Sakyo-ku, Kyoto 606-851, Japan

    So Iwata & Alexander D. Cameron

  5. Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan

    So Iwata

Authors
  1. Nien-Jen Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. So Iwata
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alexander D. Cameron
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David Drew
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.-J.H., S.I., A.D.C. and D.D. contributed to the design of the project. N.-J.H. and D.D. screened homologues, expressed and purified the protein, and carried out functional characterization. N.-J.H., S.I., A.D.C. and D.D. were involved in crystallographic experiments and analysis of data. A.C. and D.D. were responsible for overall project management and wrote the manuscript with assistance from N.-J.H. and S.I.

Corresponding authors

Correspondence to So Iwata, Alexander D. Cameron or David Drew.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Tables 1-2, Supplementary Figures 1-10 with legends and additional references. (PDF 4000 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hu, NJ., Iwata, S., Cameron, A. et al. Crystal structure of a bacterial homologue of the bile acid sodium symporter ASBT. Nature 478, 408–411 (2011). https://doi.org/10.1038/nature10450

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature10450

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