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The mechanism of sodium and substrate release from the binding pocket of vSGLT

Nature volume 468, pages 988–991 (2010)Cite this article

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Abstract

Membrane co-transport proteins that use a five-helix inverted repeat motif have recently emerged as one of the largest structural classes of secondary active transporters1,2. However, despite many structural advances there is no clear evidence of how ion and substrate transport are coupled. Here we report a comprehensive study of the sodium/galactose transporter from Vibrio parahaemolyticus (vSGLT), consisting of molecular dynamics simulations, biochemical characterization and a new crystal structure of the inward-open conformation at a resolution of 2.7 Å. Our data show that sodium exit causes a reorientation of transmembrane helix 1 that opens an inner gate required for substrate exit, and also triggers minor rigid-body movements in two sets of transmembrane helical bundles. This cascade of events, initiated by sodium release, ensures proper timing of ion and substrate release. Once set in motion, these molecular changes weaken substrate binding to the transporter and allow galactose readily to enter the intracellular space. Additionally, we identify an allosteric pathway between the sodium-binding sites, the unwound portion of transmembrane helix 1 and the substrate-binding site that is essential in the coupling of co-transport.

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Figure 1: Structures and overlay of the inward-open and inward-occluded conformations.
Figure 2: Mechanism of galactose release.
Figure 3: The potential of mean force for galactose unbinding.
Figure 4: Conformational changes in the transition from the inward-occluded to the inward-open structure.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors of the inward-open vSGLT structure have been deposited in the Protein Data Bank under accession number 2XQ2.

References

  1. Abramson, J. & Wright, E. M. Structure and function of Na+-symporters with inverted repeats. Curr. Opin. Struct. Biol. 19, 425–432 (2009)

    Article  CAS  Google Scholar 

  2. Krishnamurthy, H., Piscitelli, C. L. & Gouaux, E. Unlocking the molecular secrets of sodium-coupled transporters. Nature 459, 347–355 (2009)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  4. Wright, E. M., Hirayama, B. A. & Loo, D. F. Active sugar transport in health and disease. J. Intern. Med. 261, 32–43 (2007)

    Article  CAS  Google Scholar 

  5. Isaji, M. Sodium-glucose cotransporter inhibitors for diabetes. Curr. Opin. Investig. Drugs 8, 285–292 (2007)

    CAS  PubMed  Google Scholar 

  6. Faham, S. et al. The crystal structure of a sodium galactose transporter reveals mechanistic insights into Na+/sugar symport. Science 321, 810–814 (2008)

    Article  ADS  CAS  Google Scholar 

  7. Weyand, S. et al. Structure and molecular mechanism of a nucleobase-cation-symport-1 family transporter. Science 322, 709–713 (2008)

    Article  ADS  CAS  Google Scholar 

  8. Ressl, S., Terwisscha van Scheltinga, A. C., Vonrhein, C., Ott, V. & Ziegler, C. Molecular basis of transport and regulation in the Na+/betaine symporter BetP. Nature 458, 47–52 (2009)

    Article  ADS  CAS  Google Scholar 

  9. Fang, Y. et al. Structure of a prokaryotic virtual proton pump at 3.2 Å resolution. Nature 460, 1040–1043 (2009)

    Article  ADS  CAS  Google Scholar 

  10. Gao, X. et al. Structure and mechanism of an amino acid antiporter. Science 324, 1565–1568 (2009)

    Article  ADS  CAS  Google Scholar 

  11. Shaffer, P. L., Goehring, A., Shankaranarayanan, A. & Gouaux, E. Structure and mechanism of a Na+-independent amino acid transporter. Science 325, 1010–1014 (2009)

    Article  ADS  CAS  Google Scholar 

  12. 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 

  13. Forrest, L. R. & Rudnick, G. The rocking bundle: a mechanism for ion-coupled solute flux by symmetrical transporters. Physiology (Bethesda) 24, 377–386 (2009)

    CAS  Google Scholar 

  14. Zhou, Z. et al. LeuT-desipramine structure reveals how antidepressants block neurotransmitter reuptake. Science 317, 1390–1393 (2007)

    Article  ADS  CAS  Google Scholar 

  15. Gao, X. et al. Mechanism of substrate recognition and transport by an amino acid antiporter. Nature 463, 828–832 (2010)

    Article  ADS  CAS  Google Scholar 

  16. Singh, S. K., Piscitelli, C. L., Yamashita, A. & Gouaux, E. A competitive inhibitor traps LeuT in an open-to-out conformation. Science 322, 1655–1661 (2008)

    Article  ADS  CAS  Google Scholar 

  17. 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 

  18. Jo, S., Kim, T., Iyer, V. G. & Im, W. CHARMM-GUI: a web-based graphical user interface for CHARMM. J. Comput. Chem. 29, 1859–1865 (2008)

    Article  CAS  Google Scholar 

  19. Li, J. & Tajkhorshid, E. Ion-releasing state of a secondary membrane transporter. Biophys. J. 97, L29–L31 (2009)

    Article  CAS  Google Scholar 

  20. Quick, M., Loo, D. D. & Wright, E. M. Neutralization of a conserved amino acid residue in the human Na+/glucose transporter (hSGLT1) generates a glucose-gated H+ channel. J. Biol. Chem. 276, 1728–1734 (2001)

    Article  CAS  Google Scholar 

  21. Kumar, S., Bouzida, D., Swendsen, R. H., Kollman, P. A. & Rosenberg, J. M. The weighted histogram analysis method for free-energy calculations on biomolecules. 1. The method. J. Comput. Chem. 13, 1011–1021 (1992)

    Article  CAS  Google Scholar 

  22. Yernool, D., Boudker, O., Jin, Y. & Gouaux, E. Structure of a glutamate transporter homologue from Pyrococcus horikoshii . Nature 431, 811–818 (2004)

    Article  ADS  CAS  Google Scholar 

  23. 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 

  24. Lomize, A. L., Pogozheva, I. D., Lomize, M. A. & Mosberg, H. I. Positioning of proteins in membranes: a computational approach. Protein Sci. 15, 1318–1333 (2006)

    Article  CAS  Google Scholar 

  25. Kale, L. et al. NAMD2: Greater scalability for parallel molecular dynamics. J. Comput. Phys. 151, 283–312 (1999)

    Article  ADS  CAS  Google Scholar 

  26. Sali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993)

    Article  CAS  Google Scholar 

  27. MacKerell, A. D., Jr, Feig, M. & Brooks, C. L., III Improved treatment of the protein backbone in empirical force fields. J. Am. Chem. Soc. 126, 698–699 (2004)

    Article  CAS  Google Scholar 

  28. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996)

    Article  CAS  Google Scholar 

  29. Ryckaert, J. P., Ciccotti, G. & Berendsen, H. J. C. Numerical integration of the Cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J. Comput. Phys. 23, 327–341 (1977)

    Article  ADS  CAS  Google Scholar 

  30. Turk, E. et al. Molecular characterization of Vibrio parahaemolyticus vSGLT: a model for sodium-coupled sugar cotransporters. J. Biol. Chem. 275, 25711–25716 (2000)

    Article  CAS  Google Scholar 

  31. Turk, E., Gasymov, O. K., Lanza, S., Horwitz, J. & Wright, E. M. A reinvestigation of the secondary structure of functionally active vSGLT, the Vibrio sodium/galactose cotransporter. Biochemistry 45, 1470–1479 (2006)

    Article  CAS  Google Scholar 

  32. Veenstra, M., Lanza, S., Hirayama, B. A., Turk, E. & Wright, E. M. Local conformational changes in the Vibrio Na+/galactose cotransporter. Biochemistry 43, 3620–3627 (2004)

    Article  CAS  Google Scholar 

  33. Otwinowski, Z. & Minor, W. in Macromolecular Crystallography (eds Abelson, J. N., Simon, M. I., Carter, C. W. Jr & Sweet, R. M. ) 307–326 (Methods in Enzymology 276, Academic, 1997)

    Book  Google Scholar 

  34. 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)

    Article  ADS  CAS  Google Scholar 

  35. Qian, B. et al. High-resolution structure prediction and the crystallographic phase problem. Nature 450, 259–264 (2007)

    Article  ADS  CAS  Google Scholar 

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

    Article  Google Scholar 

  37. Adams, P. D. et al. Recent developments in the PHENIX software for automated crystallographic structure determination. J. Synchrotron Radiat. 11, 53–55 (2004)

    Article  CAS  Google Scholar 

  38. Joosten, R. P., Womack, T., Vriend, G. & Bricogne, G. Re-refinement from deposited X-ray data can deliver improved models for most PDB entries. Acta Crystallogr. D 65, 176–185 (2009)

    Article  CAS  Google Scholar 

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

    Google Scholar 

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Acknowledgements

We thank T. Vondriska and K. Philipson as well as members of the Abramson, Wright and Grabe labs for useful discussions and for reading the manuscript. We would also like to thank S. Faham for contributions at the early stages of this work, S. Iwata for advance release of the Mhp1 coordinates (Protein Data Bank ID, 2X79), and R. Roskies for assistance with the computations. Simulations were carried out through a TeraGrid grant at the Pittsburgh Supercomputing Center and the Texas Advanced Computing Center. This work was supported by NIH grants GM078844 (J.A.), RGY0069 (J.A.) and DK19567 (E.M.W.), and a grant from the Human Frontier Science Program (J.A.). M.G. is an Alfred P. Sloan Research Fellow.

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Author notes

  1. Akira Watanabe and Seungho Choe: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physiology, University of California, Los Angeles, Los Angeles, 90095-1759, California, USA

    Akira Watanabe, Vincent Chaptal, Ernest M. Wright & Jeff Abramson

  2. Department of Biological Sciences, University of Pittsburgh, Pittsburgh, 15260, Pennsylvania, USA

    Seungho Choe, John M. Rosenberg & Michael Grabe

  3. Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, 15260, Pennsylvania, USA

    John M. Rosenberg & Michael Grabe

Authors
  1. Akira Watanabe
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  3. Vincent Chaptal
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  7. Jeff Abramson
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Contributions

Experiments were carried out and diffraction data collected by A.W., V.C. and J.A. Simulations were carried out by S.C. Data were analysed and the manuscript was prepared by all authors.

Corresponding authors

Correspondence to Michael Grabe or Jeff Abramson.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-6 with legends, Supplementary Table 1 and a legend for Supplementary Movie 1. (PDF 2560 kb)

Supplementary Movie 1

This movie shows a 200 ns unrestrained MD simulation of galactose exiting from the sodium glucose transporter from Vibrio parahaemolyticus (see Supplementary Information file for full legend. (MOV 7132 kb)

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Watanabe, A., Choe, S., Chaptal, V. et al. The mechanism of sodium and substrate release from the binding pocket of vSGLT. Nature 468, 988–991 (2010). https://doi.org/10.1038/nature09580

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