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Molecular basis of transport and regulation in the Na+/betaine symporter BetP


Osmoregulated transporters sense intracellular osmotic pressure and respond to hyperosmotic stress by accumulation of osmolytes to restore normal hydration levels. Here we report the determination of the X-ray structure of a member of the family of betaine/choline/carnitine transporters, the Na+-coupled symporter BetP from Corynebacterium glutamicum, which is a highly effective osmoregulated uptake system for glycine betaine. Glycine betaine is bound in a tryptophan box occluded from both sides of the membrane with aromatic side chains lining the transport pathway. BetP has the same overall fold as three unrelated Na+-coupled symporters. Whereas these are crystallized in either the outward-facing or the inward-facing conformation, the BetP structure reveals a unique intermediate conformation in the Na+-coupled transport cycle. The trimeric architecture of BetP and the break in three-fold symmetry by the osmosensing C-terminal helices suggest a regulatory mechanism of Na+-coupled osmolyte transport to counteract osmotic stress.

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Figure 1: Structure of an N-terminally truncated, surface-engineered BetP mutant.
Figure 2: The betaine-binding site.
Figure 3: The proposed sodium binding in BetP.
Figure 4: Conformational changes in Na + -coupled transport.
Figure 5: Trimer architecture of BetP.
Figure 6: Regulatory interactions mediated by the C-terminal domains.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates for the structure reported in this work have been deposited in the Protein Data Bank under accession number 2W8A.


  1. 1

    Burg, M. B. Molecular basis of osmotic regulation. Am. J. Physiol. 268, F983–F996 (1995)

    CAS  PubMed  Google Scholar 

  2. 2

    Kinne, R. K. The role of organic osmolytes in osmoregulation: from bacteria to mammals. J. Exp. Zool. 265, 346–355 (1993)

    CAS  Article  Google Scholar 

  3. 3

    Yancey, P. H. Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. J. Exp. Biol. 208, 2819–2830 (2005)

    CAS  Article  Google Scholar 

  4. 4

    Burg, M. B. & Ferraris, J. D. Intracellular organic osmolytes: function and regulation. J. Biol. Chem. 283, 7309–7313 (2008)

    CAS  Article  Google Scholar 

  5. 5

    Wood, J. M. et al. Osmosensing and osmoregulatory compatible solute accumulation by bacteria. Comp. Biochem. Physiol. A 130, 437–460 (2001)

    CAS  Article  Google Scholar 

  6. 6

    Wood, J. M. Bacterial osmosensing transporters. Methods Enzymol. 428, 77–107 (2007)

    CAS  Article  Google Scholar 

  7. 7

    da Costa, M. S., Santos, H. & Galinski, E. A. An overview of the role and diversity of compatible solutes in Bacteria and Archaea. Adv. Biochem. Eng. Biotechnol. 61, 117–153 (1998)

    CAS  PubMed  Google Scholar 

  8. 8

    Roberts, M. F. Osmoadaptation and osmoregulation in archaea. Front. Biosci. 5, D796–D812 (2000)

    CAS  Article  Google Scholar 

  9. 9

    Pflüger, K. & Müller, V. Transport of compatible solutes in extremophiles. J. Bioenerg. Biomembr. 36, 17–24 (2004)

    Article  Google Scholar 

  10. 10

    Blomberg, A. Osmoresponsive proteins and functional assessment strategies in Saccharomyces cerevisiae . Electrophoresis 18, 1429–1440 (1997)

    CAS  Article  Google Scholar 

  11. 11

    Tuteja, N. Mechanisms of high salinity tolerance in plants. Methods Enzymol. 428, 419–438 (2007)

    CAS  Article  Google Scholar 

  12. 12

    Beck, F. X. & Neuhofer, W. Response of renal medullary cells to osmotic stress. Contrib. Nephrol. 148, 21–34 (2005)

    PubMed  Google Scholar 

  13. 13

    Lang, F. Mechanisms and significance of cell volume regulation. J. Am. Coll. Nutr. 26, 613S–623S (2007)

    CAS  Article  Google Scholar 

  14. 14

    Lim, C. H., Bot, A. G., de Jonge, H. R. & Tilly, B. C. Osmosignaling and volume regulation in intestinal epithelial cells. Methods Enzymol. 428, 325–342 (2007)

    CAS  Article  Google Scholar 

  15. 15

    Rösgen, J. Molecular basis of osmolyte effects on protein and metabolites. Methods Enzymol. 428, 459–486 (2007)

    Article  Google Scholar 

  16. 16

    Garcia-Perez, A. & Burg, M. B. Importance of organic osmolytes for osmoregulation by renal medullary cells. Hypertension 16, 595–602 (1990)

    CAS  Article  Google Scholar 

  17. 17

    Kempf, B. & Bremer, E. Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments. Arch. Microbiol. 170, 319–330 (1998)

    CAS  Article  Google Scholar 

  18. 18

    Empadinhas, N. & da Costa, M. S. Diversity and biosynthesis of compatible solutes in hyper/thermophiles. Int. Microbiol. 9, 199–206 (2006)

    CAS  PubMed  Google Scholar 

  19. 19

    Athawale, M. V., Dordick, J. S. & Garde, S. Osmolyte trimethylamine-N-oxide does not affect the strength of hydrophobic interactions: origin of osmolyte compatibility. Biophys. J. 89, 858–866 (2005)

    CAS  Article  Google Scholar 

  20. 20

    Peter, H., Burkovski, A. & Krämer, R. Isolation, characterization, and expression of the Corynebacterium glutamicum betP gene, encoding the transport system for the compatible solute betaine. J. Bacteriol. 178, 5229–5234 (1996)

    CAS  Article  Google Scholar 

  21. 21

    Kappes, R. M., Kempf, B. & Bremer, E. Three transport systems for the osmoprotectant betaine operate in Bacillus subtilis: characterization of OpuD. J. Bacteriol. 178, 5071–5079 (1996)

    CAS  Article  Google Scholar 

  22. 22

    Farwick, M., Siewe, R. M. & Krämer, R. Betaine uptake after hyperosmotic shift in Corynebacterium glutamicum . J. Bacteriol. 177, 4690–4695 (1995)

    CAS  Article  Google Scholar 

  23. 23

    Schiller, D., Krämer, R. & Morbach, S. Cation specificity of osmosensing by the betaine carrier BetP of Corynebacterium glutamicum . FEBS Lett. 563, 108–112 (2004)

    CAS  Article  Google Scholar 

  24. 24

    Peter, H., Burkovski, A. & Krämer, R. Osmo-sensing by N- and C-terminal extensions of the betaine uptake system BetP of Corynebacterium glutamicum . J. Biol. Chem. 273, 2567–2574 (1998)

    CAS  Article  Google Scholar 

  25. 25

    Ziegler, C. et al. Projection structure and oligomeric state of the osmoregulated sodium/betaine symporter BetP of Corynebacterium glutamicum . J. Mol. Biol. 337, 1137–1147 (2004)

    CAS  Article  Google Scholar 

  26. 26

    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)

    ADS  CAS  Article  Google Scholar 

  27. 27

    Chang, A. B., Lin, R., Studley, W. K., Tran, C. V. & Saier, M. H. Phylogeny as a guide to structure and function of membrane transport proteins. Mol. Membr. Biol. 21, 171–181 (2004)

    CAS  Article  Google Scholar 

  28. 28

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

    ADS  CAS  Article  Google Scholar 

  29. 29

    Saier, M. H. J. Families of transmembrane sugar transport proteins. Mol. Microbiol. 35, 699–710 (2000)

    CAS  Article  Google Scholar 

  30. 30

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

    ADS  CAS  Article  Google Scholar 

  31. 31

    Schiefner, A. et al. Cation-π interactions as determinants for binding of the compatible solutes betaine and proline betaine by the periplasmic ligand-binding protein ProX from Escherichia coli . J. Biol. Chem. 279, 5588–5596 (2004)

    CAS  Article  Google Scholar 

  32. 32

    Kempson, S. A. & Montrose, M. H. Osmotic regulation of renal betaine transport: transcription and beyond. Pflugers Arch. 449, 227–234 (2004)

    CAS  PubMed  Google Scholar 

  33. 33

    Borden, L. A. GABA transporter heterogeneity: pharmacology and cellular localization. Neurochem. Int. 29, 335–356 (1996)

    CAS  Article  Google Scholar 

  34. 34

    Rapp, M. G. E., Seppälä, S. & von Heijne, G. Identification and evolution of dual-topology membrane proteins. Nature Struct. Mol. Biol. 13, 112–116 (2006)

    CAS  Article  Google Scholar 

  35. 35

    Schiefner, A., Holtmann, G., Diederichs, K., Welte, W. & Bremer, E. Structural basis for the binding of compatible solutes by ProX from the hyperthermophilic archaeon Archaeoglobus fulgidus . J. Biol. Chem. 279, 48270–48281 (2004)

    CAS  Article  Google Scholar 

  36. 36

    Horn, C. et al. Molecular determinants for substrate specificity of the ligand-binding protein OpuAC from Bacillus subtilis for the compatible solutes betaine and proline betaine. J. Mol. Biol. 357, 592–606 (2006)

    CAS  Article  Google Scholar 

  37. 37

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

    ADS  CAS  Article  Google Scholar 

  38. 38

    Bolen, D. W. & Rose, G. D. Structure and energetics of the hydrogen-bonded backbone in protein folding. Annu. Rev. Biochem. 77, 339–362 (2008)

    CAS  Article  Google Scholar 

  39. 39

    Tanford, C. Protein denaturation. C. Theoretical models for the mechanism of denaturation. Adv. Protein Chem. 24, 1–95 (1970)

    CAS  Article  Google Scholar 

  40. 40

    Kuhlmann, S. I., Terwisscha van Scheltinga, A. C., Bienert, R., Kunte, H. J. & Ziegler, C. Osmoregulated transport of compatible solutes in the halophilic bacterium Halomonas elongata: 1.55 Å high-resolution structure of the periplasmic ectoine-binding protein from TRAP-transporter TeaABC. Biochemistry 47, 9475–9485 (2008)

    CAS  Article  Google Scholar 

  41. 41

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

    ADS  CAS  Article  Google Scholar 

  42. 42

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

    ADS  CAS  Article  Google Scholar 

  43. 43

    Lolkema, J. S. & Slotboom, D.-J. The major amino acid transporter superfamily has a similar core structure as Na+-galactose and Na+-leucine transporters. Mol. Membr. Biol. 25, 567–570 (2008)

    CAS  Article  Google Scholar 

  44. 44

    Smicun, Y., Campbell, S. D., Chen, M. A., Gu, H. & Rudnick, G. The role of external loop regions in serotonin transport. Loop scanning mutagenesis of the serotonin transporter external domain. J. Biol. Chem. 274, 36058–36064 (1999)

    CAS  Article  Google Scholar 

  45. 45

    Stephan, M. M., Chen, M. A., Penado, K. M. & Rudnick, G. An extracellular loop region of the serotonin transporter may be involved in the translocation mechanism. Biochemistry 36, 1322–1330 (1997)

    CAS  Article  Google Scholar 

  46. 46

    Palsdottir, H. & Hunte, C. Lipids in membrane protein structures. Biochim. Biophys. Acta 1666, 2–18 (2004)

    CAS  Article  Google Scholar 

  47. 47

    Ott, V., Koch, J., Späte, K., Morbach, S. & Krämer, R. Regulatory properties and interaction of the C- and N-terminal domains of BetP, an osmoregulated betaine transporter from Corynebacterium glutamicum . Biochemistry 47, 12208–12218 (2008)

    CAS  Article  Google Scholar 

  48. 48

    Schiller, D., Rübenhagen, R., Krämer, R. & Morbach, S. The C-terminal domain of the betaine carrier BetP of Corynebacterium glutamicum is directly involved in sensing K+ as an osmotic stimulus. Biochemistry 43, 5583–5591 (2004)

    CAS  Article  Google Scholar 

  49. 49

    Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993)

    CAS  Article  Google Scholar 

  50. 50

    Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006)

    Article  Google Scholar 

  51. 51

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

    ADS  CAS  Article  Google Scholar 

  52. 52

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

    CAS  Google Scholar 

  53. 53

    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)

    CAS  Article  Google Scholar 

  54. 54

    Cowtan, K. dm: An automated procedure for phase improvement by density modification. CCP4/ESF-EACBM Newslett. Protein Crystallogr. 31, 34–38 (1994)

    Google Scholar 

  55. 55

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

    Article  Google Scholar 

  56. 56

    Jones, T. A., Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for the building of protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  Google Scholar 

  57. 57

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

    CAS  Article  Google Scholar 

  58. 58

    Terwilliger, T. C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr. D 64, 61–69 (2008)

    CAS  Article  Google Scholar 

  59. 59

    Lovell, S. C. et al. Structure validation by C-alpha geometry: phi, psi, and C-beta deviation. Proteins Struct. Funct. Genet. 50, 437–450 (2003)

    CAS  Article  Google Scholar 

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The authors thank W. Kühlbrandt and R. Krämer for support and comments on the manuscript; J. Standfuß for contributions in the early stages of the project; Ö. Yildiz, T. Barros and R. Wouts for computational support; S. Schulze, S. Morbach, S. Nicklisch and L. Forrest for discussions; J. Hakulinen and J. Carrera for cloning, C. Perez for the reconstitution and freeze fracture experiments; and H. Volk for help with the figures. Special thanks are due to E. Pohl and the X10SA beamline staff at the Swiss Light Source, as well as the European Synchrotron Radiation Facility. This work is supported by the German Research Foundation, Collaborative Research Centre 807 ‘Transport and Communication across Biological Membranes’.

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Correspondence to Christine Ziegler.

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

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