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 catalytic intermediate of the maltose transporter

Abstract

The maltose uptake system of Escherichia coli is a well-characterized member of the ATP-binding cassette transporter superfamily. Here we present the 2.8-Å crystal structure of the intact maltose transporter in complex with the maltose-binding protein, maltose and ATP. This structure, stabilized by a mutation that prevents ATP hydrolysis, captures the ATP-binding cassette dimer in a closed, ATP-bound conformation. Maltose is occluded within a solvent-filled cavity at the interface of the two transmembrane subunits, about halfway into the lipid bilayer. The binding protein docks onto the entrance of the cavity in an open conformation and serves as a cap to ensure unidirectional translocation of the sugar molecule. These results provide direct evidence for a concerted mechanism of transport in which solute is transferred from the binding protein to the transmembrane subunits when the cassette dimer closes to hydrolyse ATP.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Structure of the maltose transporter in a catalytic intermediate conformation.
Figure 2: Ribbon diagram of the MalK subunits with bound ATP.
Figure 3: Architecture of the transmembrane subunits.
Figure 4: The TMD–MalK interface.
Figure 5: Transfer of the maltose from MBP to the TM binding site.
Figure 6: Comparison of the structures of two binding-protein-dependent ABC uptake systems.
Figure 7: A model for the ABC uptake system.

References

  1. Higgins, C. F. ABC transporters: from microorganisms to man. Annu. Rev. Cell Biol. 8, 67–113 (1992)

    CAS  Article  Google Scholar 

  2. Boos, W. & Lucht, J. M. in Escherichia coli and Salmonella: Cellular and Molecular Biology (eds Neidhardt, F. C. et al.) 1175–1209 (ASM Press, Washington DC, 1996)

    Google Scholar 

  3. Holland, I. B. & Blight, M. A. ABC-ATPases, adaptable energy generators fuelling transmembrane movement of a variety of molecules in organisms from bacteria to humans. J. Mol. Biol. 293, 381–399 (1999)

    CAS  Article  Google Scholar 

  4. Walker, J. E., Saraste, M., Runswick, M. J. & Gay, N. J. Distantly related sequences in the α- and β-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1, 945–951 (1982)

    CAS  Article  Google Scholar 

  5. Pinkett, H. W., Lee, A. T., Lum, P., Locher, K. P. & Rees, D. C. An inward-facing conformation of a putative metal-chelate-type ABC transporter. Science 315, 373–377 (2007)

    ADS  CAS  Article  Google Scholar 

  6. Locher, K. P., Lee, A. T. & Rees, D. C. The E. coli BtuCD structure: a framework for ABC transporter architecture and mechanism. Science 296, 1091–1098 (2002)

    ADS  CAS  Article  Google Scholar 

  7. Hollenstein, K., Frei, D. C. & Locher, K. P. Structure of an ABC transporter in complex with its binding protein. Nature 446, 213–216 (2007)

    ADS  CAS  Article  Google Scholar 

  8. Dawson, R. J. & Locher, K. P. Structure of a bacterial multidrug ABC transporter. Nature 443, 180–185 (2006)

    ADS  CAS  Article  Google Scholar 

  9. Ferenci, T. The recognition of maltodextrins by Escherichia coli. . Eur. J. Biochem. 108, 613–636 (1980)

    Article  Google Scholar 

  10. Raibaud, O., Roa, M., Braun-Breton, C. & Schwartz, M. Structure of the malB region in Escherichia coli K-12. I. Genetic map of the malK-lamB operon. Mol. Gen. Genet. 174, 241–248 (1979)

    CAS  Article  Google Scholar 

  11. Silhavy, T. J. et al. Structure of the malB region in Escherichia coli K12. II. Genetic map of the malE,F,G operon. Mol. Gen. Genet. 174, 249–259 (1979)

    CAS  Article  Google Scholar 

  12. Davidson, A. L. & Nikaido, H. Purification and characterization of the membrane-associated components of the maltose transport system from Escherichia coli . J. Biol. Chem. 266, 8946–8951 (1991)

    CAS  PubMed  Google Scholar 

  13. Davidson, A. L. & Nikaido, H. Overproduction, solubilization, and reconstitution of the maltose transport system from Escherichia coli . J. Biol. Chem. 265, 4254–4260 (1990)

    CAS  PubMed  Google Scholar 

  14. Landmesser, H. et al. Large-scale purification, dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK(2)) of Salmonella typhimurium . Biochim. Biophys. Acta 1565, 64–72 (2002)

    CAS  Article  Google Scholar 

  15. Davidson, A. L., Shuman, H. A. & Nikaido, H. Mechanism of maltose transport in Escherichia coli: Transmembrane signalling by periplasmic binding proteins. Proc. Natl Acad. Sci. USA 89, 2360–2364 (1992)

    ADS  CAS  Article  Google Scholar 

  16. Sharff, A. J., Rodseth, L. E., Spurlino, J. E. & Quiocho, F. A. Crystallographic evidence of a large ligand-induced hinge-twist motion between the two domains of the maltodextrin binding protein involved in active transport and chemotaxis. Biochemistry 31, 10657–10663 (1992)

    CAS  Article  Google Scholar 

  17. Quiocho, F. A., Spurlino, J. C. & Rodseth, L. E. Extensive features of tight oligosaccharide binding revealed in high-resolution structures of the maltodextrin transport/chemosensory receptor. Structure 5, 997–1015 (1997)

    CAS  Article  Google Scholar 

  18. Duan, X. & Quiocho, F. A. Structural evidence for the dominant role of nonpolar interactions in the binding of a transport/chemonsensory receptor to its highly polar ligands. Biochemistry 41, 706–712 (2002)

    CAS  Article  Google Scholar 

  19. Chen, J., Sharma, S., Quiocho, F. A. & Davidson, A. L. Trapping the transition state of an ATP-binding-cassette transporter: Evidence for a concerted mechanism of maltose transport. Proc. Natl Acad. Sci. USA 98, 1525–1530 (2001)

    ADS  CAS  Article  Google Scholar 

  20. Chen, J., Lu, G., Lin, J., Davidson, A. L. & Quiocho, F. A. A tweezers-like motion of the ATP-binding cassette dimer in an ABC transport cycle. Mol. Cell 12, 651–661 (2003)

    CAS  Article  Google Scholar 

  21. Lu, G., Westbrooks, J. M., Davidson, A. L. & Chen, J. ATP hydrolysis is required to reset the ATP-binding cassette dimer into the resting-state conformation. Proc. Natl Acad. Sci. USA 102, 17969–17974 (2005)

    ADS  CAS  Article  Google Scholar 

  22. Orelle, C., Dalmas, O., Gros, P., Di Pietro, A. & Jault, J. M. The conserved glutamate residue adjacent to the Walker-B motif is the catalytic base for ATP hydrolysis in the ATP-binding cassette transporter BmrA. J. Biol. Chem. 278, 47002–47008 (2003)

    CAS  Article  Google Scholar 

  23. Moody, J. E., Millen, L., Binns, D., Hunt, J. F. & Thomas, P. J. Cooperative, ATP-dependent association of the nucleotide binding cassettes during the catalytic cycle of ATP-binding cassette transporters. J. Biol. Chem. 277, 21111–21114 (2002)

    CAS  Article  Google Scholar 

  24. Smith, P. C. et al. ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer. Mol. Cell 10, 139–149 (2002)

    CAS  Article  Google Scholar 

  25. Rossmann, M. G. The molecular replacement method. Acta Crystallogr. A 46, 73–82 (1990)

    Article  Google Scholar 

  26. Covitz, K.-M. Y., Panagiotidis, C. H., Reyes, M., Treptow, N. A. & Shuman, H. A. Mutations that alter the transmembrane signalling pathway in an ATP binding cassette (ABC) transporter. EMBO J. 13, 1752–1759 (1994)

    CAS  Article  Google Scholar 

  27. Dassa, E. & Muir, S. Membrane topology of MalG, an inner membrane protein from the maltose transport system of Escherichia coli . Mol. Microbiol. 7, 29–38 (1993)

    CAS  Article  Google Scholar 

  28. Froshauer, S., Green, G. N., Boyd, D., McGovern, K. & Beckwith, J. Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli . J. Mol. Biol. 200, 501–511 (1988)

    CAS  Article  Google Scholar 

  29. Hvorup, R. N. et al. Asymmetry in the structure of the ABC transporter binding protein complex BtuCD-BtuF. Science 317, 1387–1390 (2007)

    ADS  CAS  Article  Google Scholar 

  30. Dassa, E. & Hofnung, M. Sequence of gene malG in E. coli K12: homologies between integral membrane components from binding protein-dependent transport systems. EMBO J. 4, 2287–2293 (1985)

    CAS  Article  Google Scholar 

  31. Saurin, W., Koster, W. & Dassa, E. Bacterial binding protein-dependent permeases: characterization of distinctive signatures for functionally related integral cytoplasmic membrane proteins. Mol. Microbiol. 12, 993–1004 (1994)

    CAS  Article  Google Scholar 

  32. Busch, W. & Saier, M. H. Jr. The transporter classification (TC) system, 2002. Crit. Rev. Biochem. Mol. Biol. 37, 287–337 (2002)

    CAS  Article  Google Scholar 

  33. Mourez, M., Hofnung, M. & Dassa, E. Subunit interactions in ABC transporters: a conserved sequence in hydrophobic membrane proteins of periplasmic permeases defines an important site of interaction with the ATPase subunits. EMBO J. 16, 3066–3077 (1997)

    CAS  Article  Google Scholar 

  34. Ehrle, R., Pick, C., Ulrich, R., Hofmann, E. & Ehrmann, M. Characterization of transmembrane domains 6, 7, and 8 of MalF by mutational analysis. J. Bacteriol. 178, 2255–2262 (1996)

    CAS  Article  Google Scholar 

  35. Steinke, A., Grau, S., Davidson, A., Hofmann, E. & Ehrmann, M. Characterization of transmembrane segments 3, 4, and 5 of MalF by mutational analysis. J. Bacteriol. 183, 375–381 (2001)

    CAS  Article  Google Scholar 

  36. Quiocho, F. A. Carbohydrate-binding proteins: tertiary structures and protein–sugar interactions. Annu. Rev. Biochem. 55, 287–315 (1986)

    CAS  Article  Google Scholar 

  37. Shuman, H. A. Active transport of maltose in Escherichia coli K-12: role of the periplasmic maltose binding protein and evidence for a substrate recognition site in the cytoplasmic membrane. J. Biol. Chem. 257, 5455–5461 (1982)

    CAS  PubMed  Google Scholar 

  38. Schirmer, T., Keller, T. A., Wang, Y. F. & Rosenbusch, J. P. Structural basis for sugar translocation through maltoporin channels at 3.1 Å resolution. Science 267, 512–514 (1995)

    ADS  CAS  Article  Google Scholar 

  39. Quiocho, F. A. & Ledvina, P. S. Atomic structure and specificity of bacterial periplasmic receptors for active transport and chemotaxis: variation of common themes. Mol. Microbiol. 20, 17–25 (1996)

    CAS  Article  Google Scholar 

  40. Miller, D. M., Olson, J. S., Pflugrath, J. W. & Quiocho, F. A. Rates of ligand binding to periplasmic proteins involved in bacterial transport and chemotaxis. J. Biol. Chem. 258, 13665–13672 (1983)

    CAS  PubMed  Google Scholar 

  41. Nelson, B. D. & Traxler, B. Exploring the role of integral membrane proteins in ATP-binding cassette transporters: analysis of a collection of MalG insertion mutants. J. Bacteriol. 180, 2507–2514 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Austermuhle, M. I., Hall, J. A., Klug, C. S. & Davidson, A. L. Maltose-binding protein is open in the catalytic transition state for ATP hydrolysis during maltose transport. J. Biol. Chem. 279, 28243–28250 (2004)

    CAS  Article  Google Scholar 

  43. Fetsch, E. E. & Davidson, A. L. Vanadate-catalyzed photocleavage of the signature motif of an ATP-binding cassette (ABC) transporter. Proc. Natl Acad. Sci. USA 99, 9685–9690 (2002)

    ADS  CAS  Article  Google Scholar 

  44. Sharma, S. & Davidson, A. L. Vanadate-induced trapping of nucleotide by the purified maltose transport complex requires ATP hydrolysis. J. Bacteriol. 182, 6570–6576 (2000)

    CAS  Article  Google Scholar 

  45. Urbatsch, I. L., Sankaran, B., Bhagat, S. & Senior, A. E. Both P-glycoprotein nucleotide-binding sites are catalytically active. J. Biol. Chem. 270, 26956–26962 (1995)

    CAS  Article  Google Scholar 

  46. Urbatsch, I. L., Sankaran, B., Weber, J. & Senior, A. E. P-glycoprotein is stably inhibited by vanadate-induced trapping of nucleotide at a single catalytic site. J. Biol. Chem. 270, 19383–19390 (1995)

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  48. Neuhard, J. & Nygaard, P. in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (eds Ingraham, J. L. & Neidhardt, F. C.) 445–473 (ASM Press, Washington DC, 1987)

    Google Scholar 

  49. Davidson, A. L., Laghaeian, S. S. & Mannering, D. E. The maltose transport system of Escherichia coli displays positive cooperativity in ATP hydrolysis. J. Biol. Chem. 271, 4858–4863 (1996)

    CAS  Article  Google Scholar 

  50. Sun, H. & Nathans, J. Mechanistic studies of ABCR, the ABC transporter in photoreceptor outer segments responsible for autosomal recessive Stargardt disease. J. Bioenerg. Biomembr. 33, 523–530 (2001)

    CAS  Article  Google Scholar 

  51. Davidson, A. L. & Nikaido, H. Overproduction, solubilization, and reconstitution of the maltose transport system from Escherichia coli . J. Biol. Chem. 265, 4254–4260 (1990)

    CAS  PubMed  Google Scholar 

  52. Chen, J., Lu, G., Lin, J., Davidson, A. L. & Quiocho, F. A. A tweezers-like motion of the ATP-binding cassette dimer in an ABC transport cycle. Mol. Cell 12, 651–661 (2003)

    CAS  Article  Google Scholar 

  53. Dean, D. A., Fikes, J. D., Gehring, K., Bassford, P. J. & Nikaido, H. Active transport of maltose in membrane vesicles obtained from Escherichia coli cells producing tethered maltose-binding protein. J. Bacteriol. 171, 503–510 (1989)

    CAS  Article  Google Scholar 

  54. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Macro Crystallogr. A 276, 307–326 (1997)

    CAS  Article  Google Scholar 

  55. Project, C. C. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

  56. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building 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. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  59. Painter, J. & Merritt, E. A. TLSMD web server for the generation of multi-group TLS models. J. Appl. Cryst. 39, 109–111 (2006)

    CAS  Article  Google Scholar 

  60. Howlin, B., Butler, S. A., Moss, D. S., Harris, G. W. & Driessen, H. P. C. TLSANL—TLS parameter-analysis program for segmented anisotropic refinement of macromolecular structures. J. Appl. Cryst. 26, 622–624 (1993)

    Article  Google Scholar 

  61. Ehrmann, M. & Beckwith, J. Proper insertion of a complex membrane protein in the absence of its amino-terminal export signal. J. Biol. Chem. 266, 16530–16533 (1991)

    CAS  PubMed  Google Scholar 

  62. DeLano, W.L. The PyMOL Molecular Graphics System (2002) on the World Wide Web 〈http://www.pymol.org

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

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank the beamline staff at the Advanced Photon Source beamline 23-ID for assistance with data collection, and R. MacKinnon, H. Shuman, D. Yernool and C. Orelle for discussions. This work was supported by NIH grants (J.C., A.L.D. and F.A.Q.), the Welch Foundation (F.A.Q.), the Pew Scholar Program (J.C.) and a postdoctoral fellowship from American Heart Association (M.L.O).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jue Chen.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-5 and Legends, Supplementary Tables 1-2 and additional references. (PDF 762 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Oldham, M., Khare, D., Quiocho, F. et al. Crystal structure of a catalytic intermediate of the maltose transporter. Nature 450, 515–521 (2007). https://doi.org/10.1038/nature06264

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature06264

Further reading

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