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Crystal structure of a phosphorylation-coupled saccharide transporter

Abstract

Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His 250 and Glu 334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation.

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Figure 1: Function and structure of ChbC.
Figure 2: The C-terminal sugar-binding domain.
Figure 3: Proposed conformational changes in sugar transport.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

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

References

  1. Kundig, W., Ghosh, S. & Roseman, S. Phosphate bound to histidine in a protein as an intermediate in a novel phospho-transferase system. Proc. Natl Acad. Sci. USA 52, 1067–1074 (1964)

    ADS  CAS  Article  Google Scholar 

  2. Postma, P. W., Lengeler, J. W. & Jacobson, G. R. Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. Microbiol. Rev. 57, 543–594 (1993)

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Postma, P. W. & Lengeler, J. W. Phosphoenolpyruvate:carbohydrate phosphotransferase system of bacteria. Microbiol. Rev. 49, 232–269 (1985)

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Siebold, C., Flükiger, K., Beutler, R. & Erni, B. Carbohydrate transporters of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS). FEBS Lett. 504, 104–111 (2001)

    CAS  Article  Google Scholar 

  5. Robillard, G. T. & Broos, J. Structure/function studies on the bacterial carbohydrate transporters, enzymes II, of the phosphoenolpyruvate-dependent phosphotransferase system. Biochim. Biophys. Acta 1422, 73–104 (1999)

    CAS  Article  Google Scholar 

  6. Nguyen, T. X., Yen, M. R., Barabote, R. D. & Saier, M. H., Jr Topological predictions for integral membrane permeases of the phosphoenolpyruvate:sugar phosphotransferase system. J. Mol. Microbiol. Biotechnol. 11, 345–360 (2006)

    CAS  Article  Google Scholar 

  7. Meadow, N. D., Fox, D. K. & Roseman, S. The bacterial phosphoenolpyruvate: glycose phosphotransferase system. Annu. Rev. Biochem. 59, 497–542 (1990)

    CAS  Article  Google Scholar 

  8. Rees, D. C., Johnson, E. & Lewinson, O. ABC transporters: the power to change. Nature Rev. Mol. Cell Biol. 10, 218–227 (2009)

    CAS  Article  Google Scholar 

  9. Davidson, A. L., Dassa, E., Orelle, C. & Chen, J. Structure, function, and evolution of bacterial ATP-binding cassette systems. Microbiol. Mol. Biol. Rev. 72, 317–364 (2008)

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  11. Lemieux, M. J., Huang, Y. & Wang, D. N. The structural basis of substrate translocation by the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamily. Curr. Opin. Struct. Biol. 14, 405–412 (2004)

    CAS  Article  Google Scholar 

  12. Abramson, J., Kaback, H. R. & Iwata, S. Structural comparison of lactose permease and the glycerol-3-phosphate antiporter: members of the major facilitator superfamily. Curr. Opin. Struct. Biol. 14, 413–419 (2004)

    CAS  Article  Google Scholar 

  13. Rephaeli, A. W. & Saier, M. H., Jr Regulation of genes coding for enzyme constituents of the bacterial phosphotransferase system. J. Bacteriol. 141, 658–663 (1980)

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Deutscher, J., Francke, C. & Postma, P. W. How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria. Microbiol. Mol. Biol. Rev. 70, 939–1031 (2006)

    CAS  Article  Google Scholar 

  15. Saier, M. H., Hvorup, R. N. & Barabote, R. D. Evolution of the bacterial phosphotransferase system: from carriers and enzymes to group translocators. Biochem. Soc. Trans. 33, 220–224 (2005)

    CAS  Article  Google Scholar 

  16. Saraceni-Richards, C. A. & Jacobson, G. R. Subunit and amino acid interactions in the Escherichia coli mannitol permease: a functional complementation study of coexpressed mutant permease proteins. J. Bacteriol. 179, 5171–5177 (1997)

    CAS  Article  Google Scholar 

  17. Saraceni-Richards, C. A. & Jacobson, G. R. A conserved glutamate residue, Glu-257, is important for substrate binding and transport by the Escherichia coli mannitol permease. J. Bacteriol. 179, 1135–1142 (1997)

    CAS  Article  Google Scholar 

  18. Keyhani, N. O. & Roseman, S. Wild-type Escherichia coli grows on the chitin disaccharide, N,N′-diacetylchitobiose, by expressing the cel operon. Proc. Natl Acad. Sci. USA 94, 14367–14371 (1997)

    ADS  CAS  Article  Google Scholar 

  19. Keyhani, N. O., Wang, L. X., Lee, Y. C. & Roseman, S. The chitin disaccharide, N,N′-diacetylchitobiose, is catabolized by Escherichia coli and is transported/phosphorylated by the phosphoenolpyruvate:glycose phosphotransferase system. J. Biol. Chem. 275, 33084–33090 (2000)

    CAS  Article  Google Scholar 

  20. van Montfort, B. A. et al. Mapping of the dimer interface of the Escherichia coli mannitol permease by cysteine cross-linking. J. Biol. Chem. 277, 14717–14723 (2002)

    CAS  Article  Google Scholar 

  21. Erni, B. Glucose-specific permease of the bacterial phosphotransferase system: phosphorylation and oligomeric structure of the glucose-specific IIGlc-IIIGlc complex of Salmonella typhimurium . Biochemistry 25, 305–312 (1986)

    CAS  Article  Google Scholar 

  22. Pas, H. H., Ellory, J. C. & Robillard, G. T. Bacterial phosphoenolpyruvate-dependent phosphotransferase system: association state of membrane-bound mannitol-specific enzyme II demonstrated by inactivation. Biochemistry 26, 6689–6696 (1987)

    CAS  Article  Google Scholar 

  23. Khandekar, S. S. & Jacobson, G. R. Evidence for two distinct conformations of the Escherichia coli mannitol permease that are important for its transport and phosphorylation functions. J. Cell. Biochem. 39, 207–216 (1989)

    CAS  Article  Google Scholar 

  24. Chen, Q. & Amster-Choder, O. BglF, the sensor of the bgl system and the β-glucosides permease of Escherichia coli: evidence for dimerization and intersubunit phosphotransfer. Biochemistry 37, 8714–8723 (1998)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  26. Daley, D. O. et al. Global topology analysis of the Escherichia coli inner membrane proteome. Science 308, 1321–1323 (2005)

    ADS  CAS  Article  Google Scholar 

  27. Sugiyama, J. E., Mahmoodian, S. & Jacobson, G. R. Membrane topology analysis of Escherichia coli mannitol permease by using a nested-deletion method to create mtlA-phoA fusions. Proc. Natl Acad. Sci. USA 88, 9603–9607 (1991)

    ADS  CAS  Article  Google Scholar 

  28. Buhr, A. & Erni, B. Membrane topology of the glucose transporter of Escherichia coli . J. Biol. Chem. 268, 11599–11603 (1993)

    CAS  PubMed  Google Scholar 

  29. Yagur-Kroll, S. & Amster-Choder, O. Dynamic membrane topology of the Escherichia coli β-glucoside transporter BglF. J. Biol. Chem. 280, 19306–19318 (2005)

    CAS  Article  Google Scholar 

  30. Koning, R. I. et al. The 5 Å projection structure of the transmembrane domain of the mannitol transporter enzyme II. J. Mol. Biol. 287, 845–851 (1999)

    CAS  Article  Google Scholar 

  31. 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 

  32. Weng, Q. P. & Jacobson, G. R. Role of a conserved histidine residue, His-195, in the activities of the Escherichia coli mannitol permease. Biochemistry 32, 11211–11216 (1993)

    CAS  Article  Google Scholar 

  33. Weng, Q. P., Elder, J. & Jacobson, G. R. Site-specific mutagenesis of residues in the Escherichia coli mannitol permease that have been suggested to be important for its phosphorylation and chemoreception functions. J. Biol. Chem. 267, 19529–19535 (1992)

    CAS  PubMed  Google Scholar 

  34. Opacic´, M., Vos, E. P., Hesp, B. H. & Broos, J. Localization of the substrate-binding site in the homodimeric mannitol transporter, EIImtl, of Escherichia coli . J. Biol. Chem. 285, 25324–25331 (2010)

    Article  Google Scholar 

  35. Widdas, W. F. Inability of diffusion to account for placental glucose transfer in the sheep and consideration of the kinetics of a possible carrier transfer. J. Physiol. (Lond.) 118, 23–39 (1952)

    CAS  Article  Google Scholar 

  36. Yamashita, A. et al. Crystal structure of a bacterial homologue of Na+/Cl-dependent neurotransmitter transporters. Nature 437, 215–223 (2005)

    ADS  CAS  Article  Google Scholar 

  37. Reyes, N., Ginter, C. & Boudker, O. Transport mechanism of a bacterial homologue of glutamate transporters. Nature 462, 880–885 (2009)

    ADS  CAS  Article  Google Scholar 

  38. Shi, L. et al. 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)

    CAS  Article  Google Scholar 

  39. Love, J. et al. The New York Consortium on Membrane Protein Structure (NYCOMPS): a high-throughput platform for structural genomics of integral membrane proteins. J. Struct. Funct. Genomics 11, 191–199 (2010)

    CAS  Article  Google Scholar 

  40. Zhou, M. & MacKinnon, R. A mutant KcsA K+ channel with altered conduction properties and selectivity filter ion distribution. J. Mol. Biol. 338, 839–846 (2004)

    CAS  Article  Google Scholar 

  41. Rocchia, W. et al. Rapid grid-based construction of the molecular surface and the use of induced surface charge to calculate reaction field energies: applications to the molecular systems and geometric objects. J. Comput. Chem. 23, 128–137 (2002)

    CAS  Article  Google Scholar 

  42. Punta, M. et al. Structural genomics target selection for the New York Consortium on Membrane Protein Structure. J. Struct. Funct. Genomics 10, 255–268 (2009)

    Article  Google Scholar 

  43. Levin, E. J., Quick, M. & Zhou, M. Crystal structure of a bacterial homologue of the kidney urea transporter. Nature 462, 757–761 (2009)

    ADS  CAS  Article  Google Scholar 

  44. Schaffner, W. & Weissmann, C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal. Biochem. 56, 502–514 (1973)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  47. 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)

    CAS  Article  Google Scholar 

  48. Cowtan, K. Error estimation and bias correction in phase-improvement calculations. Acta Crystallogr. D 55, 1555–1567 (1999)

    CAS  Article  Google Scholar 

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

    Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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Acknowledgements

Data for this study were measured at beamlines X4A, X4C, X25 and X29 of the National Synchrotron Light Source and the NE-CAT 24ID-C and E at the Advanced Photon Source. This work was supported by the US National Institutes of Health (DK088057, GM098878 and GM05026-sub0007 to M.Z., and T32HL087745 to E.J.L.). M.Z. is a Pew Scholar in Biomedical Sciences. The NYCOMPS central facility was supported by GM05026 to W.A.H. as part of the Protein Structure Initiative (PSI-2) established by the National Institute of General Medical Sciences. The authors would like to thank B. Honig for support and M. Saier, B. Erni, R. Kaback and D.-N. Wang for comments on the manuscript and helpful discussions. M.Z. is grateful to R. MacKinnon for advice and encouragement.

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J.L., M.P., B.R. and W.A.H. identified ChbC homologues in the database. J.L. carried out the cloning and the initial expression studies. Y.C., H.H., E.J.L., J.W. and M.Z. performed protein expression, purification, crystallization and X-ray diffraction data collection and analysis. X.J., Y.Z., E.J.L. and M.Z. solved and refined the structures. M.Q., Y.C., Y.P., J.A.J. and M.Z. characterized ChbC function. K.R.R. advised on data collection and crystallography. E.J.L. and M.Z. wrote the manuscript with input from all authors.

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Correspondence to Ming Zhou.

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Cao, Y., Jin, X., Levin, E. et al. Crystal structure of a phosphorylation-coupled saccharide transporter. Nature 473, 50–54 (2011). https://doi.org/10.1038/nature09939

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