SpringerLink
Log in

The structure of BtuB with bound colicin E3 R-domain implies a translocon

  • Article
  • Published:

From Nature Structural & Molecular Biology

View current issue Submit your manuscript

Abstract

Cellular import of colicin E3 is initiated by the Escherichia coli outer membrane cobalamin transporter, BtuB. The 135-residue 100-Å coiled-coil receptor-binding domain (R135) of colicin E3 forms a 1:1 complex with BtuB whose structure at a resolution of 2.75 Å is reported. Binding of R135 to the BtuB extracellular surface (ΔG° = −12 kcal mol−1) is mediated by 27 residues of R135 near the coiled-coil apex. Formation of the R135–BtuB complex results in unfolding of R135 N- and C-terminal ends, inferred to be important for unfolding of the colicin T-domain. Small conformational changes occur in the BtuB cork and barrel domains but are insufficient to form a translocation channel. The absence of a channel and the peripheral binding of R135 imply that BtuB serves to bind the colicin, and that the coiled-coil delivers the colicin to a neighboring outer membrane protein for translocation, thus forming a colicin translocon. The translocator was concluded to be OmpF from the occlusion of OmpF channels by colicin E3.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1: Structure of colicin E3 R-domain bound to BtuB.
Figure 2: Secondary structure changes of R-peptides upon binding to BtuB.
Figure 3: Structure changes induced in BtuB by binding of R135 or cobalamin.
Figure 4: Occlusion by colicin E3 of OmpF incorporated into planar bilayers.
Figure 5: A model for a two-receptor translocon used for import of intact colicin E3.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Keenan, R.J., Freymann, D.M., Stroud, R.M. & Walter, P. The signal recognition particle. Annu. Rev. Biochem. 70, 755–775 (2001).

    Article  CAS  Google Scholar 

  2. Rehling, P. et al. Protein insertion into the mitochondrial inner membrane by a twin-pore translocase. Science 299, 1747–1751 (2003).

    Article  CAS  Google Scholar 

  3. Schwartz, T. & Blobel, G. Structural basis for the function of the β subunit of the eukaryotic signal recognition particle receptor. Cell 112, 793–803 (2003).

    Article  CAS  Google Scholar 

  4. Breyton, C., Haase, W., Rapoport, T.A., Kuehlbrandt, W. & Collinson, I. Three-dimensional structure of the bacterial protein-translocation complex SecYEG. Nature 418, 662–665 (2002).

    Article  CAS  Google Scholar 

  5. Braun, V. Energy-coupled transport and signal transduction through the gram-negative outer membrane via TonB-ExbB-ExbD-dependent receptor proteins. FEMS Microbiol. Rev. 16, 295–307 (1995).

    Article  CAS  Google Scholar 

  6. James, R., Penfold, C.N., Moore, G.R. & Kleanthous, C. Killing of E. coli cells by E group nuclease colicins. Biochimie 84, 381–389 (2002).

    Article  CAS  Google Scholar 

  7. Bouveret, E. et al. Analysis of the Escherichia coli Tol-Pal and TonB systems by periplasmic production of Tol, TonB, colicin, or phage capsid soluble domains. Biochimie 84, 413–421 (2002).

    Article  CAS  Google Scholar 

  8. de Zamaroczy, M. & Buckingham, R.H. Importation of nuclease colicins into E. coli cells: endoproteolytic cleavage and its prevention by the Immunity protein. Biochimie 84, 423–432 (2002).

    Article  CAS  Google Scholar 

  9. Cao, Z. & Klebba, P.E. Mechanisms of colicin binding and transport through outer membrane porins. Biochimie 84, 399–412 (2002).

    Article  CAS  Google Scholar 

  10. Soelaiman, S., Jakes, K., Wu, N., Li, C.M. & Shoham, M. Crystal structure of colicin E3: implications for cell entry and ribosome inactivation. Mol. Cell 8, 1053–1062 (2001).

    Article  CAS  Google Scholar 

  11. Gernert, K.M., Surles, M.C., Labean, T.H., Richardson, J.S. & Richardson, D.C. The alacoil: a very tight, antiparallel coiled-coil of helices. Protein Sci. 4, 2252–2260 (1995).

    Article  CAS  Google Scholar 

  12. Wiener, M., Freymann, D., Ghosh, P. & Stroud, R.M. Crystal structure of colicin Ia. Nature 385, 461–464 (1997).

    Article  CAS  Google Scholar 

  13. Di Masi, D.R., White, J.C., Schnaitman, C.A. & Bradbeer, C. Transport of vitamin B12 in Escherichia coli: common receptor sites for vitamin B12 and the E colicins on the outer membrane of the cell envelope. J. Bacteriol. 115, 506–513 (1973).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Bradbeer, C., Woodrow, M.L. & Khalifah, L.I. Transport of vitamin B12 in Escherichia coli: common receptor system for vitamin B12 and bacteriophage BF23 on the outer membrane of the cell envelope. J. Bacteriol. 125, 1032–1039 (1976).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Chimento, D.P., Mohanty, A.K., Kadner, R.J. & Wiener, M. Substrate-induced transmembrane signaling in the cobalamin transporter BtuB. Nat. Struct. Biol. 10, 394–401 (2003).

    Article  CAS  Google Scholar 

  16. Buchanan, S.K. et al. Crystal structure of the outer membrane active transporter FepA from Escherichia coli. Nat. Struct. Biol. 6, 56–63 (1999).

    Article  CAS  Google Scholar 

  17. Ferguson, A.D., Hofmann, E., Coulton, J.W., Diederichs, K. & Welte, W. Siderophore-mediated iron transport: crystal structure of FhuA with bound lipopolysaccharide. Science 282, 2215–2220 (1998).

    Article  CAS  Google Scholar 

  18. Ferguson, A.D. et al. Structural basis of gating by the outer membrane transporter FecA. Science 295, 1715–1719 (2002).

    Article  CAS  Google Scholar 

  19. Taylor, R., Burgner, J.W., Clifton, J. & Cramer, W.A. Purification and characterization of monomeric Escherichia coli vitamin B12 receptor with high affinity for colicin E3. J. Biol. Chem. 273, 31113–31118 (1998).

    Article  CAS  Google Scholar 

  20. Boetzel, R. et al. Structural dynamics of the receptor-binding domain of colicin E9. Faraday Discuss. Chem. Soc. 122, 145–162 (2002).

    Article  Google Scholar 

  21. Penfold, C.N. et al. A 76-residue polypeptide of colicin E9 confers receptor specificity and inhibits the growth of vitamin B12-dependent Escherichia coli 113/3 cells. Mol. Microbiol. 38, 639–649 (2000).

    Article  CAS  Google Scholar 

  22. Walker, D., Moore, G.R., James, R. & Kleanthous, C. Thermodynamic consequences of bipartite immunity protein binding to the ribosomal ribonuclease colicin E3. Biochemistry 42, 4161–4171 (2003).

    Article  CAS  Google Scholar 

  23. Prince, S.M. et al. Detergent structure in crystals of the integral membrane light-harvesting complex LH2 from Rhodopseudomonas acidophila strain 10050. J. Mol. Biol. 326, 307–315 (2003).

    Article  CAS  Google Scholar 

  24. Larsen, R.A., Thomas, M.G. & Postle, K. Protonmotive force, ExbB, and ligand-bound FepA drive conformational changes in TonB. Mol. Microbiol. 31, 1809–1824 (1999).

    Article  CAS  Google Scholar 

  25. Jiang, X. et al. Ligand-specific opening of a gated-porin channel in the outer membrane of living bacteria. Science 276, 1261–1264 (1997).

    Article  CAS  Google Scholar 

  26. Locher, K.P. et al. Transmembrane signaling across the ligand-gated FhuA receptor: crystal structures of free and ferrichrome-bound states reveal allosteric changes. Cell 95, 771–778 (1998).

    Article  CAS  Google Scholar 

  27. Plancon, L., Chami, M. & Letellier, L. Reconstitution of FhuA, an Escherichia coli outer membrane protein, into liposomes. Binding of phage T5 to FhuA triggers the transfer of DNA into the proteoliposomes. J. Biol. Chem. 272, 16868–16872 (1997).

    Article  CAS  Google Scholar 

  28. Eroukova, V.Y. et al. Occlusion of channel activity of secondary outer membrane receptors by colicins. Biophys. J. 84, 521a (2003).

    Google Scholar 

  29. Benedetti, H. et al. Comparison of the uptake systems for the entry of various BtuB groups colicins into Escherichia coli. J. Gen. Microbiol. 135, 3413–3420 (1989).

    CAS  PubMed  Google Scholar 

  30. Schindler, H. & Rosenbusch, J.P. Matrix protein from Escherichia coli outer membranes forms voltage-controlled channels in lipid bilayers. Proc. Natl. Acad. Sci. USA 75, 3751–3755 (1978).

    Article  CAS  Google Scholar 

  31. Cowan, S.W. et al. Crystal structures explain functional properties of two E. coli porins. Nature 358, 727–733 (1992).

    Article  CAS  Google Scholar 

  32. Jeanteur, D. et al. Structural and functional alterations of a colicin-resistant mutant of OmpF porin from Escherichia coli. Proc. Natl. Acad. Sci. USA 91, 10675–10679 (1994).

    Article  CAS  Google Scholar 

  33. James, R., Kleanthous, C. & Moore, G.R. The biology of E colicins: paradigms and paradoxes. Microbiology 142, 1569–1580 (1996).

    Article  CAS  Google Scholar 

  34. Law, C.J. et al. OmpF enhances the ability of BtuB to protect susceptible Escherichia coli cells from colicin E9 cytotoxicity. FEBS Lett. 545, 127–132 (2003).

    Article  CAS  Google Scholar 

  35. Nikaido, H. & Vaara, M. In Outer Membrane in Escherichia coli and Salmonella typhimurium (ed. Neidhordt, F.C.) 7–22 (American Society of Microbiology, Washington, DC, 1987).

    Google Scholar 

  36. Collins, E.S. et al. Structural dynamics of the membrane translocation domain of colicin E9 and its interaction with TolB. J. Mol. Biol. 318, 787–804 (2002).

    Article  CAS  Google Scholar 

  37. Howard, J. Polymer mechanics. In Mechanics of Motor Proteins and the Cytoskeleton 99–116 (Sinauer, Sunderland, Massachusetts, USA, 2001).

    Google Scholar 

  38. Schindler, H. & Rosenbusch, J.P. Matrix protein in planar membranes: clusters of channels in a native environment and their functional reassembly. Proc. Natl. Acad. Sci. USA 78, 2302–2306 (1981).

    Article  CAS  Google Scholar 

  39. Sen, K., Hellman, J. & Nikaido, H. Porin channels in intact cells of Escherichia coli are not affected by Donnan potentials across the outer membrane. J. Biol. Chem. 263, 1182–1187 (1988).

    CAS  PubMed  Google Scholar 

  40. Seydel, U., Eberstein, W., Schroder, G. & Brandenburg, K. Electrostatic potential in asymmetric planar lipopolysaccharide/phospholipid bilayers probed with the valinomycin-K+ complex. Z. Naturforsch. B 47c, 757–761 (1992).

    Article  Google Scholar 

  41. Herschman, H.R. & Helinski, D.R. Purification and characterization of colicin E2 and colicin E3. J. Biol. Chem. 242, 5360–5368 (1967).

    CAS  PubMed  Google Scholar 

  42. Chen, Y.H., Yang, J.T. & Chau, K.H. Determination of the helix and β form of proteins in aqueous solution by circular dichroism. Biochemistry 13, 3350–3359 (1972).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

  45. Esnouf, R.M. An extensively modified version of MolScript that includes greatly enhanced coloring capabilities. J. Mol. Graph. 15, 132–134 (1997).

    Article  CAS  Google Scholar 

  46. Merritt, E.A. & Bacon, D.J. Raster3D: photorealistic molecular graphics. Methods Enzymol. 277, 505–524 (1997).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Lindeberg for experimental contributions in the early stages of these studies, J.T. Bolin, K. Jakes, and A. Raman for helpful discussions, P. Loll for samples of purified OmpF and W. Minor for generously sharing beam time. These studies have been supported by the US National Institutes of Health (NIH) and the Henry Koffler Professorship (WAC), a fellowship from the Japanese Ministry of Science and Education (G.K.), with NIH (M.W.), NIH/Fogarty (W.A.C. and Y.A.) and US Department of Energy and NIH National Center for Research Resources supporting, respectively, the Advanced Photon Source SBC-19BM and BioCARS 14-BMC beamlines. We thank R. Alkire, N. Duke, and G. Navrotski for assistance on the beamlines.

Author information

Author notes

  1. Genji Kurisu, Stanislav D Zakharov and Mariya V Zhalnina: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, Purdue University, Lilly Hall of Life Sciences, 915 W. State St., West Lafayette, 47907-1392, Indiana, USA

    Genji Kurisu, Stanislav D Zakharov, Mariya V Zhalnina, Sufiya Bano & William A Cramer

  2. Institute for Protein Research, Osaka University, Suita, 565-0871, Osaka, Japan

    Genji Kurisu

  3. Institute of Basic Biological Problems, Russian Academy of Sciences, Puschino, 142290, Russia

    Stanislav D Zakharov

  4. Belozersky Institute, Moscow State University, Moscow, 11999, Russia

    Veronika Y Eroukova, Tatiana I Rokitskaya & Yuri N Antonenko

  5. Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, 22908, Virginia, USA

    Michael C Wiener

Authors
  1. Genji Kurisu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stanislav D Zakharov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mariya V Zhalnina
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sufiya Bano
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Veronika Y Eroukova
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tatiana I Rokitskaya
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yuri N Antonenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Michael C Wiener
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. William A Cramer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to William A Cramer.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kurisu, G., Zakharov, S., Zhalnina, M. et al. The structure of BtuB with bound colicin E3 R-domain implies a translocon. Nat Struct Mol Biol 10, 948–954 (2003). https://doi.org/10.1038/nsb997

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsb997

  • Springer Nature America, Inc.

This article is cited by

Search

Navigation