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GroEL as a molecular scaffold for structural analysis of the anthrax toxin pore

Nature Structural & Molecular Biology volume 15, pages 754–760 (2008)Cite this article

Abstract

We analyzed the 440-kDa transmembrane pore formed by the protective antigen (PA) moiety of anthrax toxin in the presence of GroEL by negative-stain electron microscopy. GroEL binds both the heptameric PA prepore and the PA pore. The latter interaction retards aggregation of the pore, prolonging its insertion-competent state. Two populations of unaggregated pores were visible: GroEL-bound pores and unbound pores. This allowed two virtually identical structures to be reconstructed, at 25-Å and 28-Å resolution, respectively. The structures were mushroom-shaped objects with a 125-Å-diameter cap and a 100-Å-long stem, consistent with earlier biochemical data. Thus, GroEL provides a platform for obtaining initial glimpses of a membrane protein structure in the absence of lipids or detergents and can function as a scaffold for higher-resolution structural analysis of the PA pore.

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Figure 1: GroEL binds to the PA prepore and pore.
Figure 2: PA pore formed in the presence of GroEL is functional.
Figure 3: Two-dimensional average of GroEL-bound and free PA pore particles.
Figure 4: Three-dimensional reconstructions of GroEL-bound and free PA pore particles.
Figure 5: Manual fit of the PA prepore crystal structure into the PA pore EM structure.

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Acknowledgements

M.T.F. dedicates this work to the memory of Earl R. Stadtman, his mentor during his postdoctoral years at the US National Institutes of Health (NIH). Earl's influence, scientific philosophy and sense of generosity will be with M.T.F. for the rest of his days. This research was supported in part by US National Science Foundation (NSF) grant MCB-0445936 (M.T.F.), NIH grant R41 GM080074 (M.T.F.), a grant from the Kansas Technology Enterprise Corporation (M.T.F.) and NIH grant 5R37AI022021 (R.J.C.). The NSF grant supported J.J. and M.B. GroEL was a gift from EdgeBiosystems.

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Author notes

  1. Hiroo Katayama and Blythe E Janowiak: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas Life Sciences Innovation Center Building, Kansas City, 66160, Kansas, USA

    Hiroo Katayama, Marek Brzozowski, Jordan Juryck & Mark T Fisher

  2. Department of Microbiology and Molecular Genetics, Harvard Medical School, Alpert Building, Boston, 02115, Massachusetts, USA

    Blythe E Janowiak & R John Collier

  3. Department of Biology, William Jewell College, 500 College Hill, Liberty, 64068-1896, Missouri, USA

    Scott Falke

  4. School of Biological Sciences, University of Missouri-Kansas City, Kansas City, 64100, Missouri, USA

    Edward P Gogol

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  1. Hiroo Katayama
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Contributions

H.K. and B.E.J. contributed equally to the experimental design, the execution of the experiments and the interpretation of the data; they also contributed to the writing of the manuscript. Undergraduates J.J. and M.B. were involved in computer setup, network support, image scanning, data cataloging, some particle picking and initial alignment work under the guidance of H.K., S.F., E.P.G. and M.T.F. E.P.G, R.J.C. and M.T.F. contributed to experimental design, provided guidance, analysis and interpretation of the data and were responsible for writing the manuscript.

Corresponding author

Correspondence to Mark T Fisher.

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Competing interests

R.J.C. holds equity in PharmAthene, Inc., and is a consultant for CombinatoRx, Inc.

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Katayama, H., Janowiak, B., Brzozowski, M. et al. GroEL as a molecular scaffold for structural analysis of the anthrax toxin pore. Nat Struct Mol Biol 15, 754–760 (2008). https://doi.org/10.1038/nsmb.1442

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