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Crystal structure of the anthrax lethal factor

Abstract

Lethal factor (LF) is a protein (relative molecular mass 90,000) that is critical in the pathogenesis of anthrax1,2,3. It is a highly specific protease that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family near to their amino termini, leading to the inhibition of one or more signalling pathways4,5,6. Here we describe the crystal structure of LF and its complex with the N terminus of MAPKK-2. LF comprises four domains: domain I binds the membrane-translocating component of anthrax toxin, the protective antigen (PA); domains II, III and IV together create a long deep groove that holds the 16-residue N-terminal tail of MAPKK-2 before cleavage. Domain II resembles the ADP-ribosylating toxin from Bacillus cereus, but the active site has been mutated and recruited to augment substrate recognition. Domain III is inserted into domain II, and seems to have arisen from a repeated duplication of a structural element of domain II. Domain IV is distantly related to the zinc metalloprotease family, and contains the catalytic centre; it also resembles domain I. The structure thus reveals a protein that has evolved through a process of gene duplication, mutation and fusion, into an enzyme with high and unusual specificity.

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Figure 1: Stereo ribbon representation of LF, coloured by domain.
Figure 2: Sequence of LF, coloured as in Fig. 1, with secondary structure indicated.
Figure 3: Stereo representation of the active site centre of LF.
Figure 4: Molecular surface of LF coloured by charge (red, negative; blue, positive), with the model of the MAPKK-2 N-terminal peptide shown in ball-and-stick representation.

References

  1. Leppla, S. H. in Comprehensive Sourcebook of Bacterial Protein Toxins 2nd edn (eds Alouf, J. A. & Freer, J.) 243–263 (Academic, London, 1999).

    Google Scholar 

  2. Dixon, T. C., Meselson, M., Guillemin, J. & Hanna, P. C. Medical progress: anthrax. N. Engl. J. Med. 341, 815–862 (1999).

    Article  CAS  Google Scholar 

  3. Pezard, C., Berche, P. & Mock, M. Contribution of individual toxin components to virulence of Bacillus anthracis. Infect. Immun. 59, 3472–3477 (1991).

    Article  CAS  Google Scholar 

  4. Duesbury, N. S. et al. Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. Science 280, 734–737 (1998).

    Article  ADS  Google Scholar 

  5. Vitale, G. et al. Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macropahges. Biochem. Biophys. Res. Comm. 248, 706–711 (1998).

    Article  CAS  Google Scholar 

  6. Vitale, G., Bernardi, L., Napolitani, G., Mock, M. & Montecucco, C. Susceptibility of mitogen-activated protein kinase kinase family members to proteolysis by anthrax lethal factor. Biochem. J. 352, 739–745 (2000).

    Article  CAS  Google Scholar 

  7. Inglesby, T. V. et al. Anthrax as a biological weapon: medical and public health management. Working group on civilian biodefense. J. Am. Med. Assoc. 281, 1735–1745 (1999).

    Article  CAS  Google Scholar 

  8. Petosa, C., Collier, R. J., Klimpel, K. R., Leppla, S. H. & Liddington, R. C. Crystal structure of the anthrax toxin protective antigen. Nature 385, 833–838 (1997).

    Article  ADS  CAS  Google Scholar 

  9. Benson, E. L., Huynh, P. D., Finkelstein, A. & Collier, R. J. Identification of residues lining the anthrax protective antigen channel. Biochemistry 37, 3941–3948 (1998).

    Article  CAS  Google Scholar 

  10. Klimpel, K. R., Arora, N. & Leppla, S. H. Anthrax toxin lethal factor contains a zinc metalloprotease consensus sequence which is required for lethal toxin activity. Mol. Microbiol. 13, 1093–1100 (1994).

    Article  CAS  Google Scholar 

  11. Duesbery, N. S. et al. Suppression of ras-mediated transformation and inhibition of tumour growth and angiogenesis by anthrax lethal factor, a proteolytic inhibitor of multiple MEK pathways. Proc. Natl Acad. Sci. USA 98, 4089–4094 (2001).

    Article  ADS  CAS  Google Scholar 

  12. Friedlander, A. M. Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process. J. Biol. Chem. 261, 7123–7126 (1986).

    Article  CAS  Google Scholar 

  13. Hanna, P. C., Acosta, D. & Collier, R. J. On the role of macrophages in anthrax. Proc. Natl Acad. Sci. USA 90, 10198–10201 (1993).

    Article  ADS  CAS  Google Scholar 

  14. Pellizzari, R., Guidi-Rontani, C., Vitale, G., Mock, M. & Montecucco, C. Anthrax lethal factor cleaves MKK3 in macrophages and inhibits the LPS/IFNγ-induced release of NO and TNFα. FEBS Lett. 462, 199–204 (1999).

    Article  CAS  Google Scholar 

  15. Ballard, J. D., Collier, R. J. & Starnbach, M. N. Anthrax toxin-mediated delivery of a cytotoxic T-cell epitope in vivo. Proc. Natl Acad. Sci. USA 93, 12531–12534 (1996).

    Article  ADS  CAS  Google Scholar 

  16. Goletz, T. J. et al. Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120–anthrax toxin fusion protein. Proc. Natl Acad. Sci. USA 94, 12059–12064 (1997).

    Article  ADS  CAS  Google Scholar 

  17. Quinn, C. P., Singh, Y., Klimpel, K. R. & Leppla, S. H. Functional mapping of anthrax toxin lethal factor by in-frame insertion mutagenesis. J. Biol. Chem. 266, 20124–20130 (1991).

    Article  CAS  Google Scholar 

  18. Gupta, P., Singh, A., Chauhun, V. & Bhatnagar, R. Involvement of residues 147VYYEIGK153 in binding of lethal factor to protective antigen of Bacillus anthracis. Biochem. Biophys. Res. Comm. 280, 158–163 (2001).

    Article  CAS  Google Scholar 

  19. Holm, L. & Sander, C. Dali/FSSP classification of three-dimensional protein folds. Nucleic Acids Res. 25, 231–234 (1997).

    Article  CAS  Google Scholar 

  20. Carroll, S. F. & Collier, R. J. NAD binding site of diphtheria toxin: identification of a residue within the nicotinamide subsite by photochemical modification with NAD. Proc. Natl Acad. Sci. USA 81, 3307–3311 (1984).

    Article  ADS  CAS  Google Scholar 

  21. Mock, W. L. & Stanford, D. L. Arazoformyl dipeptide substrates for thermolysin. Confirmation of a reverse protonation catalytic mechanism. Biochemistry 35, 7369–7377 (1996).

    Article  CAS  Google Scholar 

  22. Park, S. & Leppla, S. H. Optimized production and purification of Bacillus anthracis lethal factor. Protein Expr. Purif. 18, 293–302 (2000).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  25. Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849–861 (1999).

    Article  CAS  Google Scholar 

  26. Roussel, A. & Cambillau, C. 86 (Silicon Graphics, Mountain View, California, 1991).

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

    Article  CAS  Google Scholar 

  28. Wang, X.-M., Mock, M., Ruysschaert, J.-M. & Cabiaux, V. Secondary structure of anthrax lethal toxin proteins and their interaction with large unilamellar vesicles (LUV): a Fourier-transform infrared spectroscopy approach. Biochemistry 35, 14939–14946 (1996).

    Article  CAS  Google Scholar 

  29. Bacon, D. J. & Anderson, W. F. A fast algorithm for rendering space-filling molecule pictures. J. Mol. Graphics 6, 219–220 (1988).

    Article  Google Scholar 

  30. Kraulis, P. J. Molscript—a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).

    Article  Google Scholar 

  31. Merrit, E. A. & Murphy, M. E. P. Raster3D version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr. D 50, 869–873 (1994).

    Article  Google Scholar 

  32. Christopher, J. A. SPOCK: The Structural Properties Observation and Calculation Kit (Program Manual) (The Center for Macromolecular Design, Texas A&M University, College Station, 1998).

    Google Scholar 

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Acknowledgements

We thank L. Bankston for discussion and D. Hsu for LF preparations. We thank and acknowledge the staff and facilities of the synchrotron sources at SSRL, Stanford; SRS, Daresbury; ESRF, Grenoble; APS, Chicago; and National Synchrotron Light Source, Brookhaven. Supported by grants from the Medical Research Council and the National Institutes of Health.

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Correspondence to Robert C. Liddington.

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Table 1. Data collection summary for Lethal Factor crystals.

Figure 1

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Pannifer, A., Wong, T., Schwarzenbacher, R. et al. Crystal structure of the anthrax lethal factor. Nature 414, 229–233 (2001). https://doi.org/10.1038/n35101998

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