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The structural basis for substrate and inhibitor selectivity of the anthrax lethal factor

Nature Structural & Molecular Biology volume 11, pages 60–66 (2004)Cite this article

Abstract

Recent events have created an urgent need for new therapeutic strategies to treat anthrax. We have applied a mixture-based peptide library approach to rapidly determine the optimal peptide substrate for the anthrax lethal factor (LF), a metalloproteinase with an important role in the pathogenesis of the disease. Using this approach we have identified peptide analogs that inhibit the enzyme in vitro and that protect cultured macrophages from LF-mediated cytolysis. The crystal structures of LF bound to an optimized peptide substrate and to peptide-based inhibitors provide a rationale for the observed selectivity and may be exploited in the design of future generations of LF inhibitors.

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Figure 1: Inhibition of LF by GM6001.
Figure 2: Structures of LF in complex with peptides and inhibitors.

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References

  1. Dixon, T.C., Meselson, M., Guillemin, J. & Hanna, P.C. Anthrax. New Engl. J. Med. 341, 815–826 (1999).

    Article  CAS  PubMed  Google Scholar 

  2. Duesbery, N.S. & Vande Woude, G.F. Anthrax toxins. Cell. Mol. Life Sci. 55, 1599–1609 (1999).

    Article  CAS  PubMed  Google Scholar 

  3. Moayeri, M., Haines, D., Young, H.A. & Leppla, S.H. Bacillus anthracis lethal toxin induces TNF-α-independent hypoxia-mediated toxicity in mice. J. Clin. Invest. 112, 670–682 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Sellman, B.R., Mourez, M. & Collier, R.J. Dominant-negative mutants of a toxin subunit: an approach to therapy of anthrax. Science 292, 695–697 (2001).

    Article  CAS  PubMed  Google Scholar 

  6. Mourez, M. et al. Designing a polyvalent inhibitor of anthrax toxin. Nat. Biotechnol. 19, 958–961 (2001).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  9. 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  PubMed  Google Scholar 

  10. 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  PubMed  PubMed Central  Google Scholar 

  11. Enslen, H. & Davis, R.J. Regulation of MAP kinases by docking domains. Biol. Cell 93, 5–14 (2001).

    Article  CAS  PubMed  Google Scholar 

  12. Agrawal, A. et al. Impairment of dendritic cells and adaptive immunity by anthrax lethal toxin. Nature 424, 329–334 (2003).

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  14. 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  CAS  PubMed  PubMed Central  Google Scholar 

  15. Park, J.M., Greten, F.R., Li, Z.W. & Karin, M. Macrophage apoptosis by anthrax lethal factor through p38 MAP kinase inhibition. Science 297, 2048–2051 (2002).

    Article  CAS  PubMed  Google Scholar 

  16. Chopra, A.P., Boone, S.A., Liang, X. & Duesbery, N.S. Anthrax lethal factor proteolysis and inactivation of MAPK kinase. J. Biol. Chem. 278, 9402–9406 (2003).

    Article  CAS  PubMed  Google Scholar 

  17. Hammond, S.E. & Hanna, P.C. Lethal factor active-site mutations affect catalytic activity in vitro. Infect. Immun. 66, 2374–2378 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Cummings, R.T. et al. A peptide-based fluorescence resonance energy transfer assay for Bacillus anthracis lethal factor protease. Proc. Natl. Acad. Sci. USA 99, 6603–6606 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Tonello, F., Seveso, M., Marin, O., Mock, M. & Montecucco, C. Screening inhibitors of anthrax lethal factor. Nature 418, 386 (2002).

    Article  CAS  PubMed  Google Scholar 

  20. Songyang, Z. et al. SH2 domains recognize specific phosphopeptide sequences. Cell 72, 767–778 (1993).

    Article  CAS  PubMed  Google Scholar 

  21. Turk, B.E., Huang, L.L., Piro, E.T. & Cantley, L.C. Determination of protease cleavage site motifs using mixture-based oriented peptide libraries. Nat. Biotechnol. 19, 661–667 (2001).

    Article  CAS  PubMed  Google Scholar 

  22. Tanoue, T., Adachi, M., Moriguchi, T. & Nishida, E. A conserved docking motif in MAP kinases common to substrates, activators and regulators. Nat. Cell Biol. 2, 110–116 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Enslen, H., Brancho, D.M. & Davis, R.J. Molecular determinants that mediate selective activation of p38 MAP kinase isoforms. EMBO J. 19, 1301–1311 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Xu, B., Stippec, S., Robinson, F.L. & Cobb, M.H. Hydrophobic as well as charged residues in both MEK1 and ERK2 are important for their proper docking. J. Biol. Chem. 276, 26509–26515 (2001).

    Article  CAS  Google Scholar 

  25. Holmquist, B. & Vallee, B.L. Metal-coordinating substrate analogs as inhibitors of metalloenzymes. Proc. Natl. Acad. Sci. USA 76, 6216–6220 (1979).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Moore, W.M. & Spilburg, C.A. Purification of human collagenases with a hydroxamic acid affinity column. Biochemistry 25, 5189–5195 (1986).

    Article  CAS  PubMed  Google Scholar 

  27. Gowravaram, M.R. et al. Inhibition of matrix metalloproteinases by hydroxamates containing heteroatom-based modifications of the P1′ group. J. Med. Chem. 38, 2570–2581 (1995).

    Article  CAS  PubMed  Google Scholar 

  28. Baxter, A.D. et al. A novel series of matrix metalloproteinase inhibitors for the treatment of inflammatory disorders. Bioorg. Med. Chem. Lett. 7, 897–902 (1997).

    Article  CAS  Google Scholar 

  29. Grobelny, D., Poncz, L. & Galardy, R.E. Inhibition of human skin fibroblast collagenase, thermolysin, and Pseudomonas aeruginosa elastase by peptide hydroxamic acids. Biochemistry 31, 7152–7154 (1992).

    Article  CAS  PubMed  Google Scholar 

  30. Levy, D.E. et al. Matrix metalloproteinase inhibitors: a structure-activity study. J. Med. Chem. 41, 199–223 (1998).

    Article  CAS  PubMed  Google Scholar 

  31. Pannifer, A.D. et al. Crystal structure of the anthrax lethal factor. Nature 414, 229–233 (2001).

    Article  CAS  PubMed  Google Scholar 

  32. Holmes, M.A. & Matthews, B.W. Binding of hydroxamic acid inhibitors to crystalline thermolysin suggests a pentacoordinate zinc intermediate in catalysis. Biochemistry 20, 6912–6920 (1981).

    Article  CAS  PubMed  Google Scholar 

  33. Grams, F. et al. X-ray structures of human neutrophil collagenase complexed with peptide hydroxamate and peptide thiol inhibitors. Implications for substrate binding and rational drug design. Eur. J. Biochem. 228, 830–841 (1995).

    Article  CAS  PubMed  Google Scholar 

  34. Gaucher, J.F. et al. Crystal structures of α-mercaptoacyldipeptides in the thermolysin active site: structural parameters for a Zn monodentation or bidentation in metalloendopeptidases. Biochemistry 38, 12569–12576 (1999).

    Article  CAS  PubMed  Google Scholar 

  35. Dhanaraj, V. et al. X-ray structure of a hydroxamate inhibitor complex of stromelysin catalytic domain and its comparison with members of the zinc metalloproteinase superfamily. Structure 4, 375–386 (1996).

    Article  CAS  PubMed  Google Scholar 

  36. Chen, L. et al. Crystal structure of the stromelysin catalytic domain at 2.0 Å resolution: inhibitor-induced conformational changes. J. Mol. Biol. 293, 545–557 (1999).

    Article  CAS  PubMed  Google Scholar 

  37. Roswell, S. et al. Crystal structure of human MMP9 in complex with a reverse hydroxamate inhibitor. J. Mol. Biol. 319, 173–181 (2002).

    Article  Google Scholar 

  38. Panchal, R. et al. Identification of small molecule inhibitors of anthrax lethal factor. Nat. Struct. Mol. Biol. 11, 67–72 (2004).

    Article  CAS  PubMed  Google Scholar 

  39. Roberts, J.E., Watters, J.W., Ballard, J.D. & Dietrich, W.F. Ltx1, a mouse locus that influences the susceptibility of macrophages to cytolysis caused by intoxication with Bacillus anthracis lethal factor, maps to chromosome 11. Mol. Microbiol. 29, 581–591 (1998).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  41. 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  PubMed  Google Scholar 

  42. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  PubMed  Google Scholar 

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

    Article  Google Scholar 

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Acknowledgements

Thanks to P. Bartlett (University of California Berkeley), D. Tronrud and B. Matthews (University of Oregon) and B. Rupp (Lawrence Livermore National Laboratory) for pointing out the canonical binding mode for Zn metalloproteases and to E. Garman (University of Oxford) and A. Gonzalez (SSRL) for discussions on the crystallography. We also thank H. Robinson and S. Vaday for collecting data at the National Synchrotron Light Source (NSLS). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), a national user facility operated by Stanford University on behalf of the US Department of Energy (DOE), Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the DOE, Office of Biological and Environmental Research, and by the NIH, National Center for Research Resources, Biomedical Technology Program and the National Institute of General Medical Sciences. Data for this work were also collected at the NSLS, Brookhaven National Laboratory, which is supported by the DOE, Division of Materials Sciences and Division of Chemical Sciences, under contract no. DE-AC02-98CH10886. This work was supported by NIH (R.J.C., R.C.L. and L.C.C.), the US National Science Foundation (L.C.C.) and the US Department of the Army (DAMD17-03-1-0062 to L.C.C.). The US Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick, Maryland 21702-5014 is the awarding and administering acquisition office. The contents of this manuscript do not necessarily reflect the position or policy of the US government, and no official endorsement should be inferred. B.E.T. is a Leukemia and Lymphoma Society special fellow.

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Author notes

  1. Benjamin E Turk and Thiang Yian Wong: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Signal Transduction, Department of Medicine, and Department of Cell Biology, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, 02215, Massachusetts, USA

    Benjamin E Turk, Emily T Jarrell & Lewis C Cantley

  2. The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, 92037, California, USA

    Thiang Yian Wong, Robert Schwarzenbacher & Robert C Liddington

  3. National Institute of Allergy and Infectious Diseases, 9000 Rockville Pike, Bethesda, 20892, Maryland, USA

    Stephen H Leppla

  4. Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, 02115, Massachusetts, USA

    R John Collier

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Correspondence to Robert C Liddington or Lewis C Cantley.

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One of the authors, R. John Collier, holds equity in the company Pharmathene, Inc.

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Turk, B., Wong, T., Schwarzenbacher, R. et al. The structural basis for substrate and inhibitor selectivity of the anthrax lethal factor. Nat Struct Mol Biol 11, 60–66 (2004). https://doi.org/10.1038/nsmb708

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