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Crystal structure of the secreted form of antigen 85C reveals potential targets for mycobacterial drugs and vaccines

Nature Structural Biology volume 7, pages 141–146 (2000)Cite this article

Abstract

The antigen 85 (ag85) complex, composed of three proteins (ag85A, B and C), is a major protein component of the Mycobacterium tuberculosis cell wall. Each protein possesses a mycolyltransferase activity required for the biogenesis of trehalose dimycolate (cord factor), a dominant structure necessary for maintaining cell wall integrity. The crystal structure of recombinant ag85C from M. tuberculosis, refined to a resolution of 1.5 Å, reveals an α/β-hydrolase polypeptide fold, and a catalytic triad formed by Ser 124, Glu 228 and His 260. ag85C complexed with a covalent inhibitor implicates residues Leu 40 and Met 125 as components of the oxyanion hole. A hydrophobic pocket and tunnel extending 21 Å into the core of the protein indicates the location of a probable trehalose monomycolate binding site. Also, a large region of conserved surface residues among ag85A, B and C is a probable site for the interaction of ag85 proteins with human fibronectin.

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Figure 1: Structure of Cα chain of the secreted form of antigen 85C.
Figure 2: Proposed binding site for TMM.
Figure 3: Schematic representation of the catalytic mechanism of mycolyl transfer.
Figure 4: Superposition of apo-ag85c with DEP-bound ag85C.
Figure 5: Fibronectin binding region and multiple sequence alignment of the three M. tuberculosis antigen 85 proteins.

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References

  1. Fukui, Y., Hirai, T., Uchida, T. & Yoneda, M. Extracellular proteins of tubercle bacilli. IV. Alpha and beta antigens as major extracellular protein products and as cellular components of a strain (H37Rv) of Mycobacterium tuberculosis. Biken J. 8, 189– 199 (1965).

    CAS  PubMed  Google Scholar 

  2. Belisle, J.T. et al. Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis. Science 276, 1420–1422 (1997).

    Article  CAS  Google Scholar 

  3. Brennan, P.J. & Nikaido, H. The envelope of mycobacteria. Annu. Rev. Biochem. 96, 29–63 (1995).

    Article  Google Scholar 

  4. Jackson, M. et al. Inactivation of the antigen 85C gene profoundly affects the mycolate content and alters the permeability of the Mycobacterium tuberculosis cell envelope. Mol. Microbiol. 31, 1573–1587 (1999).

    Article  CAS  Google Scholar 

  5. Robitzek, E.H. & Selikoff, I.J. Hydrazine derivatives of isonicotinic acid (Rimifon, Marsilid) in the treatment of active progressive caseous–pneumonic tuberculosis. Am. Rev. Tuberc. Pulm. Dis. 65, 765 (1952).

    Google Scholar 

  6. Blanchard, J.S. Molecular mechanisms of drug resistance in Mycobacterium tuberculosis. Annu. Rev. Biochem. 65, 215– 239 (1996).

    Article  CAS  Google Scholar 

  7. Banerjee, A. et al. inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science 263, 227–230 (1994).

    Article  CAS  Google Scholar 

  8. Takayama, K. & Kilburn, J.O. Inhibition of synthesis of arabinogalactan in Mycobacterium smegmatis. Antimicrob Agents Chemother. 33, 1493–1499 (1989).

    Article  CAS  Google Scholar 

  9. Silva, C.L., Ekizlerian, S.M. & Fazioli, R. Role of cord factor in the modulation of infection caused by mycobacteria. Am. J. Pathol. 118, 238 –247 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Schrag, J.D. & Cygler, M. Lipases and α/β hydrolase fold. Methods Enzymol. 284, 85– 107 (1997).

    Article  CAS  Google Scholar 

  11. Powers, J.C. et al. Proteases—structures, mechanism and inhibitors. Agents Actions Suppl. 42, 3–18 (1993).

    CAS  PubMed  Google Scholar 

  12. Kraut, J. Serine proteases: structure and mechanism of catalysis. Annu. Rev. Biochem. 46, 331–358 ( 1977).

    Article  CAS  Google Scholar 

  13. Egloff, M.P. et al. The 2.46 Å resolution structure of the pancreatic lipase–colipase complex inhibited by a C11 alkyl phosphonate. Biochemistry 34, 2751–2762 (1995).

    Article  CAS  Google Scholar 

  14. Uppenberg, J. et al. Crystallographic and molecular-modeling studies of lipase B from Candida antarctica reveal a stereospecificity pocket for secondary alcohols. Biochemistry 31, 16838– 16851 (1995).

    Article  Google Scholar 

  15. Bullock, T.L., Breddam, K. & Remington, S.J. Peptide aldehyde complexes with wheat serine carboxypeptidase II: implications for the catalytic mechanism and substrate specificity. J. Mol. Biol. 255, 714–725 (1996).

    Article  CAS  Google Scholar 

  16. Ransac, S. et al. Covalent inactivation of lipases. Methods Enzymol. 286, 190–231 ( 1997).

    Article  CAS  Google Scholar 

  17. Leuveling Tjeenk, M. et al. Inactivation of Staphylococcus hyicus lipase by hexadecylsulfonyl fluoride: evidence for an active site serine. Protein Eng. 7, 579 (1994).

    Article  CAS  Google Scholar 

  18. Maylie, M.F., Charles, M. & Desnuelle P. Action of organophosphates and sulfonyl halides on porcine pancreatic lipase. Biochim. Biophys. Acta 276, 162–175 (1972).

    Article  CAS  Google Scholar 

  19. Longhi, S., Czjzek, M., Lamzin, V., Nicolas, A. & Cambillau C. Atomic resolution (1.0 Å) crystal structure of Fusarium solani cutinase: stereochemical analysis. J. Mol. Biol. 268, 779–799 ( 1997).

    Article  CAS  Google Scholar 

  20. Martinez, C. Cutinase, a lipolytic enzyme with a preformed oxyanion hole. Biochemistry 33, 83–89 ( 1994).

    Article  CAS  Google Scholar 

  21. Abou-Zeid, C. et al. Characterization of fibronectin-binding antigens released by Mycobacterium tuberculosis and Mycobacterium bovis BCG. Infect. Immun. 56, 3046–3051 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Schlesinger L.S. & Horowitz, M.A. Phagocytosis of Mycobacterium leprae by human monocyte-derived macrophages is mediated by complement receptors CR1 (CD35), CR3 (CD11b/CD18), and CR4 (CD11c/CD18) and IFN-γ activation inhibits complement receptor function and phagocytosis of this bacterium. J. Immunol. 147, 1983 –1994 (1991).

    CAS  PubMed  Google Scholar 

  23. Schorey, J.S., Carroll, M.C. & Brown, E.J. A macrophage invasion mechanism of pathogenic mycobacteria . Science 277, 1091–1093 (1997).

    Article  CAS  Google Scholar 

  24. Naito, M., Ohara, N., Matsumoto, S. & Yamada, T. The novel fibronectin-binding motif and key residues of mycobacteria. J. Biol. Chem. 273, 2905 (1998).

  25. De Bruyn, J., Bosmans, R., Nyabenda, J. & Van Vooren, J.P. Effect of zinc deficiency on the appearance of two immunodominant protein antigens (32 kDa and 65 kDa) in culture filtrates of mycobacteria. J. Gen. 135, 79–84 ( 1989).

    CAS  Google Scholar 

  26. Fukui, Y., Hirai, T., Uchida, T. & Yoneda, M. Extracellular proteins of tubercle bacilli. IV. Alpha and beta antigens as major extracellular protein products and as cellular components of a strain (H37Rv) of Mycobacterium tuberculosis. Biken J. 8, 189– 199 (1965).

    CAS  PubMed  Google Scholar 

  27. Wiker, G.H. & Harboe, M. The antigen 85 complex: a major secretion product of Mycobacterium tuberculosis. Microbiol. Rev. 56, 648–661 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Roche, P.W., Peake, P.W., Billman-Jacobe, H., Doran, T. & Britton, W.J. T-Cell determinants and antibody binding sites on the major mycobacterial secretory protein MPB59 of Mycobacterium bovis. Infect. Immun. 62, 5319– 5326 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Horwitz, M.A., Lee, B.W., Dillon, B.J. & Harth, G. Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 92, 1530–1534 ( 1995).

    Article  CAS  Google Scholar 

  30. Orme, I.M. Characteristics and specificity of acquired immunologic memory to Mycobacterium tuberculosis infection. J. Immunol. 140, 3589–3593 (1988).

    CAS  PubMed  Google Scholar 

  31. Samanich, K.M. et al. Delineation of human antibody responses to culture filtrate antigens of Mycobacterium tuberculosis. J. Infect. Dis. 178, 1534–1538 ( 1998).

    Article  CAS  Google Scholar 

  32. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 27, 307–326 ( 1997).

    Article  Google Scholar 

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

  34. Furey, W. & Swaminathan, S. PHASES. Am. Crystallogr. Assoc. Annu. Mtg. Program. Abstr. 18, 73 (1990).

    Google Scholar 

  35. Brünger, A.T. et al. Crystallography & NMR system (version 0.9): a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 ( 1998).

    Article  Google Scholar 

  36. de La Fortelle, E. & Bricogne, G. Maximum-likelihood heavy atom parameter refinement for the multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–494 ( 1997).

    Article  CAS  Google Scholar 

  37. Cowtan, K.D. & P. Main, P. Phase combination and cross validation in iterated density-modification calculations. Acta Crystallogr. D 52, 43–48 ( 1996).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  39. Navaza, J. AMoRe: an Automated Package for Molecular Replacement. Acta Crystallogr. A 50, 157–163 ( 1994).

    Article  Google Scholar 

  40. Evans, S.V. Setor: hardware lighted three-dimensional solid model representations of macromolecules . J. Mol. Graph. 11, 134– 138 (1993).

    Article  CAS  Google Scholar 

  41. Guex, N. & Peitsch, M.C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723 ( 1997).

    Article  CAS  Google Scholar 

  42. Amundsen, S. et al. _X-POV-Team POV-Ray: persistence of vision ray-tracer. http://www.povray.org (1997).

  43. Christopher, J.A. SPOCK: the structural properties observation and calculation kit (program manual). (The Center for Macromolecular Design, Texas A&M University, College Station, Texas; 1998).

    Google Scholar 

  44. Higgins, D.G., Bleasby, A.J. & Fuchs, R. CLUSTAL V: improved software for multiple sequence alignment . Comput. Applic. Biosci. 8, 189– 191 (1992).

    CAS  Google Scholar 

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Acknowledgements

Financial support was provided by the Robert A. Welch Foundation and the NIH. We would like to thank the staff of the SBC-CAT at the APS at Argonne National laboratory, specifically, Frank Rotella for all of his help with data collection.

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Texas A&M University, College Station, 77844-2128 , Texas, USA

    Donald R. Ronning, Thomas Klabunde & James C. Sacchettini

  2. Department of Microbiology and Immunology, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH , UK

    Gurdyal S. Besra

  3. Department of Microbiology, Colorado State University, Fort Collins, 80523, Colorado, USA

    Varalakshmi D. Vissa & John T. Belisle

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  1. Donald R. Ronning
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Correspondence to James C. Sacchettini.

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Ronning, D., Klabunde, T., Besra, G. et al. Crystal structure of the secreted form of antigen 85C reveals potential targets for mycobacterial drugs and vaccines. Nat Struct Mol Biol 7, 141–146 (2000). https://doi.org/10.1038/72413

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