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Crystal structure of GyrA intein from Mycobacterium xenopi reveals structural basis of protein splicing

Nature Structural Biology volume 5, pages 31–36 (1998)Cite this article

Abstract

Several genes from prokaryotes and lower eukaryotes have been found to contain an in-frame open reading frame, which encodes for an internal protein (intein). Post-translationally, the internal polypeptide auto-splices and ligates the external sequences to yield a functional external protein (extein) and an intein. Most, but not all inteins, contain, apart from a splicing domain, a separate endonucleolytic domain that enables them to maintain their presence by a homing mechanism. We report here the crystal structure of an intein found in the gyrase A subunit from Mycobacterium xenopi at 2.2 Å resolution. The structure contains an unusual β-fold with the catalytic splice junctions at the ends of two adjacent antiparallel β-strands. The arrangement of the active site residues Ser 1, Thr 72, His 75, His 197, and Asn 198 is consistent with a four-step mechanism for the cleavage–ligation reaction. Using site-directed mutagenesis, the N-terminal cysteine, proposed as the nucleophile in the first step of the splicing reaction, was changed to a Ser 1 and Ala 0, thus capturing the intein in a pre-spliced state.

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References

  1. Perler, F.B. et al. Protein splicing elements: Inteins and exteins—a definition of terms and recommended nomenclature. Nucleic Acids Res. 22, 1125–1127 (1994).

    Article  CAS  Google Scholar 

  2. Cooper, A.A. & Stevens, T.H. Protein splicing: self-splicing of genetically mobile elements at the protein level. Trends Biochem. Sci. 20, 351–356 (1995).

    Article  CAS  Google Scholar 

  3. Guan, C. et al. Activation of glycosylasparaginase. Formation of active N-terminal threonine by intramolecular autoproteolysis. J. Biol. Chem. 271, 1732–1737 (1996).

    Article  CAS  Google Scholar 

  4. Porter, J.A. et al. Hedgehog patterning activity: role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain. Cell 86, 21–34 (1996).

    Article  CAS  Google Scholar 

  5. van Poelje, P.D. & Snell, E.E. Pyruvoyl-dependent enzymes. Annu. Rev. Biochem. 59, 29–59 (1990).

    Article  CAS  Google Scholar 

  6. Kane, P.M. et al. Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H+-adenosine triphosphatase. Science 250, 651–657 (1990).

    Article  CAS  Google Scholar 

  7. Perler, F.B., Olsen, G.J. & Adams, E. Compilation and analysis of intein sequences. Nucleic Acids Res. 25, 1087–1094 (1997).

    Article  CAS  Google Scholar 

  8. Gimble, F.S. & Thorner, J. Homing of a DNA endonuclease gene by meiotic gene conversion in Saccharomyces cerevisiae. Nature 357, 301–306 (1992).

    Article  CAS  Google Scholar 

  9. Bremer, M.C.D., Gimble, F.S., Thorner, J. & Smith, C.L. VDE endonuclease cleaves Saccharomyces cerevisiae genomic DNA at a single site: physical mapping of the VMA1 gene. Nucleic Acids Res. 20, 5484 (1992).

    Article  CAS  Google Scholar 

  10. Pietrokovski, S. Conserved sequence features of inteins (protein introns) and their use in identifying new inteins and related proteins. Protein Sci. 3, 2340–2350 (1994).

    Article  CAS  Google Scholar 

  11. Perler, F.B. et al. Intervening sequences in an archaea DNA polymerase gene. Proc. Natl. Acad. Sci. USA 89, 5577–5581 (1992).

    Article  CAS  Google Scholar 

  12. Duan, X., Gimble, F.S. & Quiocho, F.A. Crystal structure of PI-SceI, a homing endonuclease with protein splicing activity. Cell 89, 555–564 (1996).

    Article  Google Scholar 

  13. Xu, M.-Q. & Perler, F.B. The mechanism of protein splicing and its modulation by mutation. EMBO J. 15, 5146–5153 (1996).

    Article  CAS  Google Scholar 

  14. Davis, E.O. et al. Protein splicing in the maturation of M. tuberculosis recA protein: a mechanism for tolerating a novel class of intervening sequence. Cell 71, 201–210 (1992).

    Article  CAS  Google Scholar 

  15. Hirata, R. & Anraku, Y. Mutations at the putative junction sites of the yeast VMA1 protein, the catalytic subunit of the vacuolar membrane H+-ATPase, inhibit its processing by protein splicing. Biochem. Biophys. Res. Comm. 188, 40–47 (1992).

    Article  CAS  Google Scholar 

  16. Cooper, A.A., Chen, Y., Lindorfer, M.A. & Stevens, T.H. Protein splicing of the yeast TFP1 intervening protein sequence: a model of self-excision. EMBO J. 12, 2575–2583 (1993).

    Article  CAS  Google Scholar 

  17. Xu, M.-Q. et al. Protein splicing: an analysis of the branched intermediate and its resolution by succinimide formation. EMBO J. 13, 5517–5522 (1994).

    Article  CAS  Google Scholar 

  18. Dawson, P.E., Muir, T.W., Clark-Lewis, I. & Kent, S.B. Synthesis of proteins by native chemical ligation. Science 266, 776–779 (1994).

    Article  CAS  Google Scholar 

  19. Storer, A.C. & Ménard, R. Catalytic mechanism in papain family of cysteine peptidases. Meth. Enz. 244, 486–500 (1994).

    Article  CAS  Google Scholar 

  20. Drenth, J., Jansonius, J.N., Koekoek, R., Swen, H.M. & Wolthers, B.G. Structure of papain. Nature 218, 929–932 (1968).

    Article  CAS  Google Scholar 

  21. Ramachandran, G.N. & Mitra, A.K. An explanation for the rare occurrence of cis peptide units in proteins and polypeptides. J. Mol. Biol. 107, 85–92 (1976).

    Article  CAS  Google Scholar 

  22. Fsihi, H., Vincent, V. & Cole, S.T. Homing events in the gyrA gene of some mycobacteria. Proc. Natl. Acad. Sci. USA 93, 3410–3415 (1996).

    Article  CAS  Google Scholar 

  23. Oinonen, C., Tikkanen, R., Rouvinen, J. & Peltonen, L. Three-dimensional structure of human lyosomal aspartylglucosaminidase. Nature Struct. Biol. 2, 1102–1107 (1995).

    Article  CAS  Google Scholar 

  24. Smith, J.L. et al. Structure of the allosteric regulatory enzyme of purine biosynthesis. Science 264, 1427–1433 (1994).

    Article  CAS  Google Scholar 

  25. Duggleby, H.J. et al. Penicillin acylase has a single-amino-acid catalytic center. Nature 373, 264–268 (1995).

    Article  CAS  Google Scholar 

  26. Löwe, J. et al. Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 Å resolution. Science 268, 533–539 (1995).

    Article  Google Scholar 

  27. Brannigan, J.A. et al. A protein catalytic framework with an N-terminal nucleophile is capable of self-activation. Nature 378, 416–419 (1995).

    Article  CAS  Google Scholar 

  28. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data in oscillation mode. Meth. Enz. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  29. Collaborative Computational Project No. 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

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

    Google Scholar 

  31. Jones, T.A., Zou, J.-Y., Kjelgaard, M. & Cowan, S.W. 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 

  32. Brünger, A. X-PLOR Version 3.1: A System for X-ray crystallography and NMR. (Yale University Press, New Haven, Connecticut; 1992).

    Google Scholar 

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

    Article  CAS  Google Scholar 

  34. Hall, T.M.T. et al. Crystal structure of a hedgehog autoprocessing domain: homology between hedgehog and self-splicing proteins. Cell 91, 85–97 (1997).

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, 77843-2128, USA

    Thomas Klabunde, Sujata Sharma & James C. Sacchettini

  2. Institute for Medical Microbiology, University of Bern, 3010, Bern, Switzerland

    Amalio Telenti

  3. Departments of Microbiology and Immunology and Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, New York, 10461, USA

    William R. Jacobs Jr.

Authors
  1. Thomas Klabunde
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  2. Sujata Sharma
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  3. Amalio Telenti
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  4. William R. Jacobs Jr.
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  5. James C. Sacchettini
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Correspondence to James C. Sacchettini.

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Klabunde, T., Sharma, S., Telenti, A. et al. Crystal structure of GyrA intein from Mycobacterium xenopi reveals structural basis of protein splicing. Nat Struct Mol Biol 5, 31–36 (1998). https://doi.org/10.1038/nsb0198-31

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