Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Crystal structure of the apoptosis-inducing human granzyme A dimer

Nature Structural & Molecular Biology volume 10, pages 535–540 (2003)Cite this article

Abstract

Granzyme A (GzmA) belongs to a family of trypsin-like serine proteases localized in cytoplasmic granules of activated lymphocytes and natural killer (NK) cells. In contrast to the related granzyme B (GzmB), GzmA forms a stable disulfide-linked homodimer and triggers target-cell death in a caspase-independent way. Limited proteolysis of a high-molecular-mass complex containing SET (also named putative HLA-associated protein II or PHAPII), PHAPI (pp32, leucine-rich acidic nuclear protein) and HMG2 by GzmA liberates NM23-H1, a Mg2+-dependent DNase that causes single-stranded breaks in nuclear DNA. By analyzing the dimeric GzmA structure at a resolution of 2.5 Å, we determined the substrate-binding constraints and selective advantages of the two domains arranged as a unique functional tandem. The active sites of the two subunits point in opposite directions and the nearby noncatalytic surfaces can function as exosites, presenting substrates to the active site region of the adjacent partner in a manner analogous to staphylokinase or streptokinase, which present plasminogen to the cofactor–plasmin and cofactor–plasminogen complexes.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Main chain ribbon plot of the human GzmA dimer.
Figure 2: Topological alignment of GzmA and related homologs.
Figure 3: Stereo view of dimer interface.
Figure 4: Connolly surface of the GzmA homodimer.
Figure 5: Characterization of interaction between GzmA and the SET substrate.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

Protein Data Bank

References

  1. Pasternack, M.S. & Eisen, H.N. A novel serine esterase expressed by cytotoxic T lymphocytes. Nature 314, 743–745 (1985).

    Article  CAS  Google Scholar 

  2. Pasternack, M.S., Verret, C.R., Liu, M.A. & Eisen, H.N. Serine esterase in cytolytic T lymphocytes. Nature 322, 740–743 (1986).

    Article  CAS  Google Scholar 

  3. Simon, M.M., Hoschutzky, H., Fruth, U., Simon, H.G. & Kramer, M.D. Purification and characterization of a T cell specific serine proteinase (TSP-1) from cloned cytolytic T lymphocytes. EMBO J. 5, 3267–3274 (1986).

    Article  CAS  Google Scholar 

  4. Young, J.D. et al. Isolation and characterization of a serine esterase from cytolytic T cell granules. Cell 47, 183–194 (1986).

    Article  CAS  Google Scholar 

  5. Masson, D. & Tschopp, J. A family of serine esterases in lytic granules of cytolytic T lymphocytes. Cell 49, 679–685 (1987).

    Article  CAS  Google Scholar 

  6. Fruth, U. et al. A novel serine proteinase (HuTSP) isolated from a cloned human CD8+ cytolytic T cell line is expressed and secreted by activated CD4+ and CD8+ lymphocytes. Eur. J. Immunol. 17, 1625–1633 (1987).

    Article  CAS  Google Scholar 

  7. Krähenbühl, O. et al. Characterization of granzymes A and B isolated from granules of cloned human cytotoxic T lymphocytes. J. Immunol. 141, 3471–3477 (1988).

    PubMed  Google Scholar 

  8. Poe, M. et al. Human cytotoxic lymphocyte tryptase. Its purification from granules and the characterization of inhibitor and substrate specificity. J. Biol. Chem. 263, 13215–13222 (1988).

    CAS  PubMed  Google Scholar 

  9. Gershenfeld, H.K., Hershberger, R.J., Shows, T.B. & Weissman, I.L. Cloning and chromosomal assignment of a human cDNA encoding a T cell- and natural killer cell-specific trypsin-like serine protease. Proc. Natl. Acad. Sci. USA 85, 1184–118 (1988).

    Article  CAS  Google Scholar 

  10. Shi, L., Kam, C.M., Powers, J.C., Aebersold, R. & Greenberg, A.H. Purification of three cytotoxic lymphocyte granule serine proteases that induce apoptosis through distinct substrate and target cell interactions. J. Exp. Med. 176, 1521–1529 (1992).

    Article  CAS  Google Scholar 

  11. Shiver, J.W., Su, L. & Henkart, P.A. Cytotoxicity with target DNA breakdown by rat basophilic leukemia cells expressing both cytolysin and granzyme A. Cell 71, 315–322 (1992).

    Article  CAS  Google Scholar 

  12. Beresford, P.J., Xia, Z., Greenberg, A.H. & Lieberman, J. Granzyme A loading induces rapid cytolysis and a novel form of DNA damage independently of caspase activation. Immunity 10, 585–594 (1999).

    Article  CAS  Google Scholar 

  13. Beresford, P.J., Kam, C.M., Powers, J.C. & Lieberman, J. Recombinant human granzyme A binds to two putative HLA-associated proteins and cleaves one of them. Proc. Natl. Acad. Sci. USA 94, 9285–9290 (1997).

    Article  CAS  Google Scholar 

  14. Fan, Z., Beresford, P.J., Zhang, D. & Lieberman, J. HMG2 interacts with the nucleosome assembly protein SET and is a target of the cytotoxic T-lymphocyte protease granzyme A. Mol. Cell Biol. 22, 2810–2820 (2002).

    Article  CAS  Google Scholar 

  15. Fan, Z. et al. Cleaving the oxidative repair protein Ape1 enhances cell death mediated by granzyme A. Nat. Immunol. 4, 145–153 (2003).

    Article  CAS  Google Scholar 

  16. Pinkoski, M.J. & Green, D.R. Granzyme A: the road less traveled. Nat. Immunol. 4, 106–108 (2003).

    Article  CAS  Google Scholar 

  17. Chakravarti, D. & Hong, R. SET-ting the stage for life and death. Cell 112, 589–591 (2003).

    Article  CAS  Google Scholar 

  18. Fan, Z., Beresford, P.J., Oh, D.Y., Zhang, D. & Lieberman, J. Tumor suppressor NM23-H1 is a granzyme A–activated DNase during CTL-mediated apoptosis, and the nucleosome assembly protein SET is its inhibitor. Cell 112, 659–672 (2003).

    Article  CAS  Google Scholar 

  19. Beresford, P.J. et al. Granzyme A activates an endoplasmic reticulum–associated caspase-independent nuclease to induce single-stranded DNA nicks. J. Biol. Chem. 276, 43285–43293 (2001).

    Article  CAS  Google Scholar 

  20. Zhang, D., Beresford, P.J., Greenberg, A.H. & Lieberman, J. Granzymes A and B directly cleave lamins and disrupt the nuclear lamina during granule-mediated cytolysis. Proc. Natl. Acad. Sci. USA 98, 5746–5751 (2001).

    Article  CAS  Google Scholar 

  21. Zhang, D. et al. Induction of rapid histone degradation by the cytotoxic T lymphocyte protease granzyme A. J. Biol. Chem. 276, 3683–3690 (2001).

    Article  CAS  Google Scholar 

  22. Simon, M.M., Fruth, U., Simon, H.G., Gay, S. & Kramer, M.D. Evidence for multiple functions of T-lymphocytes associated serine proteinases. Adv. Exp. Med. Biol. 247A, 609–613 (1989).

    Article  CAS  Google Scholar 

  23. Kam, C.M., Hudig, D. & Powers, J.C. Granzymes (lymphocyte serine proteases): characterization with natural and synthetic substrates and inhibitors. Biochim. Biophys. Acta 1477, 307–323 (2000).

    Article  CAS  Google Scholar 

  24. Wilharm, E. et al. Generation of catalytically active granzyme K from Escherichia coli inclusion bodies and identification of efficient granzyme K inhibitors in human plasma. J. Biol. Chem. 274, 27331–27337 (1999).

    Article  CAS  Google Scholar 

  25. Hink-Schauer, C. et al. The 2.2-Å crystal structure of human pro-granzyme K reveals a rigid zymogen with unusual features. J. Biol. Chem. 277, 50923–50933 (2002).

    Article  CAS  Google Scholar 

  26. Jackson, D.S. et al. Synthesis and evaluation of diphenyl phosphonate esters as inhibitors of the trypsin-like granzymes A and K and mast cell tryptase. J. Med. Chem. 41, 2289–2301 (1998).

    Article  CAS  Google Scholar 

  27. Bode, W. et al. X-ray crystal structure of the complex of human leukocyte elastase (PMN elastase) and the third domain of the turkey ovomucoid inhibitor. EMBO J. 5, 2453–2458 (1986).

    Article  CAS  Google Scholar 

  28. Chandrasekharan, U.M., Sanker, S., Glynias, M.J., Karnik, S.S. & Husain, A. Angiotensin II-forming activity in a reconstructed ancestral chymase. Science 271, 502–505 (1996).

    Article  CAS  Google Scholar 

  29. Parry, M.A. et al. The ternary microplasmin-staphylokinase-microplasmin complex is a proteinase–cofactor–substrate complex in action. Nat. Struct. Biol. 5, 917–923 (1998).

    Article  CAS  Google Scholar 

  30. Parry, M.A., Zhang, X.C. & Bode, I. Molecular mechanisms of plasminogen activation: bacterial cofactors provide clues. Trends Biochem. Sci. 25, 53–59 (2000).

    Article  CAS  Google Scholar 

  31. Bell, J.K. et al. The oligomeric structure of human granzyme A is a determinant of its extended substrate specificity. Nat. Struct. Biol. 10, 527–534 (2003).

    Article  CAS  Google Scholar 

  32. Navaza, J. Implementation of molecular replacement in AMoRe. Acta Cryst. 57, 1367–1372 (2001).

    CAS  Google Scholar 

  33. Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D. Biol. Crystallogr. 54 (Pt 5), 905–921 (1998).

    Article  Google Scholar 

  34. Collaborative Computational Project 4. The CCP4 suite: programs for protein crystallography. Acta Cryst. D 50, 760–763 (1994).

  35. Laskowski, R., MacArthur, M., Hutchinson, E. & Thorton, J.J. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283–291 (1993).

    Article  CAS  Google Scholar 

  36. Estébanez-Perpiná, E. et al. Crystal structure of the caspase activator human granzyme B, a proteinase highly specific for an Asp-P1 residue. Biol. Chem. 381, 1203–1214 (2000).

    Article  Google Scholar 

  37. Nicholls, A., Bharadwaj, R. & Honig, B. GRASP - graphical representation and analysis of surface-properties. Biophys. J. 64, A166 (2003).

    Google Scholar 

Download references

Acknowledgements

We thank R. Friedrich, P. Fuentes-Prior, S. Steinbacher and W. Klinkert for helpful discussions and H. Wekerle and R. Huber for their continuous interest in the project. Recombinant SET was provided by J. Lieberman. This work was supported by grants of the German Research Council and the European Union.

Author information

Author notes

  1. Clara Hink-Schauer and Eva Estébanez-Perpiñá: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Neuroimmunology, Max Planck Institute of Neurobiology, Am Klopferspitz 18a, Planegg-Martinsried, D-82152, Germany

    Clara Hink-Schauer, Florian C Kurschus & Dieter E Jenne

  2. Department of Structural Research, Max Planck Institute of Biochemistry, Am Klopferspitz 18a, Planegg-Martinsried, D-82152, Germany

    Eva Estébanez-Perpiñá & Wolfram Bode

Authors
  1. Clara Hink-Schauer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eva Estébanez-Perpiñá
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Florian C Kurschus
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wolfram Bode
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dieter E Jenne
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dieter E Jenne.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hink-Schauer, C., Estébanez-Perpiñá, E., Kurschus, F. et al. Crystal structure of the apoptosis-inducing human granzyme A dimer. Nat Struct Mol Biol 10, 535–540 (2003). https://doi.org/10.1038/nsb945

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsb945

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing