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:

The oligomeric structure of human granzyme A is a determinant of its extended substrate specificity

Nature Structural & Molecular Biology volume 10, pages 527–534 (2003)Cite this article

Abstract

The cell death–inducing serine protease granzyme A (GzmA) has a unique disulfide-linked quaternary structure. The structure of human GzmA bound to a tripeptide CMK inhibitor, determined at a resolution of 2.4 Å, reveals that the oligomeric state contributes to substrate selection by limiting access to the active site for potential macromolecular substrates and inhibitors. Unlike other serine proteases, tetrapeptide substrate preferences do not correlate well with natural substrate cleavage sequences. This suggests that the context of the cleavage sequence within a macromolecular substrate imposes another level of selection not observed with the peptide substrates. Modeling of inhibitors bound to the GzmA active site shows that the dimer also contributes to substrate specificity in a unique manner by extending the active-site cleft. The crystal structure, along with substrate library profiling and mutagenesis, has allowed us to identify and rationally manipulate key components involved in GzmA substrate specificity.

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: Overall structure of dimeric human granzyme A.
Figure 2: Dimeric interface of human granzyme A.
Figure 3: The active site of human granzyme A.
Figure 4: Combinatorial substrate library results for human, mouse and H→M granzyme A.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Russell, J.H. & Ley, T.J. Lymphocyte-mediated cytotoxicity. Annu. Rev. Immunol. 20, 323–370 (2002).

    Article  CAS  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Shresta, S., Graubert, T.A., Thomas, D.A., Raptis, S.Z. & Ley, T.J. Granzyme A initiates an alternative pathway for granule-mediated apoptosis. Immunity 10, 595–605 (1999).

    Article  CAS  Google Scholar 

  5. 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 

  6. 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 

  7. 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 nulceosome assembly protein SET is its inhibitor. Cell 112, 659–672 (2003).

    Article  CAS  Google Scholar 

  8. Irmler, M. et al. Granzyme A is an interleukin 1 β-converting enzyme. J. Exp. Med. 181, 1917–1922 (1995).

    Article  CAS  Google Scholar 

  9. Suidan, H.S. et al. Granzyme A released upon stimulation of cytotoxic T lymphocytes activates the thrombin receptor on neuronal cells and astroctyes. Proc. Natl. Acad. Sci. USA 91, 8112–8116 (1994).

    Article  CAS  Google Scholar 

  10. 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 

  11. 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 

  12. 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 

  13. 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 

  14. Odake, S. et al. Human and murine cytotoxic T lymphocyte serine proteases subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins. Biochemistry 30, 2217–2227 (1991).

    Article  CAS  Google Scholar 

  15. Pasternack, M.S., Bleier, K.J. & McInerney, T.N. Granzyme A binding to target cell proteins. Granzyme A binds to and cleaves nucleolin in vitro. J. Biol. Chem. 266, 14703–14708 (1991).

    CAS  PubMed  Google Scholar 

  16. Masson, D., Zamai, M. & Tschopp, J. Identification of granzyme A isolated from cytotoxic T-lymphocyte-granules as one of the proteases encoded by CTL-specific genes. FEBS Lett. 208, 84–88 (1986).

    Article  CAS  Google Scholar 

  17. Schechter, I. & Berger, A. On the active site of protease. 3. Mapping the active of papain; specific peptide inhibitors of papain. Biochem. Biophys. Res. Comm. 32, 898–902 (1968).

    Article  CAS  Google Scholar 

  18. Thornberry, N.A. et al. A combinatorial approach defines specificity of members of the caspase family and granzyme B. Functional relationships estabilshed for key mediators of apoptosis. J. Biol. Chem. 272, 17907–17911 (1997).

    Article  CAS  Google Scholar 

  19. Harris, J.L. et al. Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Proc. Natl. Acad. Sci. USA 97, 7754–7759 (2000).

    Article  CAS  Google Scholar 

  20. Backes, B.J., Harris, J.L., Leonetti, F., Craik, C.S. & Ellman, J.A. Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin. Nat. Biotechnol. 18, 187–193 (2000).

    Article  CAS  Google Scholar 

  21. Kim, S., Narayana, S.V. & Volanakis, J.V. Crystal structure of a complement factor D mutant expressing enhanced catalytic activity. J. Biol. Chem. 270, 24399–24405 (1995).

    Article  CAS  Google Scholar 

  22. Holm, L. & Sander, C. Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993).

    Article  CAS  Google Scholar 

  23. Galvin, J.P. et al. Apoptosis induced by granzyme B-glycosaminoglycan complexes: implications for granule-mediated apoptosis in vivo. J. Immunol. 164, 5345–5350 (1999).

    Google Scholar 

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

    CAS  PubMed  Google Scholar 

  25. Barrios, A.M. & Craik, C.S. Scanning the prime-site substrate specificity of proteolytic enzymes: A novel assay based on ligand-enhanced lanthanide ion fluorescence. Bioorg. Med. Chem. Lett. 12, 3619–3623 (2002).

    Article  CAS  Google Scholar 

  26. 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 

  27. 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 

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

    CAS  PubMed  Google Scholar 

  29. Murphy, M.E. et al. Comparative molecular model building of two serine proteinases from cytotoxic T lymphocytes. Proteins 4, 190–204 (1988).

    Article  CAS  Google Scholar 

  30. Harris, J.L., Peterson, E.P., Hudig, D., Thornberry, N.A. & Craik, C.S. Definition and redesign of the extended substrate specificity of granzyme B. J. Biol. Chem. 273, 27364–27373 (1998).

    Article  CAS  Google Scholar 

  31. Waugh, S.M., Harris, J.L., Fletterick, R. & Craik, C.S. The structure of the pro-apoptotic protease granzyme B reveals the molecular determinants of its specificity. Nat. Struct. Biol. 7, 762–765 (2000).

    Article  CAS  Google Scholar 

  32. Earnshaw, W.C., Martins, L.M. & Kaufmann, S.H. Mammalian caspases: Structure, activation, substratea and functions during apoptosis. Annu. Rev. Biochem. 68, 383–424 (1999).

    Article  CAS  Google Scholar 

  33. Pereira, P.J. et al. Human β-tryptase is a ring-like tetramer with active sites facing a central pore. Nature 392, 306–311 (1998).

    Article  CAS  Google Scholar 

  34. Perona, J.J., Tsu, C.A., Craik, C.S. & Fletterick, R.J. Crystal structure of an ecotin-collagenase complex suggest a model for recognition and cleavage of the collagen triple helix. Biochemistry 36, 5381–5392 (1997).

    Article  CAS  Google Scholar 

  35. Eggers, C.T., Wang, S.X., Fletterick, R. & Craik, C.S. The role of ecotin dimerization in protease inhibition. J. Mol. Biol. 308, 975–991 (2001).

    Article  CAS  Google Scholar 

  36. Wang, S.X., Esmon, C.T. & Fletterick, R.J. Crystal structure of thrombin-ecotin reveals conformational changes and extended interactions. Biochemistry 40, 10038–10046 (2001).

    Article  CAS  Google Scholar 

  37. Tsuzuki, S. et al. Purification and identification of a binding protein for pancreatic secretory trypsin inhbitor: a novel role of the inhibitor as an anti-granzyme A. Biochem. J. 372, 227–233 (2003).

    Article  CAS  Google Scholar 

  38. Hink-Schauer, C., Estebanez-Perpina, E., Bode, W. & Jenne, D.E. Crystal structure of the apoptosis-inducing human granzyme A dimer. Nat. Struct. Biol. 10, 535–540 (2003).

    Article  CAS  Google Scholar 

  39. 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 

  40. Gill, S.C. & von Hippel, P.H. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319–326 (1989).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  42. Kissinger, C.R., Gehlhaar, D.K. & Fogel, D.B. Rapid Automated Molecular Replacement By Evolutionary Search. Acta Crystallogr. D 55, 484–491 (1999).

    Article  CAS  Google Scholar 

  43. Cowtan, K. 'dm': An automated procedure for phase improvement by density modification. Joint CCP4/ESF-EACBM Newsletter on Prot. Crystallogr. 31, 34–38 (1994).

    Google Scholar 

  44. Lamzin, V.S. & Wilson, K.S. Automated refinement of protein models. Acta Crystallogr. D 49, 129–149 (1993).

    Article  CAS  Google Scholar 

  45. 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 

  46. Murshudov, G., Vagin, A. & Dodson, E. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  47. Peitsch, M.C. ProMod and Swiss-Model: Internet-based tools for automated comparative protein modelling. Biochem. Soc. Trans. 24, 274–279 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors would like to express thanks to J. Holton for technical assistance during data collection at the ALS beam line 8.3.1. We would also like to acknowledge the Macromolecular Structure Group at the University of California, San Francisco for computational support. This work was supported in part by a US National Institutes of Health post-doctoral training grant to J.K.B. and grants from the US National Institutes of Health to C.S.C. and R.J.F.

Author information

Author notes

  1. Jessica K Bell and David H Goetz: These authors have contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, University of California, San Francisco, 94143, California, USA

    Jessica K Bell, David H Goetz, Sami Mahrus, Jennifer L Harris & Charles S Craik

  2. Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, 94143, California, USA

    Sami Mahrus & Jennifer L Harris

  3. Department of Biochemistry and Biophysics, University of California, San Francisco, 94143, California, USA

    Robert J Fletterick

Authors
  1. Jessica K Bell
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David H Goetz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sami Mahrus
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jennifer L Harris
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Robert J Fletterick
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Charles S Craik
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Charles S Craik.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bell, J., Goetz, D., Mahrus, S. et al. The oligomeric structure of human granzyme A is a determinant of its extended substrate specificity. Nat Struct Mol Biol 10, 527–534 (2003). https://doi.org/10.1038/nsb944

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsb944

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