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:

Structure of collagenase G reveals a chew-and-digest mechanism of bacterial collagenolysis

Nature Structural & Molecular Biology volume 18, pages 1109–1114 (2011)Cite this article

Subjects

Abstract

Collagen constitutes one-third of body protein in humans, reflecting its extensive role in health and disease. Of similar importance, therefore, are the idiosyncratic proteases that have evolved for collagen remodeling. The most efficient collagenases are those that enable clostridial bacteria to colonize their host tissues; but despite intense study, the structural and mechanistic basis of these enzymes has remained elusive. Here we present the crystal structure of collagenase G from Clostridium histolyticum at 2.55-Å resolution. By combining the structural data with enzymatic and mutagenesis studies, we derive a conformational two-state model of bacterial collagenolysis, in which recognition and unraveling of collagen microfibrils into triple helices, as well as unwinding of the triple helices, are driven by collagenase opening and closing.

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: Domain organization and architecture of ColG.
Figure 2: The activator domain is necessary for the degradation of collagen.
Figure 3: Mapping the peptidase active site.
Figure 4: Unified processing model of triple-helical and microfibrillar collagen.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Ottani, V., Martini, D., Franchi, M., Ruggeri, A. & Raspanti, M. Hierarchical structures in fibrillar collagens. Micron 33, 587–596 (2002).

    Article  CAS  Google Scholar 

  2. Brozek, J., Grande, F., Anderson, J.T. & Keys, A. Densitometric analysis of body composition: revision of some quantitative assumptions. Ann. NY Acad. Sci. 110, 113–140 (1963).

    Article  CAS  Google Scholar 

  3. Sweeney, S.M. et al. Candidate cell and matrix interaction domains on the collagen fibril, the predominant protein of vertebrates. J. Biol. Chem. 283, 21187–21197 (2008).

    Article  CAS  Google Scholar 

  4. Bond, M.D. & Van Wart, H.E. Purification and separation of individual collagenases of Clostridium histolyticum using red dye ligand chromatography. Biochemistry 23, 3077–3085 (1984).

    Article  CAS  Google Scholar 

  5. Matsushita, O. et al. A study of the collagen-binding domain of a 116-kDa Clostridium histolyticum collagenase. J. Biol. Chem. 273, 3643–3648 (1998).

    Article  CAS  Google Scholar 

  6. Borkakoti, N. et al. Structure of the catalytic domain of human fibroblast collagenase complexed with an inhibitor. Nat. Struct. Biol. 1, 106–110 (1994).

    Article  CAS  Google Scholar 

  7. Li, J. et al. Structure of full-length porcine synovial collagenase reveals a C-terminal domain containing a calcium-linked, four-bladed beta-propeller. Structure 3, 541–549 (1995).

    Article  CAS  Google Scholar 

  8. Stams, T. et al. Structure of human neutrophil collagenase reveals large S1′ specificity pocket. Nat. Struct. Biol. 1, 119–123 (1994).

    Article  CAS  Google Scholar 

  9. Iyer, S., Visse, R., Nagase, H. & Acharya, K.R. Crystal structure of an active form of human MMP-1. J. Mol. Biol. 362, 78–88 (2006).

    Article  CAS  Google Scholar 

  10. Adhikari, A.S., Chai, J. & Dunn, A.R. Mechanical load induces a 100-fold increase in the rate of collagen proteolysis by MMP-1. J. Am. Chem. Soc. 133, 1686–1689 (2011).

    Article  CAS  Google Scholar 

  11. Han, S. et al. Molecular mechanism of type I collagen homotrimer resistance to mammalian collagenases. J. Biol. Chem. 285, 22276–22281 (2010).

    Article  CAS  Google Scholar 

  12. Minond, D., Lauer-Fields, J.L., Nagase, H. & Fields, G.B. Matrix metalloproteinase triple-helical peptidase activities are differentially regulated by substrate stability. Biochemistry 43, 11474–11481 (2004).

    Article  CAS  Google Scholar 

  13. Chung, L. et al. Collagenase unwinds triple-helical collagen prior to peptide bond hydrolysis. EMBO J. 23, 3020–3030 (2004).

    Article  CAS  Google Scholar 

  14. Tam, E.M., Moore, T.R., Butler, G.S. & Overall, C.M. Characterization of the distinct collagen binding, helicase and cleavage mechanisms of matrix metalloproteinase 2 and 14 (gelatinase A and MT1-MMP): the differential roles of the MMP hemopexin C domains and the MMP-2 fibronectin type II modules in collagen triple helicase activities. J. Biol. Chem. 279, 43336–43344 (2004).

    Article  CAS  Google Scholar 

  15. Fecteau, K.A., Haffner, J.C. & Eiler, H. The potential of collagenase as a new therapy for separation of human retained placenta: hydrolytic potency on human, equine and bovine placentae. Placenta 19, 379–383 (1998).

    Article  CAS  Google Scholar 

  16. Shi, L. & Carson, D. Collagenase Santyl ointment: a selective agent for wound debridement. J. Wound Ostomy Continence Nurs. 36, S12–S16 (2009).

    Article  Google Scholar 

  17. Hesse, F., Burtscher, H., Popp, F. & Ambrosius, D. Recombinant enzymes for islet isolation: purification of a collagenase from Clostridium histolyticum and cloning/expression of the gene. Transplant. Proc. 27, 3287–3289 (1995).

    CAS  PubMed  Google Scholar 

  18. Ducka, P. et al. A universal strategy for high-yield production of soluble and functional clostridial collagenases in E. coli. Appl. Microbiol. Biotechnol. 83, 1055–1065 (2009).

    Article  CAS  Google Scholar 

  19. Wilson, J.J., Matsushita, O., Okabe, A. & Sakon, J. A bacterial collagen-binding domain with novel calcium-binding motif controls domain orientation. EMBO J. 22, 1743–1752 (2003).

    Article  CAS  Google Scholar 

  20. Groves, M.R. & Barford, D. Topological characteristics of helical repeat proteins. Curr. Opin. Struct. Biol. 9, 383–389 (1999).

    Article  CAS  Google Scholar 

  21. Kyrieleis, O.J., Goettig, P., Kiefersauer, R., Huber, R. & Brandstetter, H. Crystal structures of the tricorn interacting factor F3 from Thermoplasma acidophilum, a zinc aminopeptidase in three different conformations. J. Mol. Biol. 349, 787–800 (2005).

    Article  CAS  Google Scholar 

  22. Thunnissen, M.M., Nordlund, P. & Haeggstrom, J.Z. Crystal structure of human leukotriene A4 hydrolase, a bifunctional enzyme in inflammation. Nat. Struct. Biol. 8, 131–135 (2001).

    Article  CAS  Google Scholar 

  23. Matthews, B.W., Jansonius, J.N., Colman, P.M., Schoenborn, B.P. & Dupourque, D. Three-dimensional structure of thermolysin. Nat. New Biol. 238, 37–41 (1972).

    Article  CAS  Google Scholar 

  24. Jung, C.M. et al. Identification of metal ligands in the Clostridium histolyticum ColH collagenase. J. Bacteriol. 181, 2816–2822 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Wang, Y.K. et al. Mechanistic insight into the function of the C-terminal PKD domain of the collagenolytic serine protease deseasin MCP-01 from deep sea Pseudoalteromonas sp. SM9913: binding of the PKD domain to collagen results in collagen swelling but does not unwind the collagen triple helix. J. Biol. Chem. 285, 14285–14291 (2010).

    Article  CAS  Google Scholar 

  26. Sanchez-Lopez, R., Alexander, C.M., Behrendtsen, O., Breathnach, R. & Werb, Z. Role of zinc-binding- and hemopexin domain-encoded sequences in the substrate specificity of collagenase and stromelysin-2 as revealed by chimeric proteins. J. Biol. Chem. 268, 7238–7247 (1993).

    CAS  PubMed  Google Scholar 

  27. Vallee, B.L. & Auld, D.S. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 29, 5647–5659 (1990).

    Article  CAS  Google Scholar 

  28. Eckhard, U. et al. Biochemical characterization of the catalytic domains of three different Clostridial collagenases. Biol. Chem. 390, 11–18 (2009).

    Article  CAS  Google Scholar 

  29. Mookhtiar, K.A., Steinbrink, D.R. & Van Wart, H.E. Mode of hydrolysis of collagen-like peptides by class I and class II Clostridium histolyticum collagenases: evidence for both endopeptidase and tripeptidylcarboxypeptidase activities. Biochemistry 24, 6527–6533 (1985).

    Article  CAS  Google Scholar 

  30. Hu, Y. et al. Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library. J. Biol. Chem. 277, 8366–8371 (2002).

    Article  CAS  Google Scholar 

  31. French, M.F., Bhown, A. & Van Wart, H.E. Identification of Clostridium histolyticum collagenase hyperreactive sites in type I, II, and III collagens: lack of correlation with local triple helical stability. J. Protein Chem. 11, 83–97 (1992).

    Article  CAS  Google Scholar 

  32. French, M.F., Mookhtiar, K.A. & Van Wart, H.E. Limited proteolysis of type I collagen at hyperreactive sites by class I and II Clostridium histolyticum collagenases: complementary digestion patterns. Biochemistry 26, 681–687 (1987).

    Article  CAS  Google Scholar 

  33. Hulmes, D.J. Building collagen molecules, fibrils, and suprafibrillar structures. J. Struct. Biol. 137, 2–10 (2002).

    Article  CAS  Google Scholar 

  34. Wess, T.J. Collagen fibril form and function. Adv. Protein Chem. 70, 341–374 (2005).

    Article  CAS  Google Scholar 

  35. Berisio, R., Vitagliano, L., Mazzarella, L. & Zagari, A. Crystal structure of a collagen-like polypeptide with repeating sequence Pro-Hyp-Gly at 1.4 Å resolution: implications for collagen hydration. Biopolymers 56, 8–13 (2001).

    Article  CAS  Google Scholar 

  36. Chung, L. et al. Identification of the 183RWTNNFREY191 region as a critical segment of matrix metalloproteinase 1 for the expression of collagenolytic activity. J. Biol. Chem. 275, 29610–29617 (2000).

    Article  CAS  Google Scholar 

  37. Clegg, P.D., Burke, R.M., Coughlan, A.R., Riggs, C.M. & Carter, S.D. Characterisation of equine matrix metalloproteinase 2 and 9; and identification of the cellular sources of these enzymes in joints. Equine Vet. J. 29, 335–342 (1997).

    Article  CAS  Google Scholar 

  38. Itoh, Y. et al. Cell surface collagenolysis requires homodimerization of the membrane-bound collagenase MT1-MMP. Mol. Biol. Cell 17, 5390–5399 (2006).

    Article  CAS  Google Scholar 

  39. Itoh, Y., Ito, N., Nagase, H. & Seiki, M. The second dimer interface of MT1-MMP, the transmembrane domain, is essential for ProMMP-2 activation on the cell surface. J. Biol. Chem. 283, 13053–13062 (2008).

    Article  CAS  Google Scholar 

  40. Malla, N., Sjoli, S., Winberg, J.O., Hadler-Olsen, E. & Uhlin-Hansen, L. Biological and pathobiological functions of gelatinase dimers and complexes. Connect. Tissue Res. 49, 180–184 (2008).

    Article  CAS  Google Scholar 

  41. Olson, M.W. et al. Characterization of the monomeric and dimeric forms of latent and active matrix metalloproteinase-9. Differential rates for activation by stromelysin 1. J. Biol. Chem. 275, 2661–2668 (2000).

    Article  CAS  Google Scholar 

  42. Saffarian, S., Collier, I.E., Marmer, B.L., Elson, E.L. & Goldberg, G. Interstitial collagenase is a Brownian ratchet driven by proteolysis of collagen. Science 306, 108–111 (2004).

    Article  CAS  Google Scholar 

  43. Cha, H., Kopetzki, E., Huber, R., Lanzendorfer, M. & Brandstetter, H. Structural basis of the adaptive molecular recognition by MMP9. J. Mol. Biol. 320, 1065–1079 (2002).

    Article  CAS  Google Scholar 

  44. Tochowicz, A. et al. The dimer interface of the membrane type 1 matrix metalloproteinase hemopexin domain: crystal structure and biological functions. J. Biol. Chem. 286, 7587–7600 (2011).

    Article  CAS  Google Scholar 

  45. Overall, C.M. & Butler, G.S. Protease yoga: extreme flexibility of a matrix metalloproteinase. Structure 15, 1159–1161 (2007).

    Article  CAS  Google Scholar 

  46. Salamone, M. et al. A new method to value efficiency of enzyme blends for pancreatic tissue digestion. Transplant. Proc. 42, 2043–2048 (2010).

    Article  CAS  Google Scholar 

  47. Supuran, C.T., Scozzafava, A. & Clare, B.W. Bacterial protease inhibitors. Med. Res. Rev. 22, 329–372 (2002).

    Article  CAS  Google Scholar 

  48. Eckhard, U., Nüss, D., Ducka, P., Schönauer, E. & Brandstetter, H. Crystallization and preliminary X-ray characterization of the catalytic domain of collagenase G from Clostridium histolyticum. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 64, 419–421 (2008).

    Article  CAS  Google Scholar 

  49. Briers, Y., Lavigne, R., Volckaert, G. & Hertveldt, K. A standardized approach for accurate quantification of murein hydrolase activity in high-throughput assays. J. Biochem. Biophys. Methods 70, 531–533 (2007).

    Article  CAS  Google Scholar 

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

  51. Panjikar, S., Parthasarathy, V., Lamzin, V.S., Weiss, M.S. & Tucker, P.A. Auto-Rickshaw: an automated crystal structure determination platform as an efficient tool for the validation of an X-ray diffraction experiment. Acta Crystallogr. D Biol. Crystallogr. 61, 449–457 (2005).

    Article  Google Scholar 

  52. Sheldrick, G.M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008).

    Article  CAS  Google Scholar 

  53. Cowtan, K.D. & Zhang, K.Y. Density modification for macromolecular phase improvement. Prog. Biophys. Mol. Biol. 72, 245–270 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  55. Painter, J. & Merritt, E.A. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D Biol. Crystallogr. 62, 439–450 (2006).

    Article  Google Scholar 

  56. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  57. DeLano, W.L. The case for open-source software in drug discovery. Drug Discov. Today 10, 213–217 (2005).

    Article  CAS  Google Scholar 

  58. Laskowski, R.A., Moss, D.S. & Thornton, J.M. Main-chain bond lengths and bond angles in protein structures. J. Mol. Biol. 231, 1049–1067 (1993).

    Article  CAS  Google Scholar 

  59. Hooft, R.W., Vriend, G., Sander, C. & Abola, E.E. Errors in protein structures. Nature 381, 272 (1996).

    Article  CAS  Google Scholar 

  60. Weichenberger, C.X. & Sippl, M.J. NQ-Flipper: recognition and correction of erroneous asparagine and glutamine side-chain rotamers in protein structures. Nucleic Acids Res. 35, W403–W406 (2007).

    Article  Google Scholar 

Download references

Acknowledgements

We thank F. Hesse (Roche, Penzberg) for providing plasmids; P. Klemm for cloning an activator-deletion construct; S. Ginzinger and M. Sippl for help with canonical electron density expansion; L. Moroder and H. Nagase for valuable discussions; our anonymous reviewers for helpful suggestions and clarifications; staff at the synchrotron facilities ESRF, DESY and BESSY for help in data collection; and the Austrian Science Fund (FWF) for funding (H.B., project P20582).

Author information

Authors and Affiliations

  1. Division of Structural Biology, Department of Molecular Biology, University of Salzburg, Salzburg, Austria

    Ulrich Eckhard, Esther Schönauer, Dorota Nüss & Hans Brandstetter

Authors
  1. Ulrich Eckhard
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Esther Schönauer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dorota Nüss
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hans Brandstetter
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

U.E. performed experiments (protein production, enzymological measurements, crystallization, X-ray data collection and structure determination), analyzed data and prepared the manuscript; E.S. performed experiments (enzymological measurements), analyzed data and prepared the manuscript; D.N. performed experiments (crystal harvesting and X-ray data collection); H.B. devised the project, helped with structure solution, analyzed data and wrote the paper.

Corresponding author

Correspondence to Hans Brandstetter.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2 (PDF 717 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Eckhard, U., Schönauer, E., Nüss, D. et al. Structure of collagenase G reveals a chew-and-digest mechanism of bacterial collagenolysis. Nat Struct Mol Biol 18, 1109–1114 (2011). https://doi.org/10.1038/nsmb.2127

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2127

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