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.

Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation

Abstract

Many bacterial pathogens secrete proteins that activate host trypsinogen-like enzyme precursors, most notably the proenzymes of the blood coagulation and fibrinolysis systems1,2. Staphylococcus aureus, an important human pathogen implicated in sepsis and endocarditis3, secretes the cofactor staphylocoagulase, which activates prothrombin, without the usual proteolytic cleavages, to directly initiate blood clotting4,5. Here we present the 2.2 Å crystal structures of human α-thrombin and prethrombin-2 bound to a fully active staphylocoagulase variant. The cofactor consists of two domains, each with three-helix bundles; this is a novel fold that is distinct from known serine proteinase activators, particularly the streptococcal plasminogen activator streptokinase6. The staphylocoagulase fold is conserved in other bacterial plasma-protein-binding factors and extracellular-matrix-binding factors7,8,9. Kinetic studies confirm the importance of isoleucine 1 and valine 2 at the amino terminus of staphylocoagulase for zymogen activation. In addition to making contacts with the 148 loop and (pro)exosite I of prethrombin-2, staphylocoagulase inserts its N-terminal peptide into the activation pocket of bound prethrombin-2, allosterically inducing functional catalytic machinery. These investigations demonstrate unambiguously the validity of the zymogen-activation mechanism known as ‘molecular sexuality’10.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Domain organization of human prothrombin and ZAAPs.
Figure 2: Structure of the human α-thrombin•Met-SC(1–325) complex.
Figure 3: Thrombin–staphylocoagulase interface.
Figure 4: Contribution of the staphylocoagulase N-terminal peptide to zymogen activation.

References

  1. Boyle, M. D. & Lottenberg, R. Plasminogen activation by invasive human pathogens. Thromb. Haemost. 77, 1–10 (1997)

    CAS  Article  Google Scholar 

  2. Lähteenmäki, K., Kuusela, P. & Korhonen, T. K. Bacterial plasminogen activators and receptors. FEMS Microbiol. Rev. 25, 531–552 (2001)

    Article  Google Scholar 

  3. Mylonakis, E. & Calderwood, S. B. Medical progress: Infective endocarditis in adults. N. Engl. J. Med. 345, 1318–1330 (2001)

    CAS  Article  Google Scholar 

  4. Hemker, H. C., Bas, B. M. & Muller, A. D. Activation of a pro-enzyme by a stoichiometric reaction with another protein. The reaction between prothrombin and staphylocoagulase. Biochim. Biophys. Acta 379, 180–188 (1975)

    CAS  Article  Google Scholar 

  5. Kawabata, S. et al. Structure and function relationship of staphylocoagulase. J. Prot. Chem. 6, 17–32 (1987)

    CAS  Article  Google Scholar 

  6. Wang, X., Lin, X., Loy, J. A., Tang, J. & Zhang, X. C. Crystal structure of the catalytic domain of human plasmin complexed with streptokinase. Science 281, 1662–1665 (1998)

    ADS  CAS  Article  Google Scholar 

  7. Bjerketorp, J. et al. A novel von Willebrand factor binding protein expressed by Staphylococcus aureus. Microbiology 148, 2037–2044 (2002)

    CAS  Article  Google Scholar 

  8. Tettelin, H. et al. Complete genome sequence and comparative genomic analysis of an emerging human pathogen, serotype V Streptococcus agalactiae. Proc. Natl Acad. Sci. USA 99, 12391–12396 (2002)

    ADS  CAS  Article  Google Scholar 

  9. Glaser, P. et al. Genome sequence of Streptococcus agalactiae, a pathogen causing invasive neonatal disease. Mol. Microbiol. 45, 1499–1513 (2002)

    CAS  Article  Google Scholar 

  10. Bode, W. & Huber, R. Induction of the bovine trypsinogen–trypsin transition by peptides sequentially similar to the N-terminus of trypsin. FEBS Lett. 68, 231–236 (1976)

    CAS  Article  Google Scholar 

  11. Huber, R. & Bode, W. Structural basis of the activation and action of trypsin. Acc. Chem. Res. 11, 114–122 (1978)

    CAS  Article  Google Scholar 

  12. Khan, A. R. & James, M. N. Molecular mechanisms for the conversion of zymogens to active proteolytic enzymes. Protein Sci. 7, 815–836 (1998)

    CAS  Article  Google Scholar 

  13. Boxrud, P. D., Verhamme, I. M., Fay, W. P. & Bock, P. E. Streptokinase triggers conformational activation of plasminogen through specific interactions of the amino-terminal sequence and stabilizes the active zymogen conformation. J. Biol. Chem. 276, 26084–26089 (2001)

    CAS  Article  Google Scholar 

  14. Wang, S., Reed, G. L. & Hedstrom, L. Deletion of Ile1 changes the mechanism of streptokinase: evidence for the molecular sexuality hypothesis. Biochemistry 38, 5232–5240 (1999)

    CAS  Article  Google Scholar 

  15. Vijayalakshmi, J., Padmanabhan, K. P., Mann, K. G. & Tulinsky, A. The isomorphous structures of prethrombin2, hirugen-, and PPACK-thrombin: changes accompanying activation and exosite binding to thrombin. Protein Sci. 3, 2254–2271 (1994)

    CAS  Article  Google Scholar 

  16. Bode, W. et al. The refined 1.9 Å crystal structure of human α-thrombin: interaction with d-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J. 8, 3467–3475 (1989)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  18. Anderson, P. J., Nesset, A., Dharmawardana, K. R. & Bock, P. E. Characterization of proexosite I on prothrombin. J. Biol. Chem. 275, 16428–16434 (2000)

    CAS  Article  Google Scholar 

  19. Bock, P. E. Active-site-selective labeling of blood coagulation proteinases with fluorescence probes by the use of thioester peptide chloromethyl ketones II. Properties of thrombin derivatives as reporters of prothrombin fragment 2 binding and specificity of the labeling approach for other proteinases. J. Biol. Chem. 267, 14974–14981 (1992)

    CAS  PubMed  Google Scholar 

  20. Tager, M. & Drummond, M. C. Staphylocoagulase. Ann. NY Acad. Sci. 128, 92–111 (1965)

    ADS  CAS  Article  Google Scholar 

  21. Bock, P. E., Olson, S. T. & Björk, I. Inactivation of thrombin by antithrombin is accompanied by inactivation of regulatory exosite I. J. Biol. Chem. 272, 19837–19845 (1997)

    CAS  Article  Google Scholar 

  22. Cheung, A. I., Projan, S. J., Edelstein, R. E. & Fischetti, V. A. Cloning, expression, and nucleotide sequence of a Staphylococcus aureus gene (fbpA) encoding a fibrinogen-binding protein. Infect. Immun. 63, 1914–1920 (1995)

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Jeng, A. et al. Molecular genetic analysis of a group A Streptococcus operon encoding serum opacity factor and a novel fibronectin-binding protein, SfbX. J. Bacteriol. 185, 1208–1217 (2003)

    CAS  Article  Google Scholar 

  24. Joh, D., Wann, E. R., Kreikemeyer, B., Speziale, P. & Höök, M. Role of fibronectin-binding MSCRAMMs in bacterial adherence and entry into mammalian cells. Matrix Biol. 18, 211–223 (1999)

    CAS  Article  Google Scholar 

  25. Schwarz-Linek, U. et al. Pathogenic bacteria attach to human fibronectin through a tandem β-zipper. Nature 423, 177–181 (2003)

    ADS  CAS  Article  Google Scholar 

Download references

Acknowledgements

We are grateful to Diagnostica Stago for the S. aureus strain, to R. Mentele for sequencing, to G. Bourenkov for help during data collection, and to S. Iwanaga for initiating the project. We thank E. Consacro, H. Kroh and S. Stuart for excellent technical assistance. This work was supported by a National Institutes of Health (NIH) grant to P.E.B., as well as funding from SFB469, the EU project SPINE and the Fonds der Chemischen Industrie. P.P. and P.J.A were supported in part by NIH training grants.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Wolfram Bode or Paul E. Bock.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

41586_2003_BFnature01962_MOESM1_ESM.jpg

Supplementary Figure: The sequence of the crystallized SC (S. aureus Newman 2D, strain Tager 104) is compared with two other SCs and several homologs. Strictly conserved residues and conservative substitutions throughout are highlighted in red and pink, respectively; and additional conserved residues are highlighted in yellow. SC residues that contact the 148-loop or exosite I are indicated with closed or open triangles, respectively. The conserved RGD motif in SfbX is shadowed blue. (JPG 281 kb)

41586_2003_BFnature01962_MOESM2_ESM.jpg

Supplementary Figure: Stereo ribbon plot showing the symmetric dimer present in the asymmetric unit, with thrombin and SC moieties colored yellow and orange, and red and salmon, respectively. The terminal SC residues as well as single helices are labeled. (JPG 150 kb)

41586_2003_BFnature01962_MOESM3_ESM.ppt

Supplementary Figure: A superimposition of the µ-plasmin·SK (yellow) and Pre-2·SC(325) (red) complexes. Note the different arrangement around the respective proteinase. (PPT 549 kb)

41586_2003_BFnature01962_MOESM4_ESM.mov

Supplementary Movie: Zoom from standard orientation into the activation pocket of Pre-2. SC is coloured yellow, the surface of Pre-2 according to the surface potential (blue: positive, red: negative). (MOV 982 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Friedrich, R., Panizzi, P., Fuentes-Prior, P. et al. Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation. Nature 425, 535–539 (2003). https://doi.org/10.1038/nature01962

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature01962

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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