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.

Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor

Abstract

Activation of the complement system generates potent chemoattractants and leads to the opsonization of cells for immune clearance. Short-lived protease complexes cleave complement component C3 into anaphylatoxin C3a and opsonin C3b. Here we report the crystal structure of the C3 convertase formed by C3b and the protease fragment Bb, which was stabilized by the bacterial immune-evasion protein SCIN. The data suggest that the proteolytic specificity and activity depend on the formation of dimers of C3 with C3b of the convertase. SCIN blocked the formation of a productive enzyme-substrate complex. Irreversible dissociation of the complex of C3b and Bb is crucial to complement regulation and was determined by slow binding kinetics of the Mg2+-adhesion site in Bb. Understanding the mechanistic basis of the central complement-activation step and microbial immune evasion strategies targeting this step will aid in the development of complement therapeutics.

Your institute does not have access to this article

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: SCIN induces the formation of dimeric convertases.
Figure 2: Crystal structure of the C3 convertase C3bBb inhibited by SCIN.
Figure 3: Inhibition of C3bBb by SCIN.
Figure 4: The C3bBb structure derived from the C3bBb-SCIN complex.
Figure 5: The C3b-C3b interface and substrate-binding model.

Accession codes

Accessions

Protein Data Bank

References

  1. Carroll, M.C. The complement system in regulation of adaptive immunity. Nat. Immunol. 5, 981–986 (2004).

    CAS  Article  Google Scholar 

  2. Mollnes, T.E., Song, W.C. & Lambris, J.D. Complement in inflammatory tissue damage and disease. Trends Immunol. 23, 61–64 (2002).

    CAS  Article  Google Scholar 

  3. Duncan, R.C., Wijeyewickrema, L.C. & Pike, R.N. The initiating proteases of the complement system: controlling the cleavage. Biochimie 90, 387–395 (2008).

    CAS  Article  Google Scholar 

  4. Law, S.K. & Dodds, A.W. The internal thioester and the covalent binding properties of the complement proteins C3 and C4. Protein Sci. 6, 263–274 (1997).

    CAS  Article  Google Scholar 

  5. Gros, P., Milder, F.J. & Janssen, B.J. Complement driven by conformational changes. Nat. Rev. Immunol. 8, 48–58 (2008).

    CAS  Article  Google Scholar 

  6. Kerr, M.A. The human complement system: assembly of the classical pathway C3 convertase. Biochem. J. 189, 173–181 (1980).

    CAS  Article  Google Scholar 

  7. Rawal, N. & Pangburn, M.K. C5 convertase of the alternative pathway of complement. Kinetic analysis of the free and surface-bound forms of the enzyme. J. Biol. Chem. 273, 16828–16835 (1998).

    CAS  Article  Google Scholar 

  8. Kinoshita, T. et al. C5 convertase of the alternative complement pathway: covalent linkage between two C3b molecules within the trimolecular complex enzyme. J. Immunol. 141, 3895–3901 (1988).

    CAS  PubMed  Google Scholar 

  9. Rawal, N. & Pangburn, M.K. Structure/function of C5 convertases of complement. Int. Immunopharmacol. 1, 415–422 (2001).

    CAS  Article  Google Scholar 

  10. Kirkitadze, M.D. & Barlow, P.N. Structure and flexibility of the multiple domain proteins that regulate complement activation. Immunol. Rev. 180, 146–161 (2001).

    CAS  Article  Google Scholar 

  11. Pangburn, M.K. & Muller-Eberhard, H.J. The C3 convertase of the alternative pathway of human complement. Enzymic properties of the bimolecular proteinase. Biochem. J. 235, 723–730 (1986).

    CAS  Article  Google Scholar 

  12. Lambris, J.D., Ricklin, D. & Geisbrecht, B.V. Complement evasion by human pathogens. Nat. Rev. Microbiol. 6, 132–142 (2008).

    CAS  Article  Google Scholar 

  13. Rooijakkers, S.H. et al. Immune evasion by a staphylococcal complement inhibitor that acts on C3 convertases. Nat. Immunol. 6, 920–927 (2005).

    CAS  Article  Google Scholar 

  14. Rooijakkers, S.H. et al. Staphylococcal complement inhibitor: structure and active sites. J. Immunol. 179, 2989–2998 (2007).

    CAS  Article  Google Scholar 

  15. Janssen, B.J., Christodoulidou, A., McCarthy, A., Lambris, J.D. & Gros, P. Structure of C3b reveals conformational changes that underlie complement activity. Nature 444, 213–216 (2006).

    CAS  Article  Google Scholar 

  16. Wiesmann, C. et al. Structure of C3b in complex with CRIg gives insights into regulation of complement activation. Nature 444, 217–220 (2006).

    CAS  Article  Google Scholar 

  17. Torreira, E., Tortajada, A., Montes, T., de Cordoba, S.R. & Llorca, O. 3D structure of the C3bB complex provides insights into the activation and regulation of the complement alternative pathway convertase. Proc. Natl. Acad. Sci. USA 106, 882–887 (2009).

    CAS  Article  Google Scholar 

  18. Muller-Eberhard, H.J. & Gotze, O. C3 proactivator convertase and its mode of action. J. Exp. Med. 135, 1003–1008 (1972).

    CAS  Article  Google Scholar 

  19. Fishelson, Z., Pangburn, M.K. & Muller-Eberhard, H.J. C3 convertase of the alternative complement pathway. Demonstration of an active, stable C3 C3b,Bb (Ni) complex. J. Biol. Chem. 258, 7411–7415 (1983).

    CAS  PubMed  Google Scholar 

  20. Horiuchi, T., Macon, K.J., Engler, J.A. & Volanakis, J.E. Site-directed mutagenesis of the region around Cys-241 of complement component C2. Evidence for a C4b binding site. J. Immunol. 147, 584–589 (1991).

    CAS  PubMed  Google Scholar 

  21. Hourcade, D.E., Mitchell, L.M. & Oglesby, T.J. Mutations of the type A domain of complement factor B that promote high-affinity C3b-binding. J. Immunol. 162, 2906–2911 (1999).

    CAS  PubMed  Google Scholar 

  22. Tuckwell, D.S., Xu, Y., Newham, P., Humphries, M.J. & Volanakis, J.E. Surface loops adjacent to the cation-binding site of the complement factor B von Willebrand factor type A module determine C3b binding specificity. Biochemistry 36, 6605–6613 (1997).

    CAS  Article  Google Scholar 

  23. Hourcade, D.E., Mitchell, L., Kuttner-Kondo, L.A., Atkinson, J.P. & Medof, M.E. Decay-accelerating factor (DAF), complement receptor 1 (CR1), and factor H dissociate the complement AP C3 convertase (C3bBb) via sites on the type A domain of Bb. J. Biol. Chem. 277, 1107–1112 (2002).

    CAS  Article  Google Scholar 

  24. Ponnuraj, K. et al. Structural analysis of engineered Bb fragment of complement factor B: insights into the activation mechanism of the alternative pathway C3-convertase. Mol. Cell 14, 17–28 (2004).

    CAS  Article  Google Scholar 

  25. Milder, F.J. et al. Factor B structure provides insights into activation of the central protease of the complement system. Nat. Struct. Mol. Biol. 14, 224–228 (2007).

    CAS  Article  Google Scholar 

  26. Milder, F.J. et al. Structure of complement component C2a: implications for convertase formation and substrate binding. Structure 14, 1587–1597 (2006).

    CAS  Article  Google Scholar 

  27. Luo, B.H., Carman, C.V. & Springer, T.A. Structural basis of integrin regulation and signaling. Annu. Rev. Immunol. 25, 619–647 (2007).

    CAS  Article  Google Scholar 

  28. Krishnan, V., Xu, Y., Macon, K., Volanakis, J.E. & Narayana, S.V. The crystal structure of C2a, the catalytic fragment of classical pathway C3 and C5 convertase of human complement. J. Mol. Biol. 367, 224–233 (2007).

    CAS  Article  Google Scholar 

  29. Kam, C.M. et al. Human complement proteins D, C2, and B. Active site mapping with peptide thioester substrates. J. Biol. Chem. 262, 3444–3451 (1987).

    CAS  PubMed  Google Scholar 

  30. Janssen, B.J., Halff, E.F., Lambris, J.D. & Gros, P. Structure of compstatin in complex with complement component C3c reveals a new mechanism of complement inhibition. J. Biol. Chem. 282, 29241–29247 (2007).

    CAS  Article  Google Scholar 

  31. Katschke, K.J. Jr. et al. Structural and functional analysis of a C3b-specific antibody that selectively inhibits the alternative pathway of complement. J. Biol. Chem. 284, 10473–10479 (2009).

    CAS  Article  Google Scholar 

  32. Janssen, B.J. et al. Structures of complement component C3 provide insights into the function and evolution of immunity. Nature 437, 505–511 (2005).

    CAS  Article  Google Scholar 

  33. Fredslund, F. et al. Structure of and influence of a tick complement inhibitor on human complement component 5. Nat. Immunol. 9, 753–760 (2008).

    CAS  Article  Google Scholar 

  34. Rawal, N. & Pangburn, M. Formation of high-affinity C5 convertases of the alternative pathway of complement. J. Immunol. 166, 2635–2642 (2001).

    CAS  Article  Google Scholar 

  35. Hourcade, D.E., Mitchell, L.M. & Medof, M.E. Decay acceleration of the complement alternative pathway C3 convertase. Immunopharmacology 42, 167–173 (1999).

    CAS  Article  Google Scholar 

  36. Bhattacharya, A.A., Lupher, M.L., Jr., Staunton, D.E. & Liddington, R.C. Crystal structure of the A domain from complement factor B reveals an integrin-like open conformation. Structure 12, 371–378 (2004).

    CAS  Article  Google Scholar 

  37. Harris, C.L., Abbott, R.J., Smith, R.A., Morgan, B.P. & Lea, S.M. Molecular dissection of interactions between components of the alternative pathway of complement and decay accelerating factor (CD55). J. Biol. Chem. 280, 2569–2578 (2005).

    CAS  Article  Google Scholar 

  38. Pryzdial, E.L. & Isenman, D.E. Alternative complement pathway activation fragment Ba binds to C3b. Evidence that formation of the factor B-C3b complex involves two discrete points of contact. J. Biol. Chem. 262, 1519–1525 (1987).

    CAS  PubMed  Google Scholar 

  39. Lambris, J.D., Dobson, N.J. & Ross, G.D. Release of endogenous C3b inactivator from lymphocytes in response to triggering membrane receptors for β1H globulin. J. Exp. Med. 152, 1625–1644 (1980).

    CAS  Article  Google Scholar 

  40. Reeves, P.J., Callewaert, N., Contreras, R. & Khorana, H.G. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl. Acad. Sci. USA 99, 13419–13424 (2002).

    CAS  Article  Google Scholar 

  41. de Haas, C.J. et al. Chemotaxis inhibitory protein of Staphylococcus aureus, a bacterial antiinflammatory agent. J. Exp. Med. 199, 687–695 (2004).

    CAS  Article  Google Scholar 

  42. Demeler, B. & van Holde, K.E. Sedimentation velocity analysis of highly heterogeneous systems. Anal. Biochem. 335, 279–288 (2004).

    CAS  Article  Google Scholar 

  43. Demeler, B. in Analytical Ultracentrifugation: Techniques and Methods (eds. Scott, D.J., Harding S.E. & Rowe, A.J.) 210–230 (Royal Society of Chemistry, Cambridge, 2005).

    Google Scholar 

  44. Brookes, E. & Demeler, B. Parallel computational techniques for the analysis of sedimentation velocity experiments in UltraScan. Colloid Polym. Sci. 286, 139–148 (2008).

    CAS  Article  Google Scholar 

  45. Evans, P. Scaling and assessment of data quality. Acta Crystallographica Section D 62, 72–82 (2006).

    Article  Google Scholar 

  46. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007).

    CAS  Article  Google Scholar 

  47. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  48. Adams, P.D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002).

    Article  Google Scholar 

  49. Myszka, D.G. & Morton, T.A. CLAMP: a biosensor kinetic data analysis program. Trends Biochem. Sci. 23, 149–150 (1998).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank R. Romijn for help with mammalian protein expression; M. Otten and M. Daha for doing hemolytic assays; P. Lenting for help with Biacore analyses; the European Synchrotron Radiation Facility for synchrotron radiation facilities; and beamline scientists of the European Synchrotron Radiation Facility and the European Molecular Biology Laboratory for assistance. Supported by the Councils for Medical Sciences and Chemical Sciences of the Netherlands Organization for Scientific Research (S.H.M.R., J.A.G.v.S. and P.G.) and the US National Institutes of Health (J.D.L. and P.G.).

Author information

Authors and Affiliations

Authors

Contributions

J.W. expressed and purified FB and FD; M.R. and S.H.M.R. expressed and purified SCIN and chimeras; M.R. purified C3b; R.v.D., M.R. and S.H.M.R. generated and analyzed complexes and did functional assays; K.L.P. did and analyzed the analytical ultracentrifugation experiments; J.W. crystallized the complex and determined and analyzed the structure; B.J.C.J. helped with structure determination and analysis; A.T. expressed and purified Ba; D.R. did the C3b-FB and C3b-Ba binding studies; S.H.M.R., B.J.C.J., J.W., J.D.L., J.A.G.v.S. and P.G. conceived the experiments; and J.W., S.H.M.R., D.R., J.A.G.v.S. and P.G. wrote the manuscript.

Corresponding author

Correspondence to Piet Gros.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13 and Supplementary Table 1 (PDF 1183 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Rooijakkers, S., Wu, J., Ruyken, M. et al. Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor. Nat Immunol 10, 721–727 (2009). https://doi.org/10.1038/ni.1756

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.1756

Further reading

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