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* Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104;
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri, Kansas City, MO 64110;
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720;
Department of Medicine, University of Colorado Denver School of Medicine, Denver, CO 80045; and
¶ Schools of Biological Sciences and Chemistry, University of Edinburgh, Edinburgh, United Kingdom
Staphylococcus aureus possesses an impressive arsenal of complement evasion proteins that help the bacterium escape attack of the immune system. The staphylococcal complement inhibitor (SCIN) protein exhibits a particularly high potency and was previously shown to block complement by acting at the level of the C3 convertases. However, many details about the exact binding and inhibitory mechanism remained unclear. In this study, we demonstrate that SCIN directly binds with nanomolar affinity to a functionally important area of C3b that lies near the C terminus of its β-chain. Direct competition of SCIN with factor B for C3b slightly decreased the formation of surface-bound convertase. However, the main inhibitory effect can be attributed to an entrapment of the assembled convertase in an inactive state. Whereas native C3 is still able to bind to the blocked convertase, no generation and deposition of C3b could be detected in the presence of SCIN. Furthermore, SCIN strongly competes with the binding of factor H to C3b and influences its regulatory activities: the SCIN-stabilized convertase was essentially insensitive to decay acceleration by factor H and the factor I- and H-mediated conversion of surface-bound C3b to iC3b was significantly reduced. By targeting a key area on C3b, SCIN is able to block several essential functions within the alternative pathway, which explains the high potency of the inhibitor. Our findings provide an important insight into complement evasion strategies by S. aureus and may act as a base for further functional studies.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grants AI071028, AI068730, AI30040, AI072106, and CA53615 from the National Institutes of Health, and in part by the Office of Science, Office of Biological and Environmental Research, U.S. Department of Energy, under Contract DE-AC02-05CH11231 for SIBLYS beamline efforts.
2 B.V.G. and J.D.L. shared supervision of this work.
3 Address correspondence and reprint requests to Dr. John D. Lambris, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 401 Stellar Chance, 422 Curie Boulevard, Philadelphia, PA 19104. E-mail address: Lambris{at}upenn.edu
4 Abbreviations used in this paper: CR, complement receptor; SCIN, staphylococcal complement inhibitor; SPR, surface plasmon resonance; ORF, open reading frame; Efb, extracellular fibrinogen-binding protein; Ehp, Efb homologous protein; fB, factor B; fD, factor D; fH, factor H; fI, factor I; ITC, isothermal titration calorimetry; MG, macroglobulin domain; SAXS, small angle X-ray scattering; Sbi, staphylococcal Ig-binding protein; TED, thioester-containing domain.
5 The online version of this article contains supplemental material.
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