Abstract

Many pathogens target the complement system as part of their strategy of escaping the immune system. In this context, the extracellular fibrinogen-binding protein (Efb) and the Staphylococcal complement inhibitor (SCIN) from Staphylococcus aureus have been recognized as exceptionally potent complement inhibitors. While both proteins interfere at the level of C3 convertases, their molecular mechanisms and binding specificities are clearly distinct. In view of the increasingly problematic infections with S. aureus and the potential of microbial evasion proteins as templates for therapeutic complement inhibitors, more details about their mode of action are urgently needed. Here we present extensive molecular studies on the structure, interaction properties and binding specificities of Efb, and show that its target spectrum is far greater than initially expected. In particular, we found that its C-terminal domain (Efb-C) efficiently prevents the interaction of C3d with complement receptor 2 and therefore may have direct consequences for the bridging to adaptive immune responses. Using hydrogen/deuterium exchange mass spectrometry, we confirmed the induction of conformational changes in C3 upon binding of Efb-C and demonstrate that such changes may significantly alter the interaction pattern of C3b with a number of ligands and regulators. Furthermore, we extended our characterization of the binding interface between Efb-C and C3d based on structural, biophysical, and computational methods, which identified additional key residues and showed that this important interaction is largely driven by electrostatic forces. Finally, we show that SCIN not only interacts with the assembled C3 convertase but also directly binds to C3b at an area distant from the Efb-C binding site. Biophysical experiments confirmed that SCIN stabilizes the C3 convertase and indicate that the inhibitor prevents cleavage but not the initial binding of native C3 to the convertase.

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