A Structurally Dynamic N-terminal Helix Is a Key Functional Determinant in Staphylococcal Complement Inhibitor (SCIN) Proteins

Abstract

Background: SCIN proteins from S. aureus mediate immune evasion by binding to C3b. Results: A structurally dynamic region exists within N termini of SCIN proteins and affects their C3b binding and inhibitory properties. Conclusion:The N terminus is a critical functional determinant in multiple SCINs. Significance: This work provides a new model for understanding SCIN structure and function.

The Extracellular Adherence Protein from Staphylococcus aureus Inhibits the Classical and Lectin Pathways of Complement by Blocking Formation of the C3 Proconvertase

“…We next utilized a bead-based AlphaScreen assay to explore both the affinity and specificity of the C4b/Eap interaction in greater detail (20, 28). Whereas untagged Eap itself could diminish the luminescence signal generated by interaction between myc-tagged Eap and C4b-biotin in a dose-dependent manner, neither EapH1 nor EapH2 had any competitive effect even at the highest concentrations tested (Fig.…”

“…2B). To study the C4b/Eap interaction through an independent approach, we constructed an SPR biosensor wherein C4b-biotin was uniformly immobilized on a streptavidin-coated surface similarly to what we have previously reported for C3b-biotin (10, 20, 21, 28). Significantly, neither EapH1 nor EapH2 bound the C4b surface even at concentrations 10-fold higher than those which showed clear evidence of C4b binding by Eap (Fig.…”

Identification of peptidic inhibitors of the alternative complement pathway based on Staphylococcus aureus SCIN proteins

“…Since it seemed unlikely that 12-mer peptides could recapitulate the long-range contacts necessary to stabilize and inactivate the AP C3 convertase through either higher-order oligomerization or steric hindrance of convertase access to its C3 substrate as is the case for SCIN-A (Rooijakkers et al, 2009), we hypothesized that the AP inhibitory peptides identified here may instead act by disrupting convertase formation. To test this prediction directly, we employed an SPR-based method whereby the rate of AP C3 convertase formation on site specifically-immobilized C3b was monitored in real time, either in the presence or absence of 1 mM peptide (Garcia et al, 2013; Ricklin et al, 2009). In this assay, six of the seven peptides significantly inhibited AP C3 convertase formation in vitro when compared to an unrelated control peptide (Fig.…”

“…Although SCIN function is multi-faceted, a hallmark of SCIN activity is the stabilization of an inactive form of the AP C3 convertase via the formation of a ternary C3bBb/SCIN complex (Jongerius et al, 2007; Rooijakkers et al, 2005). More recently, structure/function studies have shed light on the molecular basis for complement inhibition by the SCIN family (Garcia et al, 2010; Garcia et al, 2013; Garcia et al, 2012b; Jongerius et al, 2010; Rooijakkers et al, 2009; Rooijakkers et al, 2007). These studies revealed that SCINs also participate in large, multi-protein complexes (e.g.…”

“…Interestingly, naturally occurring sequence variation for residues found in this secondary C3b contact site on SCINs augmented both the C3b-binding properties and overall function of SCIN proteins. Furthermore, it was shown that these multi-protein complexes are capable of directly blocking phagocytosis by masking the complement receptors CRIg and CR1, both of which are found on the surface of phagocytic cells (Garcia et al, 2013; Jongerius et al, 2010). Thus, by targeting a vulnerable site on C3b, SCINs simultaneously block the key amplification step in the complement cascade and impede C3b-mediated phagocytosis.…”