Structural studies on the enzymes of the complement convertases: factor B, C2a and the inhibitor SCIN

Abstract

The complement system is important for host immune defense. It recognizes and kills pathogens, presents pathogens to the adaptive immune system, and elicits inflammatory responses. Central in the complement activation process is the formation of the C3 convertase, a bimolecular enzyme complex on the cell surface of invading pathogens or altered host cells. To gain more insight into the mechanisms underlying formation and regulation of these key enzymes the studies described in this thesis focus on the proteins C2 and factor B that provide the catalytic center to the C3 convertases, and on the bacterial convertase-inhibitory protein SCIN. To prevent unwanted complement activation factor B and C2 circulate in the blood stream as pro-enzymes. The crystal structure of factor B was determined and shows how its proteolytic activity is regulated; and, how cofactor binding makes the protein susceptible to proteolytic activation. An alpha helix, formed by the linker preceding the Von Willebrand factor A (VWA) domain, plays a central role by displacing the canonical activation helix. The two helices together conformationally link the metal-ion dependent adhesion site (MIDAS), essential for factor B to bind its cofactor, and the scissile-activation peptide. Our findings suggest that cofactor binding displaces the N-terminal complement-control-protein domains, swaps the central helices and releases the scissile bond for proteolytic activation. Dissociation of activated factor B and C2 (Bb and C2a respectively) from their cofactor is irreversible and serves as an inherent stop signal in regulation of complex formation. In the crystal structure of C2a the catalytic center displays a near-active conformation surrounded by extended substrate binding loops. These loops, together with a putative substrate-induced maturation of the oxyanion hole, may contribute to the high substrate specificity of the convertases. The VWA domain displays an intermediate state of the activation helix and an open, activated MIDAS. Likely this MIDAS conformation enhances the affinity of C2a towards its ligand similar to observed in integrin "inside-out" signalling. The N-terminal residues are, unexpectedly, structurally incorporated in the VWA domain and prevent the activation helix to adopt the active conformation that putatively is required for the catalytic component to rebind its cofactor. Staphylococcus aureus is a pathogenic bacterium that blocks the complement activation pathways by excretion of the protein Staphylococcal Complement Inhibitor (SCIN). The crystal structure of this C3 convertase inhibiting protein was determined and chimeras consisting of SCIN and the structurally but non-functional homolog ORF-D were functionally characterized. The results indicate an 18 residue segment that is crucial for SCIN activity. Chimeras lacking these SCIN residues fail to inhibit production of C5a and display reduced Alternative Pathway mediated opsonization and membrane attack complex (MAC) formation. Although additional sites are involved Classical/Lectin Pathway mediated opsonization and MAC formation is reduced as well. These chimeras also display reduced capacity to stabilize the C3-convertases indicating this stabilizing effect is pivotal for SCIN activity. These data provide a starting point for the development of a second generation molecule suitable for therapeutic complement intervention.