Factor B structure provides insights into activation of the central protease of the complement system

Abstract

Factor B is the central protease of the complement system of immune defense. Here, we present the crystal structure of human factor B at 2.3-Å resolution, which reveals how the five-domain proenzyme is kept securely inactive. The canonical activation helix of the Von Willebrand factor A (VWA) domain is displaced by a helix from the preceding domain linker. The two helices conformationally link the scissile-activation peptide and the metal ion-dependent adhesion site required for binding of the ligand C3b. The data suggest that C3b binding displaces the three N-terminal control domains and reshuffles the two central helices.Reshuffling of the helices releases the scissile bond for final proteolytic activation and generates a new interface between the VWA domain and the serine protease domain. This allosteric mechanism is crucial for tight regulation of the complementamplification step in the immune response.Factor B is a tightly regulated, highly specific serine protease. In its activated form, it catalyzes the central amplification step of complement activation to initiate inflammatory responses, cell lysis, phagocytosis and B-cell stimulation 1,2 . Factor B is activated through an assembly process: it binds surface-bound C3b, or its fluid-phase counterpart C3(H 2 O), after which it is cleaved by factor D into fragments Ba (residues 1-234) and Bb (residues 235-739) 3,4 . Fragment Ba dissociates from the complex, leaving behind the alternative pathway C3 convertase complex C3b-Bb, which cleaves C3 into C3a and C3b (see Fig. 1a). This protease complex is intrinsically instable. Once dissociated from the complex, Bb cannot reassociate with C3b 5 . A similar C3 convertase complex is formed upon activation of the classical (antibody-mediated) and lectin-binding pathways, comprised of C4 and C2, which are homologous to C3 and factor B, respectively. The proenzyme factor B consists of three N-terminal complement control protein (CCP) domains, connected by a 45-residue linker to a VWA domain and a C-terminal serine protease (SP) domain, which carries the catalytic center (Fig. 1a). The VWA and SP domains form fragment Bb, and CCP1 through CCP3 and the linker form fragment Ba. Binding of factor B to C3b depends on elements in fragment Ba 6 and the Mg 2+ -dependent metal ion-dependent adhesion site (MIDAS) motif in the VWA domain of fragment Bb 7 . The VWA domain is structurally homologous to inserted (I) domains in integrins. In I domains, ligand binding to the MIDAS is coupled to a B10-Å shift of the a7 activation helix, with concomitant domain rearrangements that activate the integrins 8,9 . Structures of a truncated Bb fragment 10 and its full-length homolog C2a 11 show variable positions of the a7 activation helix affecting the orientation of the VWA and SP domains, which indicates that a related mechanism may occur in convertase formation and dissociation. These structures, however, do not reveal the regulation of the proteolytic activity of factor B. In particular, it is unclear how factor B is maintained in its in...

3D structure of the C3bB complex provides insights into the activation and regulation of the complement alternative pathway convertase

“…2B). Such differences indicate that fB undergoes substantial conformational changes when binding to C3b, as previously suggested (18).…”

“…These structural data also indicate that SCR1 probably hinders access of the ligand C3b to the MIDAS of the vWA domain, and that the triad of SCR domains is probably only weakly associated with the vWA and SP domains. Most interestingly, comparison of the fB proenzyme (18) and the Bb fragment (19) structures suggests that fB undergoes conformational changes upon binding to C3b, displacing the helix ␣L from its binding groove in the vWA domain and exposing the long linker domain between the SCR3 and the vWA domains of fB that contains the scissile bond cleaved by fD (7,18,19).…”

Deciphering complement mechanisms: The contributions of structural biology

“…Each of these proteases also carries three CCP modules at its N-terminal end. The recent crystal structure of full-length factor B revealed the position of the CCP modules to be on one side of the molecule, covering the interface between the serine protease and vWFA domains (Milder et al, 2007). For a long time, it was assumed that cofactors C3b and C4b, by associating through the MIDAS motifs present in the vWFA domains of factor B and C2, respectively, were responsible for activation of these enzymes.…”

“…However, the crystal structures of factor B (Milder et al, 2007;Ponnuraj et al, 2004), C2 (Krishnan et al, 2007;Milder et al, 2006) and their individual domains (Bhattacharya et al, 2004;Xu et al, 2000), in combination with site-directed mutagenesis and enzyme kinetic studies, revealed some interesting structural details that correlate with both the narrow substrate specificity and the unique activating mechanisms of these enzymes. These findings revealed that the four essential structural features (Fig.…”

“…We speculate that the removal of the N-terminal CCP domain from its position in the proenzyme might facilitate the approach of, and free-up space for, substrate C3 binding to the respective enzymes. In other words, once the N-terminal CCP has been removed, the relative orientation between the serine protease and vWFA domains (different between factor B and Bb (Milder et al, 2007)) might be stabilized by the bound cofactors, which can also act as 'platforms' for positioning the C3 substrate in a suitable orientation for activation of the 'disordered' active sites in factor B and C2. However, we do not know much about the possible conformational changes C3, C3b, and C4b might undergo during the binding, cleavage, and generation of C3b.…”