Structural Analysis of the C-Terminal Region (Modules 18–20) of Complement Regulator Factor H (FH)

Abstract

Factor H (FH) is a soluble regulator of the human complement system affording protection to host tissues. It selectively inhibits amplification of C3b, the activation-specific fragment of the abundant complement component C3, in fluid phase and on self-surfaces and accelerates the decay of the alternative pathway C3 convertase, C3bBb. We have determined the crystal structure of the three carboxyl-terminal complement control protein (CCP) modules of FH (FH18–20) that bind to C3b, and which additionally recognize polyanionic markers specific to self-surfaces. These CCPs harbour nearly 30 disease-linked missense mutations. We have also deployed small-angle X-ray scattering (SAXS) to investigate FH18–20 flexibility in solution using FH18–20 and FH19–20 constructs. In the crystal lattice FH18–20 adopts a “J”-shape: A ∼122-degree tilt between the structurally highly similar modules 18 and 19 precedes an extended, linear arrangement of modules 19 and 20 as observed in previously determined structures of these two modules alone. However, under solution conditions FH18–20 adopts multiple conformations mediated by flexibility between CCPs 18 and 19. We also pinpoint the locations of disease-associated missense mutations on the module 18 surface and discuss our data in the context of the C3b:FH interaction.

Incomplete inhibition by eculizumab: mechanistic evidence for residual C5 activity during strong complement activation

“…The minimized engineered version of the natural AP regulator FH, mini-FH, and the C-terminal FH fragment, FH18-20, which harbors the FH host surface recognition patch, were produced as described previously. 38,39 Hemolysis and bacterial lysis assays CP activity was measured with a standard heterologous hemolysis assay using antibody-sensitized sheep erythrocytes (shRBCs). 40 Alternatively, a very sensitive homologous assay with antibody-sensitized PNH-RBCs was adapted from the procedure of Rosse.…”

Complement genetics and susceptibility to inflammatory disease. Lessons from genotype–phenotype correlations

“…It is now well established that this FH region binds simultaneously the TED domain of C3b (or C3d) and sialylated glycans on the host cell surfaces and that this binding site involves separate domains for these molecules in SCR 19 (C3d) and SCR 20 (sialic acid) (Blaum et al, 2015;Morgan et al, 2012;Kajander et al, 2011). According to these data several mutations in FH SCR 20 associated with aHUS cause functional impairment due to faulty sialic acid recognition (Blaum et al, 2015).…”

“…Importantly, while FH binds and inactivates promptly C3b in fluid phase, the inactivation of surface-bound C3b by FH is dependent on the chemical composition of the surface to which C3b is bound. In the presence of polyanions, like sialylated glycans, glycosaminoglycans or sulphated polysaccharides (heparins), and other FH ligands, the affinity of FH for surface-bound C3b increases as a consequence of the simultaneous recognition of these FH ligands and bound C3b by the same FH molecule (Morgan et al, 2011;Blaum et al, 2015;Morgan et al, 2012;Kajander et al, 2011). The regulatory activities of the FH molecule depend on three major functional domains, an N-terminal domain (SCRs 1-4) that sustains the cofactor and decay accelerating activities and two domains in SCRs 6-7 and SCRs 19-20 which are relevant for ligand and cell surface recognition.…”

The Complement Inhibitor Factor H Generates an Anti-Inflammatory and Tolerogenic State in Monocyte-Derived Dendritic Cells

“…Thus, the region encompassing CCP1-4 of the protein (FH1-4) prevents the formation and promotes dissociation of the C3 convertase and, together with factor I, mediates proteolytic inactivation of C3b, an important opsonin and a major component of the convertase complexes that drive the complement activation cascade (4)(5)(6). Moreover, multiple mutagenesis, binding and functional reports, along with the last structural studies (7)(8)(9)(10) show that the C-terminal recognition region formed by CCP19-20 of FH (FH19-20) binds to surfacebound C3b and can efficiently discriminate polyanionic host surfaces versus foreign surfaces and protect the former from complement attack. In fact, mutations in the C terminus of FH and binding of deficiency of FH-related plasma proteins and autoantibody-positive form of hemolytic uremic syndromeassociated autoantibodies defined the C-terminal recognition region as a hot spot associated with atypical hemolytic-uremic syndrome (aHUS), leading to disturbances in the physiologic interaction of FH with host endothelial cells (11).…”