Complement factor H (FH) attenuates C3b molecules tethered via their thioester domains to self-surfaces and thereby protects host tissues. FH is a cofactor for initial C3b proteolysis that ultimately yields a surface-attached fragment (C3d), corresponding to the thioester domain. We used NMR and X-ray crystallography to study the C3d:FH19–20 complex in atomic detail. NMR further identified glycosaminoglycan-binding residues in FH module 20 of the C3d:FH19–20 complex. Mutagenesis justified the merging of the C3d:FH19–20 structure with an existing C3b:FH1–4 crystal structure. The merged structure was concatenated with the available FH6–8 crystal structure and new SAXS-derived FH1–4, FH8–15 and FH15–19 envelopes. The combined data suggests a bent-back FH molecule, binding via its termini to two sites on one C3b molecule and simultaneously to adjacent polyanionic host-surface markers.

The first eight and the last two of 20 complement control protein (CCP) modules within complement factor H (fH) encompass binding sites for C3b and polyanionic carbohydrates. These binding sites cooperate self-surface selectively to prevent C3b amplification, thus minimising complement-mediated damage to host. Intervening fH CCPs, apparently devoid of such recognition sites, are proposed to play a structural role. One suggestion is that the generally small CCPs 10–15, connected by longer-than-average linkers, act as a flexible tether between the two functional ends of fH; another is that the long linkers induce a 180° bend in the middle of fH. To test these hypotheses, we determined the NMR-derived structure of fH12–13 consisting of module 12, shown here to have an archetypal CCP structure, and module 13, which is uniquely short and features a laterally protruding helix-like insertion that contributes to a prominent electropositive patch. The unusually long fH12–13 linker is not flexible. It packs between the two CCPs that are not folded back on each other but form a shallow vee shape; analytical ultracentrifugation and X-ray scattering supported this finding. These two techniques additionally indicate that flanking modules (within fH11–14 and fH10–15) are at least as rigid and tilted relative to neighbours as are CCPs 12 and 13 with respect to one another. Tilts between successive modules are not unidirectional; their principal axes trace a zigzag path. In one of two arrangements for CCPs 10–15 that fit well with scattering data, CCP 14 is folded back onto CCP 13. In conclusion, fH10–15 forms neither a flexible tether nor a smooth bend. Rather, it is compact and has embedded within it a CCP module (CCP 13) that appears to be highly specialised given both its deviant structure and its striking surface charge distribution. A passive, purely structural role for this central portion of fH is unlikely.

The Plasmodium falciparum adhesin PfRh4 binds to complement receptor type-1 (CR1) on human erythrocytes and mediates a glycophorin-independent invasion pathway. CR1 is a complement regulator and immune-adherence receptor on erythrocytes required for shuttling of C3b/ C4b-opsonized particles to liver and spleen for phagocytosis. Using recombinant CR1 constructs, we mapped the recognition site for PfRh4 to complement control protein modules 1 to 3 (CCP1-3) at the membrane-distal amino terminus of CR1. This region of CR1 binds to C4b and C3b and accelerates decay of both classic pathway and alternative pathway C3 and C5 convertases. CCP1-3 competed for PfRh4 binding to erythroid CR1 and inhibited the PfRh4-CR1 invasion pathways across a wide range of P falciparum strains. PfRh4 did not bind significantly to other CR1 constructs, including CCP15-17, which is 85% identical to CCP1-3. PfRh4 binding to CR1 did not affect its C3b/C4b binding capability, and we show evidence for a ternary complex between CCP1-3, C4b, and PfRh4. PfRh4 binding specifically inhibited CR1's convertase decay-accelerating activity, whereas there was no effect on factor H-mediated decay-accelerating activity. These results increase our understanding of the functional implications of CR1 engagement with PfRh4 and highlight the interplay between complement regulation and infection. (Blood. 2011; 118(7): [1923][1924][1925][1926][1927][1928][1929][1930][1931][1932][1933] IntroductionThe complement system is a first line of defense against invasion by infectious agents. On pathogen entry into the host and detection of a pathogen-associated or danger-associated molecular pattern, the complement cascade is activated in seconds and results in the production of anaphylatoxins, deposition of opsonic C3 and C4 fragments, and assembly of the potentially cytolytic membrane attack complex. By ensuring that the complement system acts in a directed manner, the regulators of complement activation (RCA) protein family protect self-tissue from complement-mediated attack. 1 Interestingly, RCA family members also contribute to cell attachment or invasion strategies of disparate pathogens, including multiple viruses and bacteria. [2][3][4][5][6] Recently, complement receptor type-1 (CR1), an erythroid membrane-bound RCA protein, was shown to be a receptor used by the malaria parasite Plasmodium falciparum for invasion of human erythrocytes. 7,8 Invasion of human erythrocytes by malaria parasites depends on specific interactions between parasite adhesins and host receptors. In P falciparum, 2 gene families encode important parasite proteins that engage with erythrocyte receptors: the erythrocyte binding-like antigens (PfEBAs; these include EBA-140/BAEBL, EBA-175, EBA-181/JESEBL, and EBL-1); and reticulocyte binding-like homolog proteins (RBPs or PfRhs; these include PfRh1, PfRh2a, PfRh2b, PfRh4 and PfRh5). 9-12 During invasion these adhesins localize to the apical tip of the merozoite and interact with specific host receptors to initiate parasite entry. Invasion pat...

Wound healing is a complex homeostatic response to injury that engages numerous cellular activities, processes, and cell-to-cell interactions. The complement system, an intricate network of proteins with important roles in immune surveillance and homeostasis, has been implicated in many physiological processes; however, its role in wound healing remains largely unexplored. Here we employ a murine model of excisional cutaneous wound healing and show that C3−/− mice exhibit accelerated early stages of wound healing. Reconstitution of C3−/− mice with serum from C3+/+ mice or purified human C3 abrogated the accelerated wound healing phenotype. Wound histology of C3−/− mice revealed a reduction in inflammatory infiltrate compared to C3+/+ mice. C3 deficiency also resulted in increased accumulation of mast cells and advanced angiogenesis. We further show that mice deficient in the downstream complement effector C5 exhibit a similar wound healing phenotype, which is recapitulated in C5aR1−/− mice, but not C3aR−/− or C5aR2−/− mice. Taken together, these data suggest that C5a signaling through C5aR may in part play a pivotal role in recruitment and activation of inflammatory cells to the wound environment, which in turn could delay the early stages of cutaneous wound healing. These findings also suggest a previously underappreciated role for complement in wound healing, and may have therapeutic implications for conditions of delayed wound healing.

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.