Factor H (FH) is an abundant regulator of complement activation and protects host cells from self-attack by complement. Here we provide insights into the regulatory activity of FH by solving the crystal structure of the first four domains of FH in complex with its target C3b. FH interacts with multiple domains of C3b, covering a large, extended surface area. The structure indicated that FH destabilizes the C3 convertase by competition and electrostatic repulsion and that FH enables proteolytic degradation of C3b by providing a binding platform for the protease factor I, while stabilizing the overall domain arrangement of C3b. The results offer general models for complement regulation and provide structural explanations for disease-related mutations in both FH and C3b.

Staphylococcus aureus possesses an impressive arsenal of complement evasion proteins that help the bacterium escape attack of the immune system. The staphylococcal complement inhibitor (SCIN) protein exhibits a particularly high potency and was previously shown to block complement by acting at the level of the C3 convertases. However, many details about the exact binding and inhibitory mechanism remained unclear. In this study, we demonstrate that SCIN directly binds with nanomolar affinity to a functionally important area of C3b that lies near the C terminus of its β-chain. Direct competition of SCIN with factor B for C3b slightly decreased the formation of surface-bound convertase. However, the main inhibitory effect can be attributed to an entrapment of the assembled convertase in an inactive state. Whereas native C3 is still able to bind to the blocked convertase, no generation and deposition of C3b could be detected in the presence of SCIN. Furthermore, SCIN strongly competes with the binding of factor H to C3b and influences its regulatory activities: the SCIN-stabilized convertase was essentially insensitive to decay acceleration by factor H and the factor I- and H-mediated conversion of surface-bound C3b to iC3b was significantly reduced. By targeting a key area on C3b, SCIN is able to block several essential functions within the alternative pathway, which explains the high potency of the inhibitor. Our findings provide an important insight into complement evasion strategies by S. aureus and may act as a base for further functional studies.

Complement activation plays a critical role in controlling inflammatory responses. To assess the role of complement during ovarian cancer progression, we crossed two strains of mice with genetic complement deficiencies with transgenic mice that develop epithelial ovarian cancer (TgMISIIR-TAg). TgMISIIR-TAg mice fully or partially deficient for complement factor 3 (C3) (Tg(+)C3(KO) and Tg(+)C3(HET), respectively) or fully deficient for complement factor C5a receptor (C5aR) (Tg(+)C5aR(KO)) develop either no ovarian tumors or tumors that were small and poorly vascularized compared to wild-type littermates (Tg(+)C3(WT), Tg(+)C5aR(WT)). The percentage of tumor infiltrating immune cells in Tg(+)C3(HET) tumors compared to Tg(+)C3(WT) controls was either similar (macrophages, B cells, myeloid-derived suppressor cells), elevated (effector T cells), or decreased (regulatory T cells). Regardless of these ratios, cytokine production by immune cells taken from Tg(+)C3(HET) tumors was reduced on stimulation compared to Tg(+)C3(WT) controls. Interestingly, CD31(+) endothelial cell (EC) function in angiogenesis was significantly impaired in both C3(KO) and C5aR(KO) mice. Further, using the C5aR antagonist PMX53, tube formation of ECs was shown to be C5a-dependent, possibly through interactions with the VEGF(165) but not VEGF(121) isoform. Finally, the mouse VEGF(164) transcript was underexpressed in C3(KO) livers compare to C3(WT) livers. Thus, we conclude that complement inhibition blocks tumor outgrowth by altering EC function and VEGF(165) expression.

The complement system is a major target of immune evasion by Staphylococcus aureus. Although many evasion proteins have been described, little is known about their molecular mechanisms of action. Here we demonstrate that the extracellular fibrinogenbinding protein (Efb) from S. aureus acts as an allosteric inhibitor by inducing conformational changes in complement fragment C3b that propagate across several domains and influence functional regions far distant from the Efb binding site. Most notably, the inhibitor impaired the interaction of C3b with complement factor B and, consequently, formation of the active C3 convertase. As this enzyme complex is critical for both activation and amplification of the complement response, its allosteric inhibition likely represents a fundamental contribution to the overall immune evasion strategy of S. aureus.allosteric modulation | complement amplification | hydrogen-deuterium exchange mass spectrometry | small angle X-ray scattering | surface plasmon resonance

Compstatin is a 13-residue disulfide-bridged peptide that inhibits a key step in the activation of the human complement system. Compstatin and its derivatives have shown great promise for the treatment of many clinical disorders associated with unbalanced complement activity. To obtain more potent compstatin analogues, we have now performed an N-methylation scan of the peptide backbone and amino acid substitutions at position 13. One analogue (Ac-I[CVW(Me)QDW-Sar-AHRC](NMe)I-NH2) displayed a 1,000-fold increase in both potency (IC50=62 nM) and binding affinity for C3b (KD=2.3 nM) over that of the original compstatin. Biophysical analysis using surface plasmon resonance and isothermal titration calorimetry suggests that the improved binding originates from more favorable free conformation and stronger hydrophobic interactions. This study provides a series of significantly improved drug leads for therapeutic applications in complement-related diseases, and offers new insights into the structure-activity relationships of compstatin analogues.