Conformational activation of C5 in presence of C5 inhibitors. (A) Structure of the complex of C5 and Coversin. Crystal structure of C5:Coversin (PDB entry: 5HCC Jore et al³¹ ). Coversin addresses 3 domains in C5: CUB, TED, and the C345C domain (with the latter 2 being indicated). (B) Binding of eculizumab to C5. Crystal structure of eculizumab-Fab (pink) in complex with C5 (red) (PDB entry: 5I5K Schatz-Jakobsen et al⁴¹ ). Eculizumab binds solely to the MG-7 domain (brown) of C5. (C) Binding curves of C5 and activated C5 complexes to 440 RU of immobilized Coversin (via amine coupling onto a CMD sensor chip). C5 (red) and the activated C5 structures, C5b,6 (orange) and SC5b-9 (green), were applied to the chip at a concentration of 25 nM. (One assay was performed.) (D) Binding curves of C5 and activated C5 complexes to eculizumab immobilized via amine coupling to a level of 13,200 RUs onto a CMD sensor chip. All analytes were injected at a concentration of 25 nM. Duplicates of each sample were performed to show reproducibility. (One assay was performed.) (E) Nonconvertase-mediated hemolysis under C5 inhibition. C3b-opsonized rRBCs were exposed to a C6-C9 mixture (physiological concentrations), followed by the addition of C5 (physiological concentration) preincubated with eculizumab (5 µM, pink), Coversin (5 µM, blue), both C5 inhibitors together (green) or without inhibitor (red). Cells in water served as reference and were set to 100% lysis, incubation of cells only in PBS is shown in orange. Release of hemoglobin was measured as a marker of hemolysis. Average of 3 independent assays with SD is shown. An ordinary 1-way ANOVA using Tukey multiple comparisons test was applied. ns, not significant.