Mobility in TMH1 is essential for C9 self-association
To investigate the hypothesis that TMH1 blocks self-assembly, we designed a disulphide trap mutant (F262C/V405C, [C9mutant]) that linked TMH1 to β-strand 4 of the MACPF/CDC domain (Fig. 2a). Time resolved haemolytic assays revealed that the disulphide-trapped C9 variant is completely inactive with respect to lytic function and that addition of reducing agent resulted in restoration of lytic function (Fig. 2b). Crucially competition assays further reveal that the C9mutant competes with wild type C9 and thus is competent to bind the C5b8 or C5b89n complex to form C5b89mutant or C5b89n+mutant respectively (Fig. 2c). Together these data suggest that the sequential addition of C9 molecules to C5b891 relies on a rearrangement in TMH1.
Fig. 2 The C9 TMH1 movement is necessary for pore assembly. a Cartoon model of a C9 monomer (left) disulphide locked mutant (also called C9mutant with F262C/V405C mutations) (shown as yellow sticks), that links the TMH1 region to β-strand 4 of the MACPF domain (right). b Haemolytic activity of disulphide locked C9 against erythrocytes/antibody/complement 1–8 (EAC1-8). The TMH1 locked (no DTT) alone is inactive; however, activity can be rescued with 1 mM DTT (TMH1 locked (with DTT)). Also shown are control experiments: no C9, and wildtype C9 (with and without DTT). c Competition assay of disulphide locked mutant with wildtype C9 showing that the disulphide trapped variant competes for the elongation face with wild-type C9. A range of ratios of wildtype C9 and C9 TMH1 locked mutant used in the assays are as shown and it reveals that the disulphide locked C9 competes for the nascent MAC and stalls assembly in a dose-dependent manner. Also shown are no C9, C9 in buffer and C9 plus BSA controls. The results (b and c) are presented as the averaged turbidity measurements from three independently prepared samples (n = 3) with error reported as the standard error of the mean (SEM). See also Supplementary Fig. 7 for more detail