Discussion
Previous data suggest that both TMH1 and TMH2 of C8 fully enter the membrane prior to recruitment of C921. This finding is consistent with the observation that incomplete arc-like structures can form and penetrate the membrane4,8. In such a structure (where C8 is fully inserted) the edge strand at the elongation face is the TMH2 of C8α (Supplementary Fig. 1). Our biochemical data reveal that a C9 variant in which TMH1 is disulphide trapped is able to join C5b8, however, further elongation is not possible without the release of TMH1. We hypothesise that upon binding to C5b8 the most likely next step is for the TMH1 of C9 to add to the nascent barrel structure by forming a canonical β-hairpin with the membrane inserted TMH2 of C8α (Fig. 4). Alternatively, it is possible that TMH1 moves sufficiently to permit C9 binding, but without inserting into the membrane. However, we have no evidence for such an intermediate pre-pore like state.
Fig. 4 Schematic diagram of the unidirectional C9 assembly. We hypothesise that during the assembly, binding to the elongation face of a C9 subunit leads to the release of TMH1 which inserts to form a canonical β-hairpin. Following this, the release of TMH2 and a conformational change in the HTH region uncovers the elongation face of the newly assembled C9, allowing the next C9 subunit to join the assembly
We further suggest that prior to the next C9 subunit joining the assembly, it is highly likely that the TMH2 of C9 is also released to enter the membrane, and that this permits the HTH region to slide across the underlying β-sheet. The removal of TMH1 together with the shift in the HTH region will expose the elongation face of C5b891 and permit recruitment of the next C9 subunit into the growing MAC. Taken together these data explain how the MAC has evolved a mechanism of coupling sequential insertion with elongation. The mechanism of C9 pore formation also directly contrasts that of related molecules such as perforin and the CDCs, where it is suggested that the assembly of pre-pores (or pre-pore-like arcs) takes place prior to the simultaneous release of the membrane spanning regions6–8,12,22.