Membrane pore formation by human complement: Functional importance of the transmembrane β-hairpin (TMH) segments of C8α and C9

Human C8 and C9 have a key role in forming the pore-like “membrane attack complex” (MAC) of complement on bacterial cells. A possible mechanism for membrane insertion of these proteins was suggested when studies revealed a structural similarity between the MACPF domains of the C8α and C8β subunits and the pore-forming bacterial cholesterol-dependent cytolysins (CDCs). This similarity includes a pair of α-helical bundles that in the CDCs refold during pore formation to produce two transmembrane β-hairpins (TMH1 and TMH2). C9 is the major pore-forming component of the MAC and is also likely to contain two TMH segments because of its homology to C8α and C8β. To determine their potential for membrane insertion, the TMH sequences in C8α and those predicted to be in C9 were substituted for the TMH sequences in perfringolysin O (PFO), a well-characterized CDC. Only chimeric proteins containing TMH2 from C8α (PFO/αT2) or C9 (PFO/C9T2) could be expressed in soluble, active form. The PFO/αT2 and PFO/C9T2 chimeras retained significant hemolytic activity, formed pore-like structures on membranes, and could combine with PFO to form hemolytically active mixed complexes that were functionally similar to PFO alone. These results provide experimental evidence in support of the hypothesis that TMH segments in C8α and those predicted to be in C9 have a direct role in MAC membrane penetration and pore formation.