Functional studies of the MACPF domain of human complement protein C8α reveal sites for simultaneous binding of C8β, C8γ, and C9

Human C8 is one of five components of the membrane attack complex of complement (MAC). It contains three subunits (C8 alpha, C8 beta, C8y) arranged as a disulfide-linked C8 alpha-y dimer that is noncovalently associated with C8 beta. C8 alpha, C8 beta, and complement components C6, C7, and C9 form the MAC family of proteins. All contain N- and C-terminal modules and an intervening 40-kDa segment referred to as the membrane attack complex/perform (MACPF) domain. During MAC formation, C8 alpha binds and mediates the self-polymerization of C9 to form a pore-like structure on target cells. The C9 binding site was previously shown to reside within a 52-kDa segment composed of the C8 alpha N-terminal modules and MACPF domain (alpha MACPF). In the present study, we examined the role of the MACPF domain in binding C9. Recombinant alpha MACPF and a disulfide-linked alpha MACPF-gamma dimer were successfully produced in Escherichia coli and purified. alpha MACPF was shown to simultaneously bind C8 beta, C8 gamma, and C9 and form a noncovalent alpha MACPF(.)C8 beta(.)C8 gamma(.)C9 complex. Similar results were obtained for the recombinant alpha MACPF-gamma dimer. This dimer bound C8 beta and C9 to form a hemolytically active (alpha MACPF-gamma)(.)C8 beta(.)C9 complex. These results indicate that the principal binding site for C9 lies within the MACPF domain of C8 alpha. They also suggest this site and the binding sites for C8 beta and C8 gamma are distinct. alpha MACPF is the first human MACPF domain to be produced recombinantly and in a functional form. Such a result suggests that this segment of C8 alpha and corresponding segments of the other MAC family members are independently folded domains.