title: Functional studies of the MACPF domain of human complement protein C8alpha reveal sites for simultaneous binding of C8beta, C8gamma, and C9.
abstract: Human C8 is one of five components of the membrane attack complex of complement (MAC). It contains three subunits (C8alpha, C8beta, C8gamma) arranged as a disulfide-linked C8alpha-gamma dimer that is noncovalently associated with C8beta. C8alpha, C8beta, 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/perforin (MACPF) domain. During MAC formation, C8alpha 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 C8alpha N-terminal modules and MACPF domain (alphaMACPF). In the present study, we examined the role of the MACPF domain in binding C9. Recombinant alphaMACPF and a disulfide-linked alphaMACPF-gamma dimer were successfully produced in Escherichia coli and purified. alphaMACPF was shown to simultaneously bind C8beta, C8gamma, and C9 and form a noncovalent alphaMACPF.C8beta.C8gamma.C9 complex. Similar results were obtained for the recombinant alphaMACPF-gamma dimer. This dimer bound C8beta and C9 to form a hemolytically active (alphaMACPF-gamma).C8beta.C9 complex. These results indicate that the principal binding site for C9 lies within the MACPF domain of C8alpha. They also suggest this site and the binding sites for C8beta and C8gamma are distinct. alphaMACPF is the first human MACPF domain to be produced recombinantly and in a functional form. Such a result suggests that this segment of C8alpha and corresponding segments of the other MAC family members are independently folded domains.
authors.affiliation: Department of Chemistry and Biochemistry and School of Medicine, University of South Carolina, Columbia, South Carolina 29208, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Slade
authors.forename: Brian
authors.initials: B
authors.lastname: Chiswell
authors.forename: James M
authors.initials: JM
authors.lastname: Sodetz
mesh_terms: D001665:Binding Sites; D003185:Complement C8 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003186:Complement C9 / Q000378:metabolism*; D015870:Gene Expression; D006461:Hemolysis; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D003185:Complement C8; D003186:Complement C9; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin