An indel within the C8α subunit of human complement C8 mediates intracellular binding of C8γ and formation of C8α-γ

Human C8 is one of five complement components (C5b, C6, C7, C8, and C9) that interact to form the cytolytic membrane attack complex, or MAC. It is an oligomeric protein composed of three subunits (C8 alpha, C8 beta, C8 gamma) that are products of different genes. In C8 from serum, these are arranged as a disulfide-linked C8 alpha-gamma dimer that is noncovalently associated with csp. In this study, the site on C8 alpha that mediates intracellular binding of C8 gamma to form C8 alpha-gamma was identified. From a comparative analysis of indels (insertions/deletions) in C8 alpha and its structural homologues C8 beta, C6, C7, and C9, it was determined that C8 alpha contains a unique insertion (residues 159-175), which includes Cys(164) that farms the disulfide bond to C8 gamma. Incorporation of this sequence into csp and coexpression of the resulting construct (iC8 beta) with C8 gamma produced iC8 beta-gamma, an atypical disulfide-linked dimer. In related experiments, C8 gamma was shown to bind noncovalently to mutant forms of C8 alpha and iC8 beta in which Cys(164-->)Gly(164) substitutions were made. In addition, C8 gamma bound specifically to an immobilized synthetic peptide containing the mutant indel sequence. Together, these results indicate (a) intracellular binding of C8 gamma to C8 alpha is mediated principally by residues contained within the C8 alpha indel, (b) binding is not strictly dependent on Cys(164), and (c) C8 gamma must contain a complementary binding site for the C8 alpha indel.