Distal recognition site for classical pathway convertase located in the C345C/netrin module of complement component C5

Previous studies focused on indels in the complement C345 protein family id entified a number of potential protein-protein interaction sites in compone nts C3 and C5. Here, one of these sites in C5, near the cw-chain C terminus , was examined by alanine-scanning mutagenesis at 16 of the 18 non-alanine residues in the sequence KEALQIKYNFSFRYIYPLD. Alanine substitutions affecte d activities in the highly variable manner characteristic of binding sites. Substitutions at the lysine or either phenylalanine residue in the central KYNFSF sequence had the greatest effects, yielding mutants with <20% of th e normal activity, These three mutants were also resistant to the classical pathway (CP) C5 convertase, with sensitivities roughly proportional to the ir hemolytic activities, but had normal susceptibilities to the cobra venom factor (CVF)-dependent convertase. Synthetic peptide MGKEALQIKYNFS-NH2 was found similarly to inhibit CP but not CVF convertase activation, and the e ffects of alanine substitutions in this peptide largely reflected those of the equivalent mutations in C5, These results indicate that residues KYNFSF form a novel, distal binding site for the CP, but not CVF convertase, This site lies similar to 880 residues downstream of the convertase cleavage si te within a module that has been independently named C345C and NTR; this mo dule is found in diverse proteins including netrins and tissue inhibitors o f metalloproteinases.