Distal Recognition Site for Classical Pathway Convertase Located in the C345C/Netrin Module of Complement Component C5

Abstract

Previous studies focused on indels in the complement C345 protein family identified a number of potential protein-protein interaction sites in components C3 and C5. Here, one of these sites in C5, near the α-chain C terminus, was examined by alanine-scanning mutagenesis at 16 of the 18 non-alanine residues in the sequence KEALQIKYNFSFRYIYPLD. Alanine substitutions affected 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 the normal activity. These three mutants were also resistant to the classical pathway (CP) C5 convertase, with sensitivities roughly proportional to their 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 effects 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 ∼880 residues downstream of the convertase cleavage site within a module that has been independently named C345C and NTR; this module is found in diverse proteins including netrins and tissue inhibitors of metalloproteinases.

Formation of High-Affinity C5 Convertases of the Alternative Pathway of Complement

“…Studies by Jelezarova et al (31) have suggested C3b-C3b dimers in complex with IgG molecules to form better precursors of convertases than C3b. Recent studies by Sandoval et al (32) involving indels have identified at least two binding sites on C5 for the classical pathway C5 convertase in addition to the convertase cleavage site. Based on all these studies it appears that C5 binds with high affinity to multimeric C3b complexes through interactions of at least two distinct sites on C5 and at least two sites on C3b.…”

Functional Insights from the Structure of the Multifunctional C345C Domain of C5 of Complement

“…In that case mutations of the DE loop might disrupt the arrangement of domains within the full-length protein and exert their functional influence indirectly. Arguing against this, however, is the observation that a peptide extending from Lys 1604 to Arg 1616 inhibited complement hemolytic activity and activation of C5 by the convertase pathway C5 convertase (but not cobra venom factor) (17). Furthermore, the consequences for inhibitory activity of alanine substitution within the peptide reflected the results of alanine-scanning mutagenesis in C5.…”

“…2), and the interaction with C7, but not C6, appears to be essential for the nonreversible formation of the MAC. 2 The F1613A mutant used in the current structure determination retained the ability to bind C6 and C7 (Table I); indeed, none of several DE loop mutations in C5 influenced binding to C6 (17). Therefore, the C6/C7-binding site of C5-C345C is likely to lie elsewhere.…”

“…Thus the C5-C345C domain provides at least part of the interacting surface between C5b and C7 in formation of the MAC. The C345C domain also harbors a region that interacts with the C5 convertase (17), although the cleavage site itself lies some 800 residues away toward the N terminus of the ␣ chain of C5.…”

“…Subsequently, alanine substitutions of residues from Gly 1603 to Pro 1621 were carried out in full-length C5 (17). Most substitutions had little or no effect on the hemolytic activity of C5 or its susceptibility to proteolytic cleavage.…”