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 identified a number of potential protein-protein interaction sites in components C3 and C5. Here, one of these sites in C5, near the alpha-chain C terminus, was examined by alanine-scanning mutagenesis at 16 of the 18 non-alanine residues in the sequence KEALQIKYNFSF RYIYPLD. 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 approximately 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.
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Torrey Pines Institute for Molecular Studies, San Diego, CA 92121, USA.
Ai
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Ostresh
J M
JM
Ogata
R T
RT
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics / Q000276:immunology; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D003176:Complement C3 / Q000378:metabolism; D050577:Complement C3-C5 Convertases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003181:Complement C4 / Q000378:metabolism; D003182:Complement C5 / Q000235:genetics / Q000378:metabolism*; D003169:Complement Inactivator Proteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D003171:Complement Pathway, Classical / Q000235:genetics; D003165:Complement System Proteins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000276:immunology / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000075386:Netrin Receptors; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D011956:Receptors, Cell Surface / Q000378:metabolism*
D003176:Complement C3; D003181:Complement C4; D003182:Complement C5; D003169:Complement Inactivator Proteins; D004791:Enzyme Inhibitors; D000075386:Netrin Receptors; D010455:Peptides; D011956:Receptors, Cell Surface; C050974:complement C5b-6 complex; D003165:Complement System Proteins; D050577:Complement C3-C5 Convertases; D000409:Alanine