Active sites in complement components C5 and C3 identified by proximity to indels in the C3/4/5 protein family.
We recently suggested that sites of length polymorphisms in protein families (indels) might serve as useful guides for locating protein:protein interaction sites. This report describes additional site-specific mutagenesis and synthetic peptide inhibition studies aimed at testing this idea for the paralogous complement C3, C4, and C5 proteins. A series of C5 mutants was constructed by altering the C5 sequence at each of the 27 indels in this protein family. Mutants were expressed in COS cells and were assayed for hemolytic activity and protease sensitivity. Mutants at five indels showed relatively normal expression but substantially reduced sp. act., indicating that the mutations damaged sites important for C5 function. Twenty-three synthetic peptides with C5 sequences and 10 with C3 sequences were also tested for the ability to inhibit C hemolytic activity. Three of the C5 peptides and one of the C3 peptides showed 50% inhibition of both C hemolytic and bactericidal activities at a concentration of 100 microM. In several cases both the mutational and peptide methods implicated the same indel site. Overall, the results suggest that regions important for function of both C3 and C5 lie proximal to residues 150-200 and 1600-1620 in the precursor sequences. Additional sites potentially important for C5 function are near residue 500 in the beta-chain and at two or three sites between the N-terminus of the alpha'-chain and the C5d fragment. One of the latter sites, near residue 865, appears to be important for proteolytic activation of C5.
Low
P J
PJ
Torrey Pines Institute for Molecular Studies, San Diego, CA 92121, USA.
Ai
R
R
Ogata
R T
RT
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D003167:Complement Activation; D003176:Complement C3 / Q000235:genetics / Q000378:metabolism*; D050577:Complement C3-C5 Convertases / Q000378:metabolism; D003181:Complement C4 / Q000235:genetics / Q000378:metabolism; D003182:Complement C5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003169:Complement Inactivator Proteins / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression / Q000276:immunology; D006461:Hemolysis / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000276:immunology; D016254:Mutagenesis, Insertional; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D011487:Protein Conformation; D014357:Trypsin / Q000378:metabolism
D003176:Complement C3; D003181:Complement C4; D003182:Complement C5; D003169:Complement Inactivator Proteins; D010455:Peptides; D050577:Complement C3-C5 Convertases; D014357:Trypsin