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Active Sites in Complement Component C3 Mapped by Mutations at Indels¹

Medical Biology Institute, La Jolla, CA 92037

Engineered mutants of human complement component C3 were used to test the idea that sites of length polymorphisms in protein families (indels) can guide a search for protein:protein interaction sites. Sequence changes were introduced at each of the 27 indels in the C3/4/5 protein family, and mutants at 26 indels were expressed by transiently transfected COS cells. Expressed proteins were assayed 1) for concentration, by ELISA and by autoradiography of radiolabeled protein; 2) for classical pathway hemolytic activity; 3) for susceptibility to proteolytic activation by the alternative pathway and cobra venom factor C3 convertases; and 4) for susceptibility to complement factor I in the presence of factor H. Most of the mutations did not appreciably alter expression or activity relative to wild-type C3, consistent with the idea that most indels occur at the protein surface. Mutations at four indels severely damaged C3 functional activity, but did not affect the stability or structure of the protein, as assessed by their effects on expression by COS cells and on susceptibility to cleavage by C3 convertases and factor I. These indels are therefore near functionally important amino acid residues; they represent good candidates for sites of protein:protein interactions. Mutation of the sequence at a fifth indel altered the equilibrium between the latent and reacted C3 conformations, and mutations at 4 other indels substantially decreased both protein activity and expression. The mutants provided an overview of the structural and functional roles played by different parts of C3.

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