Structural requirements for the complement regulatory activities of C4BP

C4b-binding protein (C4BP) is a regulator of the classical complement pathw ay C3 convertase (C4bC2a complex). It is a disulfide-linked polymer of seve n cr-chains and a unique beta -chain; the alpha- and beta -chains are compo sed of eight and three complement control protein (CCP) domains, respective ly. To elucidate the importance of the polymeric nature of C4BP and the str uctural requirements for the interaction between C4b and the alpha -chain, 19 recombinant C4BP variants were created. Six truncated monomeric variants , nine polymeric variants in which individual CCPs were deleted, and finall y, four variants in which double alanine residues were introduced between C CPs were functionally characterized. The smallest truncated C4BP variant st ill active in regulating fluid phase C4b comprised CCPI-3, The monomeric va riants were less efficient than polymeric C4BP in degrading C4b on cell sur faces. All three N-terminal CCP domains contributed to the binding of C4b a nd mere important for full functional activity; CCP2 and CCPS were the most important. The spatial arrangements of the first CCPs were found to be imp ortant, as introduction of alanine residues between CCPs 1 and 2, CCPs 2 an d 3, and CCPs 3 and 4 resulted in functional impairment. The results presen ted here elucidate the structural requirements of individual CCPs of C4BP, as well as their spatial arrangements within and between subunits for expre ssion of full functional activity.