A cluster of positively charged amino acids in the C4BP α-chain is crucial for C4b binding and factor I cofactor function

C4b-binding protein (C4BP) is a regulator of the classical complement pathway, acting as a cofactor to factor I in the degradation of C4b. Computer modeling and structural analysis predicted a cluster of positively charged amino acids at the interface between complement control protein modules 1 and 2 of the C4BP cu-chain to be involved in C4b binding. Three C4BP mutants, R39Q, R64Q/R66Q, and R39Q/R64Q/R66Q, were ex pressed and assayed for their ability to bind Gib and to function as factor I cofactors. The apparent affinities of R39Q, R64Q/R66Q, and R39Q/R64Q/R66Q for immobilized Gib were 15-, 50-, and 140-fold lower, respectively, than that of recombinant wild type C4BP. The Gib binding site demonstrated herein was also found to be a specific heparin binding site. In C4b degradation, the mutants demonstrated decreased ability to serve as factor I cofactors. In particular, the R39Q/R64Q/R66Q mutant was inefficient as cofactor for cleavage of the Arg(937)-Thr(938) peptide bond in Gib. In contrast, the factor I mediated cleavage of Arg(1317)-Asn(1318) bond was less affected by the C4BP mutations. In conclusion, we identify a cluster of amino acids that is part of a C4b binding site involved in the regulation of the complement system.