Two clusters of acidic amino acids near the NH2 terminus of complement component C4 alpha'-chain are important for C2 binding.
Previous work has indicated a role for the NH2-terminal segment of the C3 alpha'-chain in the binding interactions of C3b with a number of its protein ligands. In particular, we have identified two clusters of acidic residues, namely, E736 and E737 and to a lesser extent D730 and E731, as being important in the binding of C3b to factor B and complement receptor 1 and the binding of iC3b to complement receptor 3. Whereas human C3 and C4 have an overall sequence identity of 29%, over a segment near the NH2 termini of their respective alpha'-chains the sequence identity is 56% (70% chemical similarity). Given the functional similarity between the C4b-C2 and C3b-B interactions in the respective formation of the classical and alternative pathway C3 convertases, as well as the sequence conservation of two acidic clusters, we hypothesized that residues 744EED and 749DEDD within the NH2-terminal segment of the C4 alpha'-chain would mediate in part the binding of C2 to C4b. We tested this hypothesis using three independent approaches. Site-directed mutagenesis experiments revealed that replacing subsets of the charged residues by their isosteric amides within either acidic cluster resulted in molecules having reduced C2 binding activity. Moreover, a synthetic peptide (C4 residues 740-756) encompassing the two acidic clusters was a specific inhibitor of the binding of C2 to red cell-associated C4b. Finally, Ab raised against the above peptide was able to block the interaction between red cell-associated C4b and fluid phase C2. Taken together, these results strongly suggest that the NH2-terminal acidic residue-rich segment of C4 alpha'-chain contributes importantly to the interaction of C4b with C2.