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Two Clusters of Acidic Amino Acids Near the NH₂ Terminus of Complement Component C4 '-Chain Are Important for C2 Binding¹

Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

Previous work has indicated a role for the NH₂-terminal segment of the C3 '-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 '-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 '-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 '-chain contributes importantly to the interaction of C4b with C2.

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