Evidence for the involvement of arginine 462 and the flanking sequence of human C4 beta-chain in mediating C5 binding to the C4b subcomponent of the classical complement pathway C5 convertase.

Abstract

Replacement of human C4 beta-chain residue arginine 458 by tryptophan, a substitution that occurs naturally in the hemolytically inactive A6 allotype of C4, totally abrogates the molecule's ability to act as a C5 binding subunit of the classical pathway C5 convertase. Hydropathy plots predict R458 to be within a hydrophilic segment extending from residue 455 to 469 and having the sequence SIERPDSRPPRVGDT. To further assess the potential involvement of this segment in the C5 binding function of C4, we have engineered "ala-scan" mutants through this segment, concentrating predominantly on charged residues, and analyzed their functional profiles. C4B isotype mutant proteins S455A (0.7), E457A (1.1), R458A (0.3), D460A (0.2), R462A (0.0), R465A (0.6), and D468A (0.3) displayed the relative to wild-type hemolytic activities indicated in the parentheses. In all cases, the hemolytic defect was accounted for solely at the C5 convertase stage. The total absence of C5 binding activity in the R462A mutant suggests a requirement for the guanidinium group per se, because mutants with a charge-conservative lysine or a relatively isosteric methionine at this position were also completely inactive. In contrast, the inactivity of the C4A6-like R458W mutant is probably caused by the intolerance of tryptophan in a hydrophilic segment, as substitution of R458 by alanine or methionine yielded recombinant molecules that retained 30% and 60% of wild-type hemolytic activity, respectively. Taken together, the mutagenesis results strongly imply that residues in the 455-469 segment contribute to the C5 binding site in C4; however, the conformational context of the segment appears to be crucial, as a synthetic peptide corresponding to this segment displayed no ability to inhibit C5 binding to surface-bound C4b.