Reengineering immunoglobulin domain interactions by introduction of charged residues

The formation of the antibody variable domain binding unit (Fv) is the net result of three competing assembly reactions. The affinities of concurrent homologous interactions of heavy and light chain variable domains limits the heterologous interaction leading to productive formation of the Fv. To address the possible role of light chain dimerization in this phenomenon, the Gln38 residue at the dimer interface of an immunoglobulin light chain variable domain (VL) was replaced by charged amino acids. The effects of these mutations on VL homodimer formation were monitored by small-zone size exclusion HPLC and the affinities of interaction were determined by computer simulation. Reduced VL homodimerization was observed in three of the four mutants, Q38R, Q38D and Q38K. The association constants for the Q38R and Q38D homodimers were 1.2 x 10(4) and 3.2 x 10(3) M(-1), respectively. This corresponded to a 20-75-fold reduction in the homodimer association constant relative to the wild-type VL, which had an association constant of 2.4 x 10(5) M(-1). Surprisingly, the fourth charge mutant, Q38E, had a higher association constant than the wild-type VL. The potential for charged residues to facilitate heterodimeric assembly of immunoglobulin domains was also tested. Heterodimerization was observed between the Q38D and Q38R V(L)s, but with an association constant of 4.7 x 10(4) M(-1), approximately fivefold lower than that obtained for homodimerization of the native V(L). In addition, replacement of the neutral, solvent-accessible Gln38 residue with either Asp or Arg was found to be significantly destabilizing. These results suggest that charged residues could be introduced at immunoglobulin domain interfaces to guide heterodimer formation and to minimize unfavorable competing homologous associations. Nonetheless, these apparently simple modifications may also result in unintended consequences that are likely to depend upon structural features of particular variable domains.