Protein Engineering, Vol 11, 303-309, Copyright © 1998 by Oxford University Press
R Raffen, PW Stevens, C Boogaard, M Schiffer and FJ Stevens
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.
ARTICLES
Reengineering immunoglobulin domain interactions by introduction of charged residues
Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, IL 60439, USA.
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