Antigen–Antibody
Interactions and Structural
Flexibility of a Femtomolar-Affinity Antibody
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Abstract
The femtomolar-affinity mutant antibody (4M5.3) generated
by directed
evolution is interesting because of the potential of antibody engineering.
In this study, the mutant and its wild type (4-4-20) were compared
in terms of antigen–antibody interactions and structural flexibility
to elucidate the effects of directed evolution. For this purpose,
multiple steered molecular dynamics (SMD) simulations were performed.
The pulling forces of SMD simulations elucidated the regions that
form strong attractive interactions in the binding pocket. Structural
analysis in these regions showed two important mutations for improving
attractive interactions. First, mutation of Tyr102(H) to Ser (sequence
numbering of Protein Data Bank entry 1FLR) played a role in resolving the steric
hindrance on the pathway of the antigen in the binding pocket. Second,
mutation of Asp31(H) to His played a role in resolving electrostatic
repulsion. Potentials of mean force (PMFs) of both the wild type and
the mutant showed landscapes that do not include obvious intermediate
states and go directly to the bound state. These landscapes were regarded
as funnel-like binding free energy landscapes. Furthermore, the structural
flexibility based on the fluctuations of the positions of atoms was
analyzed. It was shown that the fluctuations in the positions of the
antigen and residues in contact with antigen tend to be smaller in
the mutant than in the wild type. This result suggested that structural
flexibility decreases as affinity is improved by directed evolution.
This suggestion is similar to the relationship between affinity and
flexibility for in vivo affinity maturation, which was suggested by
Romesberg and co-workers [Jimenez, R., et al. (2003) <i>Proc.
Natl. Acad. Sci. U.S.A.</i> <i>100</i>, 92–97].
Consequently, the relationship was found to be applicable up to femotomolar
affinity levels