The recombinant antibody fragment scFv(F8), which recognizes the coat protein of the plant virus AMCV, is characterized by peculiar high in vitro stability and functional folding even in reducing environments, making it fit for designing stable antibodies with desired properties. Mutagenesis and functional analysis evidenced two residues, at positions 47 and 58 of the VH chain, playing a crucial role in the antigen binding recognition. Here, we used a computational procedure to assess the effects of these mutations on the stability, structure and dynamics of the antigen–binding site. Structural models of the wild type scFv(F8) and of its H47 and H58 mutants were built by homology modelling and assessed by multiple 15.5 ns of molecular dynamics sim- ulations. Computational results indicate that the 47H substitution strongly affects the CDR–H2 conformation, destabilizes the VH /VL interface and confers high con- formational flexibility to the antigen–binding site, leading the mutant to functional loss. The mutation at position H58 strenghtens the binding site, bestowing a high antigen specificity on the mutant. The essential dynamics and the analysis of the protein–solvent interface further corroborate the correspondence between the ex- tent of the structurally–determined flexibility of the binding site with the different functional behaviours proved by the wild-type and its mutants. These results may have useful implications for structure–based design of antibody combining site.

Structure and dynamics of the anti-AMCV scFv(F8): effects of selected mutations on the antigen combining site

Rosato, Vittorio;Galeffi, Patrizia;Cantale, Cristina;Arcangeli, Caterina
2008

Abstract

The recombinant antibody fragment scFv(F8), which recognizes the coat protein of the plant virus AMCV, is characterized by peculiar high in vitro stability and functional folding even in reducing environments, making it fit for designing stable antibodies with desired properties. Mutagenesis and functional analysis evidenced two residues, at positions 47 and 58 of the VH chain, playing a crucial role in the antigen binding recognition. Here, we used a computational procedure to assess the effects of these mutations on the stability, structure and dynamics of the antigen–binding site. Structural models of the wild type scFv(F8) and of its H47 and H58 mutants were built by homology modelling and assessed by multiple 15.5 ns of molecular dynamics sim- ulations. Computational results indicate that the 47H substitution strongly affects the CDR–H2 conformation, destabilizes the VH /VL interface and confers high con- formational flexibility to the antigen–binding site, leading the mutant to functional loss. The mutation at position H58 strenghtens the binding site, bestowing a high antigen specificity on the mutant. The essential dynamics and the analysis of the protein–solvent interface further corroborate the correspondence between the ex- tent of the structurally–determined flexibility of the binding site with the different functional behaviours proved by the wild-type and its mutants. These results may have useful implications for structure–based design of antibody combining site.
single–chain fragment, molecular dynamics simulation, essential dynamics analysis, structure-function relationship, solvent role
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/20.500.12079/3228
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