EFFECTS OF SUBSTITUTIONS IN THE BINDING SURFACE OF AN ANTIBODY ON ANTIGEN AFFINITY

The interactions between the Fab and single-chain Fv (scFv) fragments of an antibody (NC10) and its antigen, influenza virus neuraminidase, were analysed in the crystal structures of the Fab-neuraminidase and s cFv--neuraminidase complexes. To investigate the contribution to bindi ng made by cavities, salt links and hydrogen bonds in the antibody-ant igen interface, 14 single amino acid replacements were made at six con tact residues in the scFv fragment by site-directed mutagenesis. The b inding affinity of each mutant scFv antibody for neuraminidase was det ermined with a BLAcore(TM) optical biosensor. Four of the mutations re sulted in large changes in the free energy of binding to neuraminidase (Delta Delta G > 1 kcal/mol) and together may account for similar to 70% of the free energy of binding. Hence these data support the theory that a small number of residues form the 'functional epitope' and are most important for binding of NC10 to neuraminidase. The salt link be tween antibody residue (Asp)H56 and (Lys)N432 from neuraminidase was d emonstrated to be important for affinity, since substitution of (Asp)H 56 with Asn caused a large reduction in the free energy of binding (De lta Delta G = +2.8 kcal/mol). Hydrogen bonds provided by (Tyr)L32 and (Asp)H56 were also important for binding: mutation of (Tyr)L32 to Phe resulted in a significant reduction in binding affinity (MG = +1.7 kca l/mol). Disruption of hydrophobic interactions (van der Waals contacts ) led to significant reductions in affinity also ((Tyr)H99 to Ala, Del ta Delta G = +1.5 kcal/mol; (Leu)L94 to Ala, Delta Delta G > +3.0 kcal /mol). An attempt to increase binding affinity by filling a cavity in the interface with a larger antibody side chain was unsuccessful, as t he free energy gained by new antibody-antigen interactions did not com pensate for the removal of cavity-bound water molecules.