Spectroscopic, calorimetric, and kinetic demonstration of conformational adaptation in peptide antibody recognition

Little is known about the extent to which protein flexibility contributes to antigen-antibody recognition and cross-reactivity. Using short coiled coil peptides (leucine zippers) as model antigens, we demonstrate that a monoclonal antibody can force a noncognate peptide into a conformation that is similar to the conformation of the cognate peptide against which the monoclonal antibody is directed. Monoclonal antibodies 29AB and 13AD were raised against the 29-residue peptide LZ (Ac-EYEALEKKLAALE-AKLQALEKKLEALEHG-amide) that forms a very stable coiled coil. The two antibodies cross-reacted strongly with the random coil analogue LZ(7P14P) that contains Lys-->Pro and Ala-->Pro substitutions in positions 7 and 14, respectively. The antibody-bound peptide LZ(7P14P) adopted an altered conformation that possibly was coiled coil-like, as shown by CD difference spectroscopy and fluorescence quenching experiments on coumarin-labeled peptides. Isothermal titration calorimetry revealed that the cross-reaction of antibodies 13AD and 29AB with the random coil peptide LZ(7P14P) exhibited a large unfavorable entropy. This, however, was strongly compensated by a more favorable enthalpy, resulting in only a small difference between the association constants for peptide LZ and LZ(7P14P), respectively. To investigate the opposite type of cross-reaction, monoclonal antibody 42PF was raised against the random coil peptide LZ(7P14P). 42PF cross-reacted with coiled coil peptide LZ by forcing it to dissociate into single chains. Enthalpy/entropy compensation again enabled the cross-reaction, which now was entropically favored and enthalpically disfavored. The rate of reaction of antibody 42PF with peptide LZ was controlled by the rate of dissociation of LZ into single chains. This observation, as well as the generally much slower reaction rate with the noncognate peptides, indicated that the cross-reactivity occurred because the antibody selected the conformer of the antigen that binds the strongest, a mechanism we call ''induced fit by conformational selection''.