Conformational changes in the antibody constant domains upon hapten-binding

Abstract

Bacterial proteins A and G (SpA and SpG) are immunoglobulin receptors that can be used as probes for monitoring change in the conformation of heavy chain constant (C H) domains. Interaction of anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody (Ab) with SpA and SpG were measured by isothermal titration calorimetry and surface plasmon resonance in order to address the question of whether hapten-binding induces a conformational change in the C H domain. The interactions of IgG2a or its enzymatic fragments with SpA were measured in the presence or absence of the hapten. Although binding of Fab and F(ab′) 2 fragments were not observed to free SpA, they did bind to immobilized SpA. In addition, the association constant ( K a) for interaction of IgG2a with immobilized SpA was approximately 20-fold higher than that with free SpA. This was explained in terms of high avidity resulting from multivalent interaction between IgG2a and immobilized SpA on the chip. Interestingly, the hapten-binding weakened the interaction between the F(ab′) 2 fragment and SpA. Furthermore, approximately half of the IgG2a was incapable of binding to immobilized SpA in the presence of hapten. These results were explained using a model which assumed the formation of two kinds of SpA/IgG complexes; one through sites on F(ab′) 2 arms and the other through sites on the Fc region. The former type dissociated as a result of hapten-binding, as did the F(ab′) 2 fragment and suggested that a conformational change had occurred around the Fab arms, while the latter type did not dissociate because of the higher avidity of the Fc region. However, using a mutant SpA with a lower K a value for the interaction with IgG2a, it was shown that hapten-binding induced long range conformational changes in the Fc region of IgG2a. Similar evidence of conformational change upon hapten-binding was also obtained using SpG as a probe.