Conformational changes in the antibody constant domains upon hapten-binding

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 (CH) 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 CH 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 (Ka) 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 Ka 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.