Participation of the N-Terminal Region of C epsilon 3 in the Binding of Human IgE to Its High-Affinity Receptor Fc epsilon RI

The binding of immunoglobulin E (IgE) to its high-affinity receptor (Fc epsilon RI) expressed on mast cells and basophils is central to the development of an allergic reaction. Previous studies have implicated the third constant domain of IgE-Fc (C epsilon 3) as the site of the interaction with Fc epsilon RI. We have prepared a series of site-directed mutants of human IgE-Fc, particularly focusing on the N-terminal "linker" region and AB loop of C epsilon 3. The kinetics of binding IgE and its Fc fragments to the immobilized receptor were determined by surface plasmon resonance (SPR), and two phases of binding were observed. We identified one mutation in the N-terminal linker region, R334S, that has a dramatic effect on binding. R334S lowers the affinity of IgE-Fc for Fc epsilon RI by 120-fold, principally through an increase in the dissociation rate of the slower phase of the interaction. This mutation has a similar effect in Fc epsilon 3-4, a truncated form of IgE-Fc which lacks the C epsilon 2 domain pair, and thus it does not exert its effect through altering the quaternary structure of IgE-Fc, firmly implicating Arg334 as a contact residue in the complex. However R334S has no effect on the binding of Fc epsilon RII (CD23), the low-affinity receptor for IgE, demonstrating the structural integrity of the mutated IgE-Fc. Circular dichroism spectroscopy and thermal stability studies further indicate that the R334S mutation does not disorder or destabilize the structure of IgE-Fc or Fc epsilon 3-4. These results demonstrate the importance of the N-terminal linker region of C epsilon 3 in the interaction of IgE with Fc epsilon RI.