Participation of the N-terminal region of Cε3 in the binding of human IgE to its high-affinity receptor FcεRI

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 Pc 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.