Identification of contact residues in the IgE binding site of human FcεRIα

The high-affinity receptor for immunoglobulin E (IgE), Fc epsilon RI, is an alpha beta gamma(2) tetramer found on mast cells, basophils, and several other types of immune effector cells. The interaction of IgE with the alpha-subunit of Fc epsilon RI is central to the pathogenesis of allergy. Detailed knowledge of the mode of interaction of Fc epsilon RI with IgE may facilitate the development of inhibitors for general use in the treatment of allergic disease. To this end we have performed site-directed mutagenesis on a soluble form of the Fc epsilon RI alpha-chain (sFc epsilon RI alpha). The effects of four mutations in the second immunoglobulin-like domain of sFc epsilon RI alpha upon the kinetics of binding to IgE and fragments of IgE have been analyzed using surface plasmon resonance. As described in the preceding paper of this issue [Henry, A. J., et al. (1997) Biochemistry 36, 15568-15578], biphasic binding kinetics was observed. Two of the mutations had significant effects on binding: K117D reduced the affinity of sFc epsilon RI alpha for IgE by a factor of 30, while D159K increased the affinity for IgE by a factor of 7, both principally through changes in the rates of dissociation of the slower phase of the interaction. Circular dichroism spectra of sFc epsilon RI alpha incorporating either of these mutations were indistinguishable from those of wild-type sFc epsilon RI alpha, demonstrating that the native conformation had not been disrupted. Our results, together with those from site-directed mutagenesis on fragments of IgE presented in the accompanying paper, define the contact surfaces in the IgE:sFc epsilon RI alpha complex.