Conformation of the Isolated Cε3 Domain of IgE and Its Complex with the High-Affinity Receptor, FcεRI

Publisher Website
Google Scholar
Abstract
Immunoglobulin E (IgE) exhibits a uniquely high affinity for its receptor, FcεRI, on the surface of mast cells and basophils. Previous work has implicated the third domain of the constant region of the ε-heavy chain (Cε3) in binding to FcεRI, but the smallest fragment of IgE that is known to bind with full affinity is a covalent dimer of the Cε3 and Cε4 domains. We have expressed the isolated Cε3 in Escherichia coli, measured its affinity for FcεRI, and examined its conformation alone and in the complex with FcεRI. Sedimentation equilibrium in the analytical centrifuge reveals that this product is a monomer. The kinetics of binding to an immobilized fragment of the FcεRI α-chain, measured by surface plasmon resonance, yields an affinity constant Ka = 5 × 106 M-1, as compared with 4 × 109 M-1 for IgE. The circular dichroism spectrum and measurements of fluorescence as a function of the concentration of a denaturant do not reveal any recognizable secondary structure or hydrophobic core. On binding to the FcεRI α-chain fragment, there is no change in the circular dichroism spectrum, indicating that the conformation of Cε3 is unchanged in the complex. Thus the isolated Cε3 domain is sufficient for binding to FcεRI, but with lower affinity than IgE. This may be due to the loss of its native immunoglobulin domain structure or to the requirement for two Cε3 domains to constitute the complete binding site for FcεRI or to a combination of these factors.