title: Conformation of the isolated cepsilon3 domain of IgE and its complex with the high-affinity receptor, FcepsilonRI.
abstract: Immunoglobulin E (IgE) exhibits a uniquely high affinity for its receptor, FcepsilonRI, on the surface of mast cells and basophils. Previous work has implicated the third domain of the constant region of the epsilon-heavy chain (Cepsilon3) in binding to FcepsilonRI, but the smallest fragment of IgE that is known to bind with full affinity is a covalent dimer of the Cepsilon3 and Cepsilon4 domains. We have expressed the isolated Cepsilon3 in Escherichia coli, measured its affinity for FcepsilonRI, and examined its conformation alone and in the complex with FcepsilonRI. Sedimentation equilibrium in the analytical centrifuge reveals that this product is a monomer. The kinetics of binding to an immobilized fragment of the FcepsilonRI alpha-chain, measured by surface plasmon resonance, yields an affinity constant K(a) = 5 x 10(6) M(-)(1), as compared with 4 x 10(9) 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 FcepsilonRI alpha-chain fragment, there is no change in the circular dichroism spectrum, indicating that the conformation of Cepsilon3 is unchanged in the complex. Thus the isolated Cepsilon3 domain is sufficient for binding to FcepsilonRI, 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 Cepsilon3 domains to constitute the complete binding site for FcepsilonRI or to a combination of these factors.
authors.affiliation: The Randall Centre for Molecular Mechanisms of Cell Function, King's College London, UK.
authors.forename: A J
authors.initials: AJ
authors.lastname: Henry
authors.forename: J M
authors.initials: JM
authors.lastname: McDonnell
authors.forename: R
authors.initials: R
authors.lastname: Ghirlando
authors.forename: B J
authors.initials: BJ
authors.lastname: Sutton
authors.forename: H J
authors.initials: HJ
authors.lastname: Gould
mesh_terms: D002460:Cell Line; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D007073:Immunoglobulin E / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D011487:Protein Conformation; D017510:Protein Folding; D017455:Receptors, IgE / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D017455:Receptors, IgE; D011994:Recombinant Proteins; D007073:Immunoglobulin E