One of the critical steps in allergic reactions is the binding of IgE to its high affinity receptor (FcepsilonRI). The extracellular domain of the alpha-chain (alpha-t) of the FcepsilonRI is solely responsible for the binding to IgE, and the Fc portion of IgE is sufficient for binding to FcepsilonRI. For a complete understanding of the interaction between FcepsilonRI and IgE the structure of the two proteins and of their complex has to be determined. This project is aimed to obtain the structure of alpha-t and of its complex with Fc. Knowledge of the alpha-t structure or of its complex with Fc would allow the design of novel therapeutic agents. We have expressed alpha-t capable of binding IgE in CHO cells. However, glycosylation heterogeneity prevented crystallization. On the other hand the deglycosylated alpha-t produced in E.Coli was unstable and had tendency to aggregate. For this reason we have produced a preparation of alpha-t with limited glycosylation. These proteins lead to crystal that could diffract to a resolution of 3.5 angstroms. A data set 83% complete at 4 angstroms resolution was collected using a synchrotron as an X-ray source. Screening for heavy atoms derivatives and molecular replacement studies are in progress. He have expressed the Fc as a soluble protein and in inclusion body in E.coli. We hope to be able to produce milligrams amounts of this Fc for making cocrystal with alpha-t. In addition we have found that a major conformational rearrangements occurs on the molecule of IgE after binding to alpha-t.
