CHARACTERIZATION OF THE FC EPSILON RI ALPHA CHAIN BY CRYSTALLOGRAPHY

通过晶体学表征 FC EPSILON RI ALPHA 链

• 批准号: 5200559
• 负责人: J P KINET
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5200559
• 关键词: Escherichia coli; X ray crystallography; antibody receptor; glycosylation; human tissue; immunoglobulin E; intermolecular interaction; molecular cloning; protein structure; receptor binding

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.

过敏反应中的关键步骤之一是IgE与 其高亲和性受体（FcepsilonRI）。 的胞外结构域 Fc ε RI的α链（α-t）仅负责 与IgE的结合，并且IgE的Fc部分足以结合 到FcepsilonRI。为了完整地理解 Fc ε RI和IgE这两种蛋白质的结构以及它们的分子结构。 复杂性必须确定。该项目旨在获得 α-t及其与Fc复合物结构。关于阿尔法t的知识 结构或其与Fc的复合物将允许设计新的 治疗剂。 我们已经表达了能够结合IgE的α-t 在CHO细胞中。然而，糖基化异质性阻止了 晶化另一方面， 在大肠杆菌中不稳定，有聚集的倾向。因此我们 已经制备了具有有限糖基化的α-T制剂。这些 蛋白质导致晶体，可以将分辨率提高到3.5 埃。在4埃分辨率下完成了83%的数据集， 使用同步加速器作为X射线源收集的。筛选重型 原子衍生物和分子置换研究正在进行中。他 已将Fc表达为可溶性蛋白并在包涵体中 E.coli.我们希望能够生产毫克数量的这种Fc， 与alpha-t形成共晶。此外，我们还发现， 结合后IgE分子发生构象重排 到阿尔法-T。