Conformation of the isolated Cε3 domain of IgE and its complex with the high-affinity receptor, FcεRI
Conformation of the isolated Cε3 domain of IgE and its complex with the high-affinity receptor, FcεRI
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DOI：
10.1021/bi9928391
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发表时间：
2000-06-27
期刊：
影响因子：
2.9
通讯作者：
Gould, HJ
中科院分区：
文献类型：
--
作者：
Henry, AJ;McDonnell, JM;Gould, HJ
免疫球蛋白E（IgE）对其在肥大细胞和嗜碱性粒细胞表面上的受体Fc ε RI表现出独特的高亲和力。先前的工作已经暗示ε-重链恒定区的第三结构域（C ε 3）与FceRI结合，但是已知以完全亲和力结合的IgE的最小片段是C ε 3和C ε 4结构域的共价二聚体。我们已经在大肠杆菌中表达了分离的C β 3，测量了其对Fc β RI的亲和力，并检查了其单独和与Fc β RI复合的构象。在分析离心机中的沉降平衡显示该产物是单体。通过表面等离子体共振测量，与Fc ε RI α链的固定片段结合的动力学产生亲和常数K-a = 5 x 10（6）M-1，而IgE为4 x 10（9）M-1。圆二色光谱和荧光作为变性剂浓度的函数的测量没有揭示任何可识别的二级结构或疏水核心。在与Fc ε RI α链片段结合时，圆二色光谱没有变化，表明复合物中C ε 3的构象没有变化。因此，分离的C β 3结构域足以结合Fc β RI，但亲和力低于IgE。这可能是由于其天然免疫球蛋白结构域结构的丧失，或由于需要两个C β 3结构域来构成Fc β RI的完整结合位点，或由于这些因素的组合。
Immunoglobulin E (IgE) exhibits a uniquely high affinity for its receptor, Fc epsilon RI, on the surface of mast cells and basophils. Previous work has implicated the third domain of the constant region of the epsilon-heavy chain (C epsilon 3) in binding to FceRI, but the smallest fragment of IgE that is known to bind with full affinity is a covalent dimer of the C epsilon 3 and C epsilon 4 domains. We have expressed the isolated C epsilon 3 in Escherichia coli, measured its affinity for Fc epsilon RI, and examined its conformation alone and in the complex with Fc epsilon RI. Sedimentation equilibrium in the analytical centrifuge reveals that this product is a monomer. The kinetics of binding to an immobilized fragment of the Fc epsilon RI 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 Fc epsilon RI alpha-chain fragment, there is no change in the circular dichroism spectrum, indicating that the conformation of C epsilon 3 is unchanged in the complex. Thus the isolated C epsilon 3 domain is sufficient for binding to Fc epsilon RI, 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 C epsilon 3 domains to constitute the complete binding site for Fc epsilon RI or to a combination of these factors.