Padavattan Sivaraman, Flicker Sabine, Schirmer Tilman, Madritsch Christoph, Randow Stefanie, Reese Gerald, Vieths Stefan, Lupinek Christian, Ebner Christof, Valenta Rudolf, Markovic-Housley Zora
Division of Structural Biology, Biozentrum, University of Basel, Basel, Switzerland.
J Immunol. 2009 Feb 15;182(4):2141-51. doi: 10.4049/jimmunol.0803018.
We report the three-dimensional structure of the complex between the major respiratory grass pollen allergen Phl p 2 and its specific human IgE-derived Fab. The Phl p 2-specific human IgE Fab has been isolated from a combinatorial library constructed from lymphocytes of a pollen allergic patient. When the variable domains of the IgE Fab were grafted onto human IgG1, the resulting Ab (huMab2) inhibited strongly the binding of allergic patients' IgE to Phl p 2 as well as allergen-induced basophil degranulation. Analysis of the binding of the allergen to the Ab by surface plasmon resonance yielded a very low dissociation constant (K(D) = 1.1 x 10(-10) M), which is similar to that between IgE and Fcepsilon;RI. The structure of the Phl p 2/IgE Fab complex was determined by x-ray crystallography to 1.9 A resolution revealing a conformational epitope (876 A(2)) comprised of the planar surface of the four-stranded anti-parallel beta-sheet of Phl p 2. The IgE-defined dominant epitope is discontinuous and formed by 21 residues located mostly within the beta strands. Of the 21 residues, 9 interact directly with 5 of the 6 CDRs (L1, L3, H1, H2, H3) of the IgE Fab predominantly by hydrogen bonding and van der Waals interactions. Our results indicate that IgE Abs recognize conformational epitopes with high affinity and provide a structural basis for the highly efficient effector cell activation by allergen/IgE immune complexes.
我们报道了主要呼吸道草花粉过敏原Phl p 2与其特异性人IgE衍生Fab片段之间复合物的三维结构。Phl p 2特异性人IgE Fab片段是从一名花粉过敏患者淋巴细胞构建的组合文库中分离得到的。当将IgE Fab的可变区嫁接到人IgG1上时，所得抗体（huMab2）强烈抑制过敏患者IgE与Phl p 2的结合以及过敏原诱导的嗜碱性粒细胞脱颗粒。通过表面等离子体共振分析过敏原与抗体的结合，得到了非常低的解离常数（K(D)=1.1×10(-10) M），这与IgE和Fcepsilon;RI之间的解离常数相似。通过X射线晶体学确定了Phl p 2/IgE Fab复合物的结构，分辨率为1.9 Å，揭示了一个由Phl p 2的四链反平行β折叠平面表面组成的构象表位（876 Å(2)）。IgE定义的主要表位是不连续的，由主要位于β链内的21个残基组成。在这21个残基中，9个主要通过氢键和范德华相互作用直接与IgE Fab的6个CDR（L1、L3、H1、H2、H3）中的5个相互作用。我们的结果表明，IgE抗体以高亲和力识别构象表位，并为过敏原/I gE免疫复合物高效激活效应细胞提供了结构基础。
