Tsumoto K, Ueda Y, Maenaka K, Watanabe K, Ogasahara K, Yutani K, Kumagai I
Department of Chemistry and Biotechnology, Faculty of Engineering, University of Tokyo, Japan.
J Biol Chem. 1994 Nov 18;269(46):28777-82.
For elucidating the contribution of structurally perturbed antigenic residues upon antibody binding to antigen-antibody interaction, the interaction between hen egg white lysozyme (HEL) and HyHEL10 Fv fragment, which is one of several monoclonal antibodies against HEL and structurally well defined (Padlan, E.A., Silverton, E. W., Sheriff, S., Cohen, G. H., Smith-Gill, S. J., and Davies, D. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5938-5942), was investigated. Asp-101 and Trp-62 of HEL, whose conformations are perturbed by the binding of antibody HyHEL10 in this interaction, were replaced with Gly, and the resulting interactions were studied by assay of the inhibition of the lysozyme activity with the Fv fragment and by titration calorimetry. The results can be summarized as follows. 1) It was possible to prepare the fully functional Fv fragment of HyHEL10 using a secretory expression system in Escherichia coli. Its inhibition profile for HEL activity was almost indistinguishable from that of HyHEL10 IgG, and the contribution of enthalpy to driving the interaction was shown to be significant. 2) A thermodynamic study of the interaction between the D101G mutant HEL and the Fv fragment revealed that, although the negative enthalpy change was smaller than that for the wild type, the Gibbs energy was almost identical to that of the wild type, which resulted from the smaller entropy loss. 3) Study of the interaction between the W62G mutant HEL and this Fv fragment indicated that the rotation of the Trp-62 indole ring upon binding of the antibody made an enthalpic contribution to antibody-antigen interaction, although Trp-62 of HEL was proposed not to be the direct contact residue in the HyHEL10.HEL complex. 4) From these results, it was confirmed experimentally that structural perturbations of antigenic residues upon antibody binding of antigen would contribute to the gain of enthalpic energy, in spite of partial offset by entropic loss, and to driving the interaction.
为阐明结构扰动的抗原残基在抗体与抗原-抗体相互作用结合时的作用，研究了鸡蛋清溶菌酶（HEL）与HyHEL10 Fv片段之间的相互作用，HyHEL10是几种抗HEL单克隆抗体之一，其结构已得到很好的界定（帕德兰，E.A.，西尔弗顿，E.W.，谢里夫，S.，科恩，G.H.，史密斯-吉尔，S.J.，和戴维斯，D.R.（1989年）《美国国家科学院院刊》86，5938-5942）。在这种相互作用中，HEL的Asp-101和Trp-62的构象因抗体HyHEL10的结合而受到扰动，将它们替换为甘氨酸，并通过用Fv片段抑制溶菌酶活性的测定和滴定热分析法研究由此产生的相互作用。结果可总结如下。1）使用大肠杆菌中的分泌表达系统能够制备出功能完全正常的HyHEL10 Fv片段。其对HEL活性的抑制谱与HyHEL10 IgG几乎没有区别，并且焓对驱动相互作用的贡献显示出是显著的。2）对D101G突变型HEL与Fv片段之间相互作用的热力学研究表明，尽管负焓变比野生型小，但吉布斯自由能与野生型几乎相同，这是由较小的熵损失导致的。3）对W62G突变型HEL与该Fv片段之间相互作用的研究表明，尽管在HyHEL10.HEL复合物中HEL的Trp-62不被认为是直接接触残基，但抗体结合时Trp-62吲哚环的旋转对抗体-抗原相互作用有焓贡献。4）从这些结果可以通过实验证实，抗原与抗体结合时抗原残基的结构扰动尽管会被熵损失部分抵消，但仍会有助于焓能的增加并驱动相互作用。
