Structure and dynamics of the anti-AMCV scFv(F8): Effects of selected mutations on the antigen combining site
Structure and dynamics of the anti-AMCV scFv(F8): Effects of selected mutations on the antigen combining site
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DOI：
10.1016/j.jsb.2008.06.013
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发表时间：
2008-10-01
影响因子：
3
通讯作者：
Rosato, Vittorio
中科院分区：
文献类型：
--
作者：
Arcangeli, Caterina;Cantale, Cristina;Rosato, Vittorio
重组抗体片段scFv（F8）识别植物病毒AMCV的外壳蛋白，具有高的体外稳定性和在还原环境下的功能性折叠，适合设计具有所需性能的稳定抗体。突变和功能分析表明，在V-H链的47和58位有两个残基，在抗原结合识别中起着至关重要的作用。在这里，我们使用计算程序来评估这些突变对抗原结合位点的稳定性、结构和动力学的影响。通过同源性建模建立野生型scFv（F8）及其H47和H58突变体的结构模型，并进行15.5 ns的多次分子动力学模拟评估。计算结果表明，47H取代强烈影响CDR-H-2构象，使V-H/V-L界面不稳定，并使抗原结合位点具有高构象灵活性，导致突变体功能丧失。H58位置的突变加强了结合位点，赋予突变体高抗原特异性。本质动力学和对蛋白质-溶剂界面的分析进一步证实了由结构决定的结合位点柔韧性的程度与野生型及其突变体所证明的不同功能行为之间的对应关系。这些结果对基于结构的抗体结合位点设计具有重要的指导意义。(c) 2008爱思唯尔公司版权所有。
The recombinant antibody fragment scFv(F8), which recognizes the coat protein of the plant virus AMCV, is characterized by peculiar high in vitro stability and functional folding even in reducing environments, making it fit for designing stable antibodies with desired properties. Mutagenesis and functional analysis evidenced two residues, at positions 47 and 58 of the V-H chain, playing a crucial role in the antigen binding recognition.Here, we used a computational procedure to assess the effects of these mutations on the stability, structure and dynamics of the antigen-binding site. Structural models of the wild type scFv(F8) and of its H47 and H58 mutants were built by homology modelling and assessed by multiple 15.5 ns of molecular dynamics simulations. Computational results indicate that the 47H substitution strongly affects the CDR-H-2 conformation, destabilizes the V-H/V-L interface and confers high conformational flexibility to the antigen-binding site, leading the mutant to functional loss. The mutation at position H58 strenghtens the binding site, bestowing a high antigen specificity on the mutant. The essential dynamics and the analysis of the protein-solvent interface further corroborate the correspondence between the extent of the structurally-determined flexibility of the binding site with the different functional behaviours proved by the wild-type and its mutants. These results may have useful implications for structure-based design of anti-body combining site. (c) 2008 Elsevier Inc. All rights reserved.