Roberts V A, Getzoff E D
Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037.
FASEB J. 1995 Jan;9(1):94-100. doi: 10.1096/fasebj.9.1.7821766.
Metal-binding sites were designed within the antigen-binding pocket of the catalytic antibody 43C9 based on a 3-dimensional antibody model and crystallographic structures of Zn-binding metalloenzymes. These tetrahedral Zn-binding sites were designed to mimic both secondary and tertiary structural characteristics of catalytic metal sites in enzymes. Each site was planned to have two His ligands across from each other on adjacent antiparallel beta-strands. Sites were selected to sequester the metal ion from bulk solvent and place an open metal coordination position next to the antigen or potential substrates. Three distinct metal-site designs, with ligands in the variable light domain, in the variable heavy domain, and in both domains, were later implemented experimentally and shown spectroscopically to bind metal ions as predicted. These results demonstrate the success of our design approach, the versatility of the antibody structure for metalloprotein design, and the validity of the 3-dimensional model. The ability to predictably design multiple metal sites in the ordered antigen-recognition region at the bottom of the pocket allows tuning of metal ion placement and enhances the likelihood of interaction with putative substrates.
基于三维抗体模型和锌结合金属酶的晶体结构，在催化抗体43C9的抗原结合口袋内设计了金属结合位点。这些四面体锌结合位点旨在模拟酶中催化金属位点的二级和三级结构特征。每个位点计划在相邻的反平行β链上彼此相对地具有两个组氨酸配体。选择这些位点以从大量溶剂中螯合金属离子，并在抗原或潜在底物旁边放置一个开放的金属配位位置。后来通过实验实施了三种不同的金属位点设计，配体分别位于可变轻链结构域、可变重链结构域以及两个结构域中，并通过光谱学证明其能如预测的那样结合金属离子。这些结果证明了我们设计方法的成功、抗体结构在金属蛋白设计中的多功能性以及三维模型的有效性。在口袋底部有序的抗原识别区域中可预测地设计多个金属位点的能力，使得能够调整金属离子的位置，并增加与假定底物相互作用的可能性。
