Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA.
J Struct Biol. 2014 Feb;185(2):215-22. doi: 10.1016/j.jsb.2013.05.003. Epub 2013 May 13.
Engineering antibodies to utilize non-canonical amino acids (NCAA) should greatly expand the utility of an already important biological reagent. In particular, introducing crosslinking reagents into antibody complementarity determining regions (CDRs) should provide a means to covalently crosslink residues at the antibody-antigen interface. Unfortunately, finding the optimum position for crosslinking two proteins is often a matter of iterative guessing, even when the interface is known in atomic detail. Computer-aided antibody design can potentially greatly restrict the number of variants that must be explored in order to identify successful crosslinking sites. We have therefore used Rosetta to guide the introduction of an oxidizable crosslinking NCAA, l-3,4-dihydroxyphenylalanine (l-DOPA), into the CDRs of the anti-protective antigen scFv antibody M18, and have measured crosslinking to its cognate antigen, domain 4 of the anthrax protective antigen. Computed crosslinking distance, solvent accessibility, and interface energetics were three factors considered that could impact the efficiency of l-DOPA-mediated crosslinking. In the end, 10 variants were synthesized, and crosslinking efficiencies were generally 10% or higher, with the best variant crosslinking to 52% of the available antigen. The results suggest that computational analysis can be used in a pipeline for engineering crosslinking antibodies. The rules learned from l-DOPA crosslinking of antibodies may also be generalizable to the formation of other crosslinked interfaces and complexes.
利用非天然氨基酸（NCAA）来工程化抗体将极大地扩展这一重要生物试剂的应用范围。特别是，将交联试剂引入抗体互补决定区（CDR）应该提供一种在抗体-抗原界面上共价交联残基的方法。不幸的是，即使已知界面的原子细节，找到交联两个蛋白质的最佳位置通常也是反复猜测的问题。计算机辅助抗体设计有可能大大限制为确定成功交联位点而必须探索的变体数量。因此，我们使用 Rosetta 将可氧化交联 NCAA，L-3,4-二羟基苯丙氨酸（L-DOPA），引入抗保护性抗原 scFv 抗体 M18 的 CDR 中，并测量了其与同源抗原炭疽保护性抗原结构域 4 的交联。计算的交联距离、溶剂可及性和界面能是三个可能影响 L-DOPA 介导交联效率的因素。最终，合成了 10 个变体，交联效率通常在 10%或更高，最佳变体可交联到 52%的可用抗原。结果表明，计算分析可用于交联抗体的工程设计。从抗体的 L-DOPA 交联中获得的规则也可能适用于形成其他交联界面和复合物。
