Structural Biochemistry, Bijvoet Centre for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, the Netherlands.
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands.
J Mol Biol. 2024 Aug 15;436(16):168649. doi: 10.1016/j.jmb.2024.168649. Epub 2024 Jun 8.
The FLAG-tag/anti-FLAG system is a widely used biochemical tool for protein detection and purification. Anti-FLAG M2 is the most popular antibody against the FLAG-tag, due to its ease of use, versatility, and availability in pure form or as bead conjugate. M2 binds N-terminal, C-terminal and internal FLAG-tags and binding is calcium-independent, but the molecular basis for the FLAG-tag specificity and recognition remains unresolved. Here we present an atomic resolution (1.17 Å) structure of the FLAG peptide in complex with the Fab of anti-FLAG M2, revealing key binding determinants. Five of the eight FLAG peptide residues form direct interactions with paratope residues. The FLAG peptide adopts a 3 helix conformation in complex with the Fab. These structural insights allowed us to rationally introduce point mutations on both the peptide and antibody side. We tested these by surface plasmon resonance, leading us to propose a shorter yet equally binding version of the FLAG-tag for the M2 antibody.
FLAG 标签/抗 FLAG 系统是一种广泛用于蛋白质检测和纯化的生化工具。抗 FLAG M2 是针对 FLAG 标签最常用的抗体，因其易于使用、多功能性以及可获得纯形式或珠状缀合物而备受青睐。M2 结合 N 端、C 端和内部 FLAG 标签，结合不依赖于钙，但 FLAG 标签特异性和识别的分子基础仍未解决。在这里，我们展示了 FLAG 肽与抗 FLAG M2 的 Fab 复合物的原子分辨率（1.17 Å）结构，揭示了关键的结合决定因素。FLAG 肽的 8 个残基中有 5 个与互补位残基形成直接相互作用。FLAG 肽在与 Fab 结合时采用 3 螺旋构象。这些结构见解使我们能够在肽和抗体侧合理引入点突变。我们通过表面等离子体共振进行了测试，这使我们提出了针对 M2 抗体的更短但同样具有结合能力的 FLAG 标签版本。
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