Zhou Tao, Zhang Huiling, Yu Xiaoting, Pan Kangliang, Yao Xiaojun, Shen Xing, Lei Hongtao
Guangdong Provincial Key Laboratory of Food Quality and Safety and Nation-Local Joint Engineering Research Center for Machining and Safety of Livestock and Poultry Products, South China Agricultural University, Guangzhou 510642, China.
College of Mathematics and Informatics & College of Software Engineering, South China Agricultural University, Guangzhou 510642, China.
Foods. 2025 Apr 15;14(8):1360. doi: 10.3390/foods14081360.
Allostery is a fundamental biological phenomenon that occurs when a molecule binds to a protein's allosteric site, triggering conformational changes that regulate the protein's activity. However, allostery in antibodies remains largely unexplored, and only a few reports have focused on allostery from antigen-binding fragments (Fab) to crystallizable fragments (Fc). But this study, using anti-phenobarbital antibodies-which are widely applied for detecting the potential health food adulterant phenobarbital-as a model and employing multiple computational methods, is the first to identify a cyclopeptide (cyclo[Link-M-WFRHY-K]) that induces allostery from Fc to Fab in antibody and elucidates the underlying antibody allostery mechanism. The combination of molecular docking and multiple allosteric site prediction algorithms in these methods identified that the cyclopeptide binds to the interface of heavy chain region-1 (CH) in antibody Fab and heavy chain region-2 (CH) in antibody Fc. Meanwhile, molecular dynamics simulations combined with other analytical methods demonstrated that cyclopeptide induces global conformational shifts in the antibody, which ultimately alter the Fab domain and enhance its antigen-binding activity from Fc to Fab. This result will enable cyclopeptides as a potential Fab-targeted allosteric modulator to provide a new strategy for the regulation of antigen-binding activity and contribute to the construction of novel immunoassays for food safety and other applications using allosteric antibodies as the core technology. Furthermore, graph theory analysis further revealed a common allosteric signaling pathway within the antibody, involving residues Q123, S207, S326, C455, A558, Q778, D838, R975, R1102, P1146, V1200, and K1286, which will be very important for the engineering design of the anti-phenobarbital antibodies and other highly homologous antibodies. Finally, the non-covalent interaction analysis showed that allostery from Fc to Fab primarily involves residue signal transduction driven by hydrogen bonds and hydrophobic interactions.
变构是一种基本的生物学现象，当一个分子与蛋白质的变构位点结合时就会发生，从而引发构象变化，进而调节蛋白质的活性。然而，抗体中的变构现象在很大程度上仍未得到充分探索，仅有少数报告关注了从抗原结合片段（Fab）到可结晶片段（Fc）的变构。但本研究以广泛应用于检测潜在健康食品掺假物苯巴比妥的抗苯巴比妥抗体为模型，并采用多种计算方法，首次鉴定出一种环肽（环[Link-M-WFRHY-K]），该环肽可诱导抗体中从Fc到Fab的变构，并阐明了潜在的抗体变构机制。这些方法中分子对接与多种变构位点预测算法相结合，确定该环肽与抗体Fab中的重链区域1（CH）和抗体Fc中的重链区域2（CH）的界面结合。同时，分子动力学模拟结合其他分析方法表明，环肽可诱导抗体发生全局构象变化，最终改变Fab结构域并增强其从Fc到Fab的抗原结合活性。这一结果将使环肽作为一种潜在的靶向Fab的变构调节剂，为调节抗原结合活性提供新策略，并有助于构建以变构抗体为核心技术的用于食品安全及其他应用的新型免疫测定方法。此外，图论分析进一步揭示了抗体内部一条共同的变构信号通路，涉及残基Q123、S207、S326、C455、A558、Q778、D838、R975、R1102、P1146、V1200和K1286，这对于抗苯巴比妥抗体及其他高度同源抗体的工程设计非常重要。最后，非共价相互作用分析表明，从Fc到Fab的变构主要涉及由氢键和疏水相互作用驱动的残基信号转导。
