Structural Insights into the Stability and Recognition Mechanism of the Antiquinalphos Nanobody for the Detection of Quinalphos in Foods.
Structural Insights into the Stability and Recognition Mechanism of the Antiquinalphos Nanobody for the Detection of Quinalphos in Foods.
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DOI：
10.1021/acs.analchem.3c01370
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发表时间：
2023-07
影响因子：
7.4
通讯作者：
Jiadong Li;Guangyao Wu;Li-hua Li-Li-hua-Li-2107973403;Lanteng Wang;Yifan Liang;Ru Fang;Qiu-Ling Zhang;Ling-Ling Xie-Ling;Xing Shen;Yu-Dong Shen;Zhenlin Xu;Hong Wang;B. Hammock
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文献类型：
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作者：
Jiadong Li;Guangyao Wu;Li-hua Li-Li-hua-Li-2107973403;Lanteng Wang;Yifan Liang;Ru Fang;Qiu-Ling Zhang;Ling-Ling Xie-Ling;Xing Shen;Yu-Dong Shen;Zhenlin Xu;Hong Wang;B. Hammock
纳米体由于其特殊的物理化学性质，在免疫分析中具有巨大的潜力。随着Nbs的不朽特性和利用蛋白质工程操纵其结构的能力，了解Nbs的高稳定性、亲和力和选择性的结构特征将变得越来越有价值。本研究以一种抗醌类化合物Nb为模型，阐述了Nbs独特理化性质的结构基础和识别机制。结果表明，nb - 11a配体复合物表现出由CDR1、CDR2和FR3形成的“隧道”结合模式。小配体的取向和疏水性是其对Nb-11A不同亲和力的主要决定因素。此外，导致Nb-11A在高温和有机溶剂中稳定性有限的主要因素是氢键网络的重排和结合腔的扩大。重要的是，活性空腔底部的Ala 97和Ala 34以及入口的Arg 29和Leu 73在半抗原识别中起着至关重要的作用，突变体Nb-F3进一步证实了这一点。因此，我们的发现有助于更深入地了解抗半抗原Nbs的识别和稳定性机制，并为新型半抗原的合理设计和定向进化产生高性能抗体提供新的思路。
Nanobodies (Nbs) have great potential in immunoassays due to their exceptional physicochemical properties. With the immortal nature of Nbs and the ability to manipulate their structures using protein engineering, it will become increasingly valuable to understand what structural features of Nbs drive high stability, affinity, and selectivity. Here, we employed an anti-quinalphos Nb as a model to illustrate the structural basis of Nbs' distinctive physicochemical properties and the recognition mechanism. The results indicated that the Nb-11A-ligand complexes exhibit a "tunnel" binding mode formed by CDR1, CDR2, and FR3. The orientation and hydrophobicity of small ligands are the primary determinants of their diverse affinities to Nb-11A. In addition, the primary factors contributing to Nb-11A's limited stability at high temperatures and in organic solvents are the rearrangement of the hydrogen bonding network and the enlargement of the binding cavity. Importantly, Ala 97 and Ala 34 at the active cavity's bottom and Arg 29 and Leu 73 at its entrance play vital roles in hapten recognition, which were further confirmed by mutant Nb-F3. Thus, our findings contribute to a deeper understanding of the recognition and stability mechanisms of anti-hapten Nbs and shed new light on the rational design of novel haptens and directed evolution to produce high-performance antibodies.