Guangdong Provincial Key Laboratory of Food Quality and Safety, National-Local Joint Engineering Research Center for Processing and Safety Control of Livestock and Poultry Products, College of Food Science, South China Agricultural University, Guangzhou 510642, China.
Future Technology Institute, South China Normal University, Guangzhou 510631, China.
Anal Chem. 2023 Aug 1;95(30):11306-11315. doi: 10.1021/acs.analchem.3c01370. Epub 2023 Jul 10.
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.
纳米抗体（Nbs）因其独特的物理化学性质，在免疫分析中具有巨大的潜力。由于 Nbs 的不朽性质和使用蛋白质工程操纵其结构的能力，了解哪些结构特征驱动 Nbs 的高稳定性、亲和力和选择性将变得越来越有价值。在这里，我们使用抗克百威 Nb 作为模型来说明 Nbs 独特的物理化学性质和识别机制的结构基础。结果表明，Nb-11A-配体复合物呈现由 CDR1、CDR2 和 FR3 形成的“隧道”结合模式。小分子配体的取向和疏水性是决定它们与 Nb-11A 不同亲和力的主要因素。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，活性腔底部的 Ala97 和 Ala34 以及入口处的 Arg29 和 Leu73 在半抗原识别中起着重要作用，这一点通过突变体 Nb-F3 进一步得到证实。因此，我们的研究结果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产生高性能抗体的定向进化提供新的思路。
