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<p>Nanobodies (Nbs) have shown great potential for use in immunoassays targeting small-molecule contaminants in food safety monitoring. However, a limited understanding of their recognition mechanisms has hindered the development of high-performance Nbs and the improvement of assay performance. Herein, a previously developed nanobody (Nb) 3F9 against tenuazonic acid (TeA) was selected as a model to resolve its X-ray crystal structure. Notably, Nb3F9 adopts a typical immunoglobulin fold, with TeA deeply inserted into the complementary-determining region 3 (CDR3) and buried in a binding pocket formed by Phe37, Ser99, Tyr107, Arg110, Asp112, Met113, Asp114, Pro115, Arg117, and Gly118. Based on this insight, integrating computational prediction with site-directed mutagenesis, a mutant Nb3F9-Y107K was obtained, achieving an 8.6-fold increase in sensitivity while maintaining excellent stability and high specificity compared with the wild-type. It is believed that this work provides a rational framework for improving the binding activity of Nbs and expanding their applications in food safety.</p> Graphical abstract <p></p>

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Structural basis of a CDR3-embedded binding mechanism in a nanobody for sensitivity enhancement toward tenuazonic acid

  • 期刊论文

  • 发表日期：

    2026年5月28日

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  • Xiao-Feng Wei,
  • Ru-Yu Fang,
  • Cheng-Long Wang,
  • Yi-Fan Liang,
  • Jia-Dong Li,
  • Lan-Teng Wang,
  • Yu-Dong Shen,
  • Zhen-Lin Xu,
  • Zhi-Li Xiao,
  • Rudolf J. Schneider,
  • Hong Wang

摘要

Nanobodies (Nbs) have shown great potential for use in immunoassays targeting small-molecule contaminants in food safety monitoring. However, a limited understanding of their recognition mechanisms has hindered the development of high-performance Nbs and the improvement of assay performance. Herein, a previously developed nanobody (Nb) 3F9 against tenuazonic acid (TeA) was selected as a model to resolve its X-ray crystal structure. Notably, Nb3F9 adopts a typical immunoglobulin fold, with TeA deeply inserted into the complementary-determining region 3 (CDR3) and buried in a binding pocket formed by Phe37, Ser99, Tyr107, Arg110, Asp112, Met113, Asp114, Pro115, Arg117, and Gly118. Based on this insight, integrating computational prediction with site-directed mutagenesis, a mutant Nb3F9-Y107K was obtained, achieving an 8.6-fold increase in sensitivity while maintaining excellent stability and high specificity compared with the wild-type. It is believed that this work provides a rational framework for improving the binding activity of Nbs and expanding their applications in food safety.

Graphical abstract