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

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.