Insights into the recognition mechanism of shark-derived single-domain antibodies with high affinity and specificity targeting fluoroquinolones

In this study, we investigated the molecular recognition mechanisms of shark-derived single-domain antibodies (ssdAbs) targeting fluoroquinolones using an integrated approach that combines in silico homologous modeling, molecular dynamics simulations, molecular docking, and alanine scanning mutagenesis. Three ssdAbs—2E6, 1N9, and 1O17—specific to enrofloxacin, norfloxacin, and ofloxacin, respectively, were selected based on previous work. Through AlphaFold2 and GalaxyWEB, the protein structures of these ssdAbs were predicted and optimized, followed by molecular dynamics simulations to emulate realistic protein behavior in a solvent environment. Molecular docking, alanine scanning mutagenesis, and subsequent verifications identified 30N and 93W of 2E6; 30N, 89R, 98Y, and 99D of 1N9; 100W and 101R of 1O17, all located within the complementarity determining region 3 loop, as critical for antigen binding. These residues primarily interact with their targets through hydrogen bonds, salt bridges, π–π stackings, and cation–π interactions. This study revealed, for the first time, the binding mechanism of ssdAbs to fluoroquinolones from a theoretical perspective, emphasizing the importance of aromatic and polar residues in recognizing characteristic epitopes, such as the carboxyl group at the C3 position and the 1-piperazinyl group at the C7 position. Our findings provide valuable insights for the rational design and enhancement of ssdAbs for detecting small molecule hazards in aquaculture.