Contact Occupancy and Per-Residue Energetic Contributions Distinguish Aflatoxin B1 Nanobodies with Different Binding Affinities.

Aflatoxin B1 (AFB1) is a potent foodborne mycotoxin for which nanobodies offer compact, engineerable detection reagents, yet the molecular basis of their affinity differences remains poorly defined. Here, closely related nanobodies G8, NB28, and NB26 were compared. Fluorescence correlation spectroscopy showed G8 had the lowest dissociation constant for AFB1-BSA, followed by NB28 and NB26. To resolve this affinity separation, we performed triplicate 3 μs molecular dynamics simulations for each complex. While all complexes remained stable, G8 maintained broader, persistent aromatic and hydrophobic contacts extending into its elongated CDR3, whereas NB28 and NB26 relied on localized contacts centered on PHE47 and TRP101. Per-residue MM/GBSA decomposition confirmed favorable thermodynamic contributions from these persistent G8 residues. Ultimately, integrating contact occupancy, energy decomposition, and pairwise co-occurrence identified distinct cooperative contact networks (six in G8, versus two in NB28/NB26). This workflow demonstrates how mapping dynamic, cooperative binding networks rather than static proximity can systematically prioritize candidate residues for engineering high-affinity detection reagents in food-safety surveillance.