Impact of alanine mutations on buffalo nasal odorant binding protein–ligand interactions: implications for estrus biosensing

Buffaloes are economically important livestock; however, their reproductive efficiency remains low due to poor estrus detection, leading to missed opportunities for artificial insemination. Buffaloes release volatile pheromonal cues during estrus, which are recognized by nasal odorant-binding proteins (OBPs). Taking advantage of this mechanism, buffalo nasal odorant-binding protein (BunOBP) can be utilized as a biosensor element for estrus detection. In this study, a three-dimensional model of BunOBP was constructed and key binding-site residues (N55, F98, E99, Y118, and E131) were systematically mutated to alanine to evaluate their role in ligand binding. Molecular docking identified E131A_2879 (E131A with pCresol) and ASM_445070 (All-Site Mutation with Farnesol) as top-scoring complexes. These complexes were further investigated using 200 ns molecular dynamic simulations. The ASM_445070 complex exhibited superior stability, with an average RMSD of 1.04 ± 0.10 nm, stable radius of gyration (∼1.5–1.6 nm), and consistent solvent-accessible surface area (∼97.68 nm2). The less stable complexes showed ligand dissociation characterized by high ligand RMSD, loss of hydrogen bonds, and increased protein–ligand separation. Hydrogen bond analysis revealed sustained interactions in ASM_445070, supporting its strong binding affinity. Replicate simulations confirmed the reproducibility of these observations. Overall, the results demonstrate that ASM_445070 forms a stable protein-ligand complex, and represents a promising candidate for developing future OBP-based biosensors for estrus detection in buffaloes.