Subtype-selective binding of milk β-casomorphins across the opioid receptor family revealed by molecular docking and molecular dynamics simulation.

β-Casomorphins (BCM) are opioid-like peptides produced during the digestion of β-casein in the gastrointestinal tract. They are also involved in other gastrointestinal and neurophysiological processes. Although the effect of BCM7 on the μ-opioid receptor (MOR) has been extensively studied, the molecular interaction mechanisms of other BCM with different opioid receptors have not been elucidated. This study aimed to investigate the receptor-specific binding behavior of BCM3-BCM11 across the μ-, δ-, κ-, and nociceptin/orphanin FQ peptide (NOP) opioid receptors using structure-based computational approaches. Three-dimensional structures of BCM peptides were generated and subjected to a peptide docking analysis for each opioid receptor. Comparative analyses of docking scores and binding modes were performed to identify receptor-preferred BCM variants. The selected high-affinity complexes for each receptor were further evaluated using molecular dynamics simulations to assess their structural stability and dynamic behavior. Protein-ligand contact analyses were conducted to characterize the residue-level interaction patterns and interaction persistence over time. Docking analysis revealed pronounced receptor- and peptide-dependent binding profiles. The BCM7 exhibited the strongest affinity for MOR, consistent with previous reports. Meanwhile, other receptors showed a preference for distinct BCM variants, including BCM6, BCM9, BCM10, and BCM11, depending on the receptor subtype. Molecular dynamics simulations indicated that the selected BCM-receptor complexes reached stable conformational states after equilibration. Hydrogen bonding, hydrophobic interactions, and water-mediated contacts collectively contribute to peptide stabilization; notable differences are observed in contact composition among receptor subtypes, reflecting subtype-specific recognition features. Overall, this study provides a comparative molecular framework for understanding how structurally diverse BCM interact with members of the opioid receptor family. These findings demonstrate that BCM7 dominance is largely MOR-specific and that other BCM variants may preferentially engage non-MOR opioid receptors. These results highlight the importance of looking beyond BCM7 and considering both peptide diversity and receptor subtype specificity when investigating BCM-mediated signaling mechanisms.