Formation Mechanism and Antibacterial Activity of Natural Antimicrobial Lysozyme with Antibiotics Doxycycline and Tigecycline.

The combination of lysozyme with antibiotics has emerged as a promising strategy to combat bacterial resistance; however, the lack of mechanistic understanding regarding their molecular-level interactions remains a critical knowledge gap, hindering the rational design of optimized synergistic therapies. Through integrated spectroscopic techniques (FTIR, CD, UV-vis, fluorescence), thermodynamic analyses, molecular docking, and 200-ns molecular dynamics simulation, we established that doxycycline/tigecycline bind lysozyme via moderate-affinity hydrophobic interactions (Ka ∼ 104 L mol-1) but with distinct binding patterns-tigecycline forms additional hydrogen bonds through its glycylamino side chain, yielding higher stability and lower free drug fraction (fu) compare to doxycycline. Additionally, doxycycline and tigecycline have a relatively mild effect on lysozyme conformation, but doxycycline has a greater impact on lysozyme conformation than tigecycline. Key findings reveal a paradoxical efficacy relationship: while doxycycline exhibits stronger intrinsic antibacterial activity, the lysozyme-tigecycline complex demonstrates synergistic enhancement (particularly against E. coli) through stable binding-mediated protection and optimized release kinetics, whereas lysozyme-doxycycline's weaker binding leads to premature release and reduced activity. These results fundamentally advance protein-drug interaction paradigms by demonstrating that optimal therapeutic outcomes require balanced binding stability, with broader implications for designing lysozyme-based delivery systems where moderately strong interactions enable synergistic effects while preserving structural integrity, offering a template for developing combination therapies against antimicrobial resistance.