C-terminal–mediated modulation of Holin function from bacteriophage K: implications for phage-derived antimicrobial design

Among phage-derived antimicrobial molecules, holins are membrane-active proteins that play a central role in bacterial lysis; however, the contribution of their individual structural domains to antibacterial activity remains poorly understood. In this study, we investigated the functional role of the extended C-terminal region of the phage K holin protein and its contribution to antibacterial activity. Structural predictions and molecular simulations were combined with recombinant protein expression and synthetic peptide production to evaluate the individual and cooperative roles of the transmembrane domain (TMD) and C-terminal segments. Our findings demonstrate that recombinant Holin (rHolin) and rTMD disrupt bacterial growth, induce membrane lysis in both intracellular expression and external application assays. Among the synthetic peptides, only pIII displayed minimal activity when applied externally (MIC, 1000 µg/mL), highlighting the essential synergistic role of the TMD core in lytic activity. Among the recombinant derivatives, rTMD-pI showed the most favorable antibacterial profile (20–40 µg/mL). Growth inhibition, membrane integrity analyses, and computational simulations consistently indicated that antibacterial activity depends on the coordinated action of the TMD and C-terminal regions rather than the C-terminus alone. Molecular modeling further suggested that C-terminal interactions promote conformational rearrangements and localized membrane destabilization that facilitate pore formation. Low cytotoxicity toward human fibroblasts was observed within the tested concentration range. Overall, these findings provide new insights into the structure–function relationships underlying holin-mediated antibacterial activity. MD simulations reveal local membrane thinning and C-terminal folding–driven perturbations. Recombinant Holin expression triggers early bacterial lysis, limiting production yields. rHolin derivatives show selective anti-S. aureus activity with low human cytotoxicity.