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Abstract Amphipathic α-helical peptides (AHPs) have shown potential as a therapeutic approach against multi-drug-resistant bacterial infections due to their broad-spectrum antimicrobial activity by disrupting bacterial membranes. However, their nonspecific interactions with membranes often result in cytotoxicity toward mammalian cells. Previous studies have shown that a PxxP motif near the middle of cathelicidin-derived antimicrobial peptides contributes to potent and selective antibacterial activity. In this study, we compared KL18 with KL-PxxP to examine the effects of the central PxxP motif in AHPs on their structure, antibiotic activity, and mode of action. In a membrane-mimetic environment, we observed that KL18 had a much higher helical content compared to KL-PxxP. In aqueous buffer, KL18 adopted a highly ordered α-helical conformation, while KL-PxxP exhibited a disordered conformation. We found that KL-PxxP exhibited 4–16 times higher antibacterial activity than KL18 and significantly reduced the hemolytic activity. These findings suggest that the dynamic conformational behaviors caused by the central PxxP motif conferred the antibacterial selectivity of AHPs. Additionally, KL-PxxP showed strong binding to anionic liposomes and weak binding to zwitterionic liposomes, explaining its selectivity for bacteria over mammalian cells. Despite having a low ability to dissipate the bacterial membrane potential, KL-PxxP translocated efficiently across lipid membranes. Therefore, we propose that the central PxxP motif in AHPs provides dynamic conformational behavior in aqueous and membrane-mimetic environments, enhances binding to anionic membranes, and facilitates translocation across lipid bilayers, resulting in improved antibacterial potency and selectivity. Understanding the unique structural characteristics and functional roles of the PxxP motif in the antimicrobial mechanism of action holds great potential for advancing the development of novel peptide antibiotics.

Abstract Antimicrobial peptides (AMPs) that selectively permeabilize bacterial membranes are promising alternatives to conventional antibiotics. Dimerization of AMP is considered an attractive strategy to enhance antimicrobial and membrane-lytic activity, but it also increases undesired hemolytic and cytotoxic activity. Here, we prepared Lys-linked homodimers of membrane-permeabilizing magainin II and cell-penetrating buforin II. Dimerization did not significantly alter conformational behavior, but it had a substantial impact on antimicrobial properties. We found that while the magainin II dimer showed increased antimicrobial and cytotoxic effects, the buforin II dimer conferred much greater antibacterial potency without exhibiting cytotoxic activity. Interestingly, the buforin II dimer was highly effective against several antibiotic-resistant bacterial isolates. Membrane permeabilization experiments indicated that the magainin II dimer rapidly disrupted both anionic and zwitterionic membranes, whereas the buforin II dimer selectively disrupted anionic membranes. Like the monomeric form, the buforin II dimer was efficiently translocated across lipid bilayers. Therefore, our results suggest that the dimerization of cell-penetrating buforin II not only disrupts the bacterial membrane, but also translocates it across the membrane to target intracellular components, resulting in effective antimicrobial activity. We propose that dimerization of intracellular targeting AMPs may present a superior strategy for therapeutic control of pathogenic bacteria.