Effect of central PxxP motif in amphipathic alpha-helical peptides on antimicrobial activity and mode of action

Abstract

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.

Keywords

• Amphipathic alpha-helical peptides
• Antimicrobial activity
• PXXP motif
• Helix-hinge-helix
• Peptide-membrane interaction