Carboxy-Amidated AamAP1-Lys has Superior Conformational Flexibility and Accelerated Killing of Gram-Negative Bacteria

C-terminal amidation of antimicrobial peptides (AMPs) is a frequent minor modification used to improve antibacterial potency, commonly ascribed to increased positive charge, protection from proteases, and a stabilized secondary structure. Although the activity of AMPs is primarily associated with the ability to penetrate bacterial membranes, hitherto the effect of amidation on this interaction has not been understood in detail. Here, we show that amidation of the scorpion-derived membranolytic peptide AamAP1-Lys produces a potent analog with faster bactericidal activity, increased membrane permeabilization, and greater Gram-negative membrane penetration associated with greater conformational flexibility. AamAP1-lys-NH₂ has improved antibiofilm activity against Acinetobacter baumannii and Escherichia coli, benefits from a two- to 3-fold selectivity improvement, and provides protection against A. baumannii infection in a Galleria mellonella burn wound model. Circular dichroism spectroscopy shows both peptides adopt α-helix conformations in the steady state. However, molecular dynamics (MD) simulations reveal that, during initial binding, AamAP1-Lys-NH₂ has greater conformation heterogeneity, with substantial polyproline-II conformation detected alongside α-helix, and penetrates the bilayer more readily than AamAP1-Lys. AamAP1-Lys-NH₂ induced membrane permeabilization of A. baumannii occurs only above a critical concentration with slow and weak permeabilization and slow killing occurring at its lower MIC but causes greater and faster permeabilization than AamAP1-Lys, and kills more rapidly, when applied at equal concentrations. Therefore, while the increased potency of AamAP1-Lys-NH₂ is associated with slow bactericidal killing, amidation, and the conformational flexibility it induces, affords an improvement in the AMP pharmacodynamic profile and may need to be considered to achieve improved therapeutic performance.