C-Terminal Amidation: Structural Insights into Enhanced Antimicrobial Peptide Efficacy and Amyloidogenesis

Post-translational modifications, such as C-terminal amidation, are energetically costly but critical for membrane-active antimicrobial peptides (AMPs). This study examined the impact of C-terminal amidation on uperin 3.5 (U3.5, an amyloid-forming AMP) interactions with POPE:DOPG (3:1) lipid bilayers using molecular simulations. Whereas monomeric U3.5-NH₂ rapidly attached to the lipid bilayer surface, forming a stable α-helix, U3.5-OH exhibited weaker interactions. Simulations of U3.5 tetramers, derived from the amyloid cryoelectron microscopy structure, revealed that amidation enhanced peptide-bilayer and peptide-peptide interactions, initially stabilizing β-sheet structures and facilitating embedding into the bilayer. The β-sheet tetramer gradually dissociated into monomeric U3.5-NH₂ peptides on the bilayer surface. Following dissociation, the monomers formed stable, amphipathic α-helices that were strongly embedded in the bilayer, consistent with a carpet-like antimicrobial mechanism. Enhanced peptide-lipid interactions resulted in lipid redistribution and changes to membrane curvature, potentially leading to membrane rupture. These findings highlight the role of U3.5-NH₂ amyloid as a "carrier vehicle" for antimicrobial action at the bilayer interface, emphasizing the crucial function of C-terminal amidation in stabilizing peptide structure and promoting antimicrobial activity.