Structure-Function Relationships of Antimicrobial Piscidins 1 and 3 bound to Cholesterol-Containing Lipid Membranes

Abstract

Isolated in the mast cells of hybrid-striped sea bass, piscidin 1 (p1) and piscidin 3 (p3) are antimicrobial, cationic, and amphipathic peptides that have demonstrated broad-spectrum activity against bacteria, fungi, viruses, and cancer cells. Both p1 and p3 adopt an alpha-helical structure when bound to phospholipid membranes. p1 is the more active of the two isoforms, exhibiting higher lytic activity on different bacterial strains, as well as erythrocytes. This research uses various biophysical methods to investigate the differences in the backbone structure and bilayer location of piscidin bound to different lipid bilayers of biological relevance.To mimic the composition of human erythrocytes, a mixture of 4:1 palmitoyl-oleoyl-phosphatidylcholine (POPC):cholesterol (CHL) at pH 7.4 was used. Oriented bilayer samples prepared with 1:40 peptide:lipid were analyzed using solid-state NMR yielding 15N-1H dipolar couplings as well as 15N amide chemical shifts. These data were used to compute a high-resolution atomic-level structure of p1 and p3 bound to zwitterionic bilayers. The peptide's backbone structure is largely conserved across various bacterial and mammalian membrane mimics, suggesting that the difference in a peptide's activity on various membranes is more reliant on the peptide's side-chain and its position within the membrane.The structural results were supplemented with dye leakage assays in order to probe the peptide's activity on lipid vesicles, mimicking bacterial and mammalian cells. These results lend insight into how specific changes in peptide's structure may result in varying activity on different membrane systems. Molecular dynamics (MD) simulations were used to predict the peptide's bilayer location and depth of insertion. Membrane thinning, an event known to precede pore formation, was shown using MD. Overall, these experiments help obtain principles to design novel antibiotic pharmaceuticals with low hemolytic effects and high lytic activity on bacteria.