Determining the Structures and Dynamics of Antimicrobial Piscidins 1 and 3 Bound to Cholesterol-Containing Lipid Membranes using Solid-State NMR Techniques

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, and virally infected cells. Both p1 and p3 adopt an alpha-helical structure when bound to phospholipid membranes. Interestingly, p1 is the more active of the two isoforms, exhibiting higher lytic activity on different bacterial strains, as well as erythrocytes. This research uses solid-state NMR to investigate the differences in structure and dynamics of p1 and p3 bound to different lipid bilayers of biological relevance.Our current study aims at elucidating both the structure and dynamics of p1 and p3 in mammalian mimetic membranes containing cholesterol. Here, we use 4:1 palmitoyl-oleoyl-phosphatidylcholine (POPC):cholesterol (CHL) at a pH of 7.4, to closely mimic the composition of human erythrocytes at physiological pH. Oriented bilayer samples were prepared with 1:30 peptide:lipid and were analyzed using high-resolution solid-state NMR that yielded an 15N-1H dipolar coupling as well as 15N and 1H chemical shifts for the amide NH bond of each residue. Together, these data can be used to compute high-resolution atomic structures of the two isoforms when bound to the surface of the bilayer. Dynamics of p1 and p3 have also been investigated using one-dimensional 15N solid-state NMR. Differences in structure and dynamics for p1 and p3 when they are bound to mammalian and bacterial lipid systems provide the basis for identifying structural moieties of the peptides that are directly related to their respective activities. Enhancing our understanding of how these peptides interact with different lipid systems will aid in the design of novel antibiotic pharmaceuticals that exhibit low hemolytic effects, while maximizing inhibitory activity on bacteria.