Folded Structure and Insertion Depth of the Frog-Skin Antimicrobial Peptide Esculentin-1b(1–18) in the Presence of Differently Charged Membrane-Mimicking Micelles

Antimicrobial peptides (AMPs) are effectors of the innate immunity of most organisms. Their role in the defense against pathogen attack and their high selectivity for bacterial cells make them attractive for the development of a new class of antimicrobial drugs. The N-terminal fragment of the frog-skin peptide esculentin-1b (Esc(1–18)) has shown broad-spectrum antimicrobial activity. Similarly to most cationic AMPs, it is supposed to act by binding to and damaging the negatively charged plasma membrane of bacteria. Differently from many other AMPs, Esc(1–18) activity is preserved in biological fluids such as serum. In this work, a structural investigation was performed through NMR spectroscopy. The 3D structure was obtained in the presence of either zwitterionic or negatively charged micelles as membrane models for eukaryotic and prokaryotic membranes, respectively. Esc(1–18) showed a higher affinity for and deeper insertion into the latter and adopted an amphipathic helical structure characterized by a kink at the residue G8. These findings were confirmed by measuring penetration into lipid monolayers. The presence of negatively charged lipids in the bilayer appears to be necessary for Esc(1–18) to bind, to fold in the right three-dimensional structure, and, ultimately, to exert its biological role as an AMP

Circular Dichroism analysis.

<p><b>(A)</b> Conventional CD spectra of both SB056-lin and β-SB056-lin in the presence of POPC/POPG (1/1 mol/mol) SUVs. <b>(B)</b> SRCD spectra of both SB056-lin and β-SB056-lin in the presence of POPC/POPG (1/1 mol/mol) SUVs. SRCD spectra of <b>(C)</b> SB056-lin and <b>(D)</b> β-SB056-lin in the presence of differently charged SUVs.</p

MIC values of SB056-lin and β-SB056-lin against two Gram-negative and two Gram-positive bacterial strains.

<p>MIC values of SB056-lin and β-SB056-lin against two Gram-negative and two Gram-positive bacterial strains.</p

Summary of peptide-lipid interactions.

<p>The proportion of anionic lipids in the vesicles is increased from top to bottom. The behavior of the original SB056-lin peptide is represented on the left hand side, and the sequence-optimized β-SB056-lin on the right. Black arrows of different length and thickness are used to indicate the different binding equilibria. SB056-lin binds only to anionic bilayers, and in a not so well-ordered β-stranded conformation. The sequence optimized β-SB056-lin, on the other hand, forms regular β-strands that self-assemble into extended β-sheets when the negative charge of the bilayer exceeds electro-neutrality of the peptide-lipid system.</p

Schematic representation of the peptides.

<p><b>(A)</b> Original SB056-lin, and <b>(B)</b> sequence-optimized β-SB056-lin. Yellow circles indicate hydrophobic residues, blue ones positively charged amino acids, and cyan indicates the polar serine residue.</p