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

Abstract

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, fold in the right threedimensional structure and, ultimately, to exert its biological role as an AMP. 3The lack of a new class of antibiotics is an urgent problem, as many important pathogens are rapidly developing resistance to most of the clinically usable antibiotics. 1,2 Clinical impact of antibiotic resistance is immense, with increasing length of hospital stay, costs and mortality. 3,4 Antimicrobial peptides (AMPs) have attracted the interest of the scientific community as promising candidates for the development of a new class of antimicrobial drugs. They are part of the innate immune system of the majority of the multicellular organisms, and are typically characterized by a wide spectrum of antimicrobial activity, from Gram-negative to Gram-positive bacteria, fungi, protozoa and enveloped viruses. [5][6][7] Despite amino acid sequence homology is not high, AMPs share some common features. They are relatively short (< 60 residues), cationic, have a large content (≥ 50%) of hydrophobic residues and adopt an amphipathic three-dimensional folding when interacting with pathogens" membranes. 6 Generally, AMPs destroy or permeate the microbial plasma-membrane. The membrane is formed through evolutionarily well conserved biosynthetic processes and represents a non-selective target, making modifications by the microorganisms difficult and, thus, resistance onset is unlikely. 8 Several recent articles have reviewed in detail the different modes of action postulated for AMPs. [9][10][11] The amphipathic conformation adopted by the AMP, with the hydrophilic residues interacting with the polar head groups of the phospholipids and the water molecules, and the hydrophobic residues being protected from water contacts by inserting into the lipid bilayer core, is considered essential for it to exert the antimicro...

Frog Skin Innate Immune Defences: Sensing and Surviving Pathogens

“…The native structure and biochemistry of frog AMPs is particularly important as it dictates AMP function (108) and allows for intrinsic interactions with anionic membranes, such as those found on bacteria, fungi, viruses, and parasites (109). The association of the frog skin AMPs with anionic membranes, and the mechanisms by which they disrupt membrane integrity, are well-studied (110–114). The mechanisms responsible for disrupting membrane integrity are heavily influenced by lipid composition (111, 115, 116), and include lipid flip-flop, leakage, or transmembrane integration (111, 115, 117).…”

d-Amino acids incorporation in the frog skin-derived peptide esculentin-1a(1-21)NH2 is beneficial for its multiple functions

“…Solution-state NMR experiments in the presence of anisotropic membrane models, i.e., the negatively charged sodium dodecylsulfate/dodecylphosphocholine detergent micelles, revealed that Esc-1b(1-18)NH 2 consists of two helical segments separated by a kink at Gly 8 (Manzo et al 2014). In addition, it was suggested that the cationic residues located at the N-terminal half of this peptide are important not only for peptide-bacteria binding but also for facilitating a deeper insertion of Esc-1b(1-18)NH 2 between the detergent headgroups.…”

“…In addition, it was suggested that the cationic residues located at the N-terminal half of this peptide are important not only for peptide-bacteria binding but also for facilitating a deeper insertion of Esc-1b(1-18)NH 2 between the detergent headgroups. Furthermore, in the presence of zwitterionic micelles, which mimic the electrically-neutral membrane of mammalian cells only the C-terminal fragment was expected to fold in a helical conformation (Manzo et al 2014). Therefore, with the aim of optimizing the biostability of Esc(1-21) without affecting its antimicrobial activity, we synthesized a diastereomer of Esc(1-21), named Esc(1-21)-1c, by replacing two l-amino acids in the C-terminal portion, i.e.…”

Fighting microbial infections: A lesson from amphibian skin-derived esculentin-1 peptides

“…charged sodium dodecylsulfate/dodecylphosphocholine detergent micelles, Esc-1b(1-18)NH 2 consists of two helical segments separated by a kink at Gly 8. Note that the cationic residues mostly "concentrated" at the N-terminal half of this peptide may be important not only to favor its binding to anionic membranes, but also to help a deeper insertion of the peptide between the polar lipid headgroups [78].…”