Bombinin H4

Skin secretions of the European frog Bombina variegata contain a family of hydrophobic peptides, called bombinins H, which probably play a role in the defense against microbes. These peptides are rich in glycine (25%), which may allow structural polymorphism. Indeed, there is increasing evidence that bombinin H can, dependent on the environment, adopt different conformations. Moreover, some of these peptides contain a d-amino acid; the bombinins H2 and H4 differ from each other in that they contain either L-Ile or D-allo-Ile at position 2. In this paper we report the solution structure obtained by using NMR techniques of the mainly helical conformation, which these peptides adopt upon binding to the bilayer mimetics SDS and DPC. A glycine ridge is exposed at one side of the helix and may provide a helix-helix interaction site. In this respect, the structure of bombinin H resembles the influenza hemagglutinin fusion peptide and the helical conformer of Alzheimer peptide Abeta(25-40). Neither structure nor orientation of bombinin H are affected by the chiral inversion. Environmental conditions can trigger self-aggregation of bombinin H in solution due to hydrophobic interaction. Under these conditions the stereochemistry of the randomly ordered N-terminal segment modulates the preference to fold into a particular conformation.

Bombinin H2 and H4 are peptides isolated from the skin of the frog Bombina variegata that exhibit antimicrobial activity against Leishmania as well as bacteria. H4 is an isomer of H2 that has d-allo-Ile at position 2 from the N-terminus. Although H4 exhibits higher antimicrobial activity than that of H2, the molecular mechanism has remained unclear. In this study, we tried to reveal the molecular mechanism in terms of lipid membrane disruption through pore formation, using electrophysiological measurements. Based on our experiments, we estimated the pore-forming structure, pore size, and the kinetics in a bacteria model membrane. Stochastic analysis of the current data indicated that peptide isomerization enables us to accelerate the pore formation owing to the higher affinity between the peptide and lipid membrane. Additionally, the H2/H4 mixture was studied with 31P NMR and cross-linking experiment with mass spectrometry. It was found that heterogeneous pore formation with H2 and H4 was indicated. This electrophysiological approach will likely be promising as a useful tool for analyzing the molecular mechanism of pore-forming peptides.

Bombinin H4 is an antimicrobial peptide that was isolated from the toad Bombina variegata. Bombinin H family peptides are active against gram-positive, gram-negative bacteria, and fungi as well as the parasite Leishmania. Among them, bombinin H4 (H4), which contains d-allo-isoleucine (d-allo-Ile) as the second residue in its sequence, is the most active, and its l-isomer is bombinin H2 (H2). H4 has a significantly lower LC50 than H2 against Leishmania. However, the atomic-level mechanism of the membrane interaction and higher activity of H4 has not been clarified. In this work, we investigated the behavior of the conformations and interactions of H2 and H4 with the Leishmania membrane using 31P solid-state nuclear magnetic resonance (NMR), vibrational circular dichroism (VCD) spectroscopy, and molecular dynamics (MD) simulations. The generation of isotropic 31P NMR signals depending on the peptide concentration indicated the abilities of H2 and H4 to exert antimicrobial activity via membrane disruption. The VCD experiment and density functional theory calculation confirmed the different stability and conformations of the N-termini of H2 and H4. MD simulations revealed that the N-terminus of H4 is more stable than that of H2 in the membrane, in line with the VCD experiment data. VCD and MD analyses demonstrated that the first l-Ile and second d-allo-Ile of H4 tend to take a cis conformation. These residues function as an anchor and facilitate the easy winding of the helical conformation of H4 in the membrane. It may assist to quickly reach to the threshold concentration of H4 on the Leishmania membrane. This article is part of a Special Issue entitled: d-Amino acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca.