Abstract
Antimicrobial peptides play important roles in the host innate defense mechanism of many plants and animals. A series of short Trp-rich peptides derived from the C-terminal region of Bothrops asper myothoxin II, a Lys49 phospholipase A2 (PLA2), were found to reproduce the antimicrobial activities of their parent molecule. Of these peptides, KKWRWWLKALAKK—designated PEM-2—was found to display improved activity against both Gram-positive and Gram-negative bacteria. To improve the antimicrobial activity of PEM-2 for potential clinical applications further, we determined the solution structure of PEM-2 bound to membrane-mimetic dodecylphosphocholine (DPC) micelles by two-dimensional NMR methods. The DPC micelle-bound structure of PEM-2 adopts an α-helical conformation and the positively charged residues are clustered together to form a hydrophilic patch. The surface electrostatic potential map indicates that two of the three tryptophan residues are packed against the peptide backbone and form a hydrophobic face with Leu7, Ala9, and Leu10. A variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that PEM-2 interacted with negatively charged phospholipid vesicles and efficiently induced dye release from these vesicles, suggesting that the antimicrobial activity of PEM-2 could be due to interactions with bacterial membranes. Potent analogues of PEM-2 with enhanced antimicrobial and less pronounced hemolytic activities were designed with the aid of these structural studies. However, the efficacies of many antimicrobial peptides are greatly reduced under high salt concentrations limiting their development as pharmaceutical compounds. Here, we also describe an easy strategy to increase salt resistance of antimicrobial peptides by replacing tryptophan or histidine residues with the bulky amino acids β-naphthylalanine and 4-4’-biphenylalanine. The activities of the salt-sensitive peptide P-113 were diminished at high salt concentrations, whereas the activities of its β-naphthylalanine and 4-4’-biphenylalanine substituted variants were less affected. Moreover, we develop a series of strategy to investigate the relationship between orientation and salt resistance of tryptophan-rich antimicrobial peptides by NMR methods. PEM-2-W5K/A9W, a short Trp-rich antimicrobial peptide developed based on the structural studies of PEM-2, has been shown to be highly active against various bacterial strains with less hemolytic activity. Here, correlations between membrane immersion depth, orientation, hydrophobicity, and salt-resistance of PEM-2 and PEM-2-W5K/A9W have been investigated via solution structure and paramagnetic resonance enhancement studies. The results indicated that the strong salt-resistance of PEM-2-W5K/A9W may arise from the peptide position itself deeply into microbial cell membranes, making it more efficient in disrupting the membranes, hence compensate the competition from the cations to the negatively charged microbial cell surface. The antimicrobial activities of PEM-2-W5K/A9W against various bacterial and fungal strains including multidrug-resistant and clinical isolates under high salt conditions were tested. The activities of the salt-sensitive peptide P-113 and PEM-2 were reduced and diminished at high salt concentrations, whereas the activities of PEM-2-W5K/A9W were less affected.