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Background: Human ␣-defensin HD5 is a multifunctional antimicrobial peptide whose functional determinants have yet to be elucidated. Results: Alanine scanning mutagenesis aided by x-ray crystallography identified Leu 29 at the dimer interface as crucial; N-methylation of Glu 21 to debilitate HD5 dimerization also affected activity. Conclusion: Dimerization and hydrophobicity are important for HD5 function. Significance: The molecular basis of ␣-defensin function is better understood.

Crotamine is a highly basic peptide from the venom of Crotalus durissus terrificus rattlesnake. Its common gene ancestry and structural similarity with the β-defensins, mainly due to an identical disulfide bond pattern, stimulated us to assess the antimicrobial properties of native, recombinant, and chemically synthesized crotamine. Antimicrobial activities against standard strains and clinical isolates were analyzed by the colorimetric microdilution method showing a weak antibacterial activity against both Gram-positive and Gram-negative bacteria [MIC (Minimum Inhibitory Concentration) of 50->200 μg/mL], with the exception of Micrococcus luteus [MIC ranging from 1 to 2 μg/mL]. No detectable activity was observed for the filamentous fungus Aspergillus fumigatus and Trichophyton rubrum at concentrations up to 125 μg/mL. However, a pronounced antifungal activity against Candida spp., Trichosporon spp., and Cryptococcus neoformans [12.5-50.0 μg/mL] was observed. Chemically produced synthetic crotamine in general displayed MIC values similar to those observed for native crotamine, whereas recombinant crotamine was overridingly more potent in most assays. On the other hand, derived short linear peptides were not very effective apart from a few exceptions. Pronounced ultrastructure alteration in Candida albicans elicited by crotamine was observed by electron microscopy analyses. The peculiar specificity for highly proliferating cells was confirmed here showing potential low cytotoxic effect of crotamine against nontumoral mammal cell lines (HEK293, PC12, and primary culture astrocyte cells) compared to tumoral B16F10 cells, and no hemolytic activity was observed. Taken together these results suggest that, at low concentration, crotamine is a potentially valuable anti-yeast or candicidal agent, with low harmful effects on normal mammal cells, justifying further studies on its mechanisms of action aiming medical and industrial applications.

We performed a comprehensive alanine scan of human ␣-defensin HNP1 and tested the ability of the resulting analogs to kill Staphylococcus aureus, inhibit anthrax lethal factor, and bind human immunodeficiency virus-1 gp120. By far, the most deleterious mutation for all of these functions was W26A. The activities lost by W26A-HNP1 were restored progressively by replacing W26 with non-coded, straight-chain aliphatic amino acids of increasing chain length. The hydrophobicity of residue 26 also correlated with the ability of the analogs to bind immobilized wild type HNP1 and to undergo further self-association. Thus, the hydrophobicity of residue 26 is not only a key determinant of the direct interactions of HNP1 with target molecules, but it also governs the ability of this peptide to form dimers and more complex quaternary structures at micromolar concentrations. Although all defensin peptides are cationic, their amphipathicity is at least as important as their positive charge in enabling them to participate in innate host defense.

Mammalian ␣-defensins, expressed primarily in leukocytes and epithelia, play important roles in innate and adaptive immune responses to microbial infection. Six invariant cysteine residues forming three indispensable disulfide bonds and one Gly residue required structurally for an atypical ␤-bulge are totally conserved in the otherwise diverse sequences of all known mammalian ␣-defensins. In addition, a pair of oppositely charged residues (Arg/Glu), forming a salt bridge across a protruding loop in the molecule, is highly conserved. To investigate the structural and functional roles of the conserved Arg 6 -Glu 14 salt bridge in human ␣-defensin 5 (HD5), we chemically prepared HD5 and its precursor proHD5 as well as their corresponding salt bridge-destabilizing analogs E14Q-HD5 and E57Q-proHD5. The Glu-to-Gln mutation, whereas significantly reducing the oxidative folding efficiency of HD5, had no effect on the folding of proHD5. Bovine trypsin productively and correctly processed proHD5 in vitro but spontaneously degraded E57Q-proHD5. Significantly, HD5 was resistant to trypsin treatment, whereas E14Q-HD5 was highly susceptible. Further, degradation of E14Q-HD5 by trypsin was initiated by the cleavage of the Arg 13 -Gln 14 peptide bond in the loop region, a catastrophic proteolytic event resulting directly in quick digestion of the whole defensin molecule. The E14Q mutation did not alter the bactericidal activity of HD5 against Staphylococcus aureus but substantially enhanced the killing of Escherichia coli. By contrast, proHD5 and E57Q-proHD5 were largely inactive against both strains at the concentrations tested. Our results confirm that the primary function of the conserved salt bridge in HD5 is to ensure correct processing of proHD5 and subsequent stabilization of mature ␣-defensin in vivo.Defensins are expressed predominantly in leukocytes and epithelial cells as a family of cationic antimicrobial peptides that play important roles in innate and adaptive immune responses to microbial infection (1-5). In humans, defensins are classified into ␣-and ␤-families, differing by amino acid composition, cellular origin, and disulfide connectivity. To date, six human ␣-defensins have been identified, including four primarily from neutrophils, also known as human neutrophil peptides 1-4 or HNP1-4 2 (6 -10) and two mainly from intestinal Paneth cells, also known as human defensins 5-6 or 12). Although more than 30 putative ␤-defensin genes have been described using genomics tools (13-15), only a handful of ␤-defensins have been isolated and characterized at the protein level (16).Human ␣-defensins are synthesized in vivo as significantly larger precursor molecules consisting of an N-terminal pro domain of 43-49 amino acid residues and a C-terminal Cysrich defensin domain of 29 -33 amino acid residues. The pro domain in proHNPs, important for correct folding, sorting, and trafficking of defensins (17-20), is proteolytically excised by yet-to-be-identified enzyme(s) prior to the storing of mature HNPs in the azurophilic g...