Solution Structures of the Antifungal Heliomicin and a Selected Variant with both Antibacterial and Antifungal Activities^,

Abstract

In response to an experimental infection, the lepidopteran Heliothis virescens produces an antifungal protein named heliomicin. Heliomicin displays sequence similarities with antifungal plant defensins and antibacterial or antifungal insect defensins. To gain information about the structural elements required for either antifungal or antibacterial activity, heliomicin and selected point-mutated variants were expressed in yeast as fusion proteins. The effects of mutations, defined by comparing the primary structure of heliomicin with the sequences of members of the insect defensin family, were analyzed using antibacterial and antifungal assays. One of the variants shows significant activity against Gram-positive bacteria while remaining efficient against fungi. The three-dimensional structures of this variant and of the wild-type protein were determined by two-dimensional (1)H NMR to establish a correlation between structure and antibacterial or antifungal activity. Wild-type and mutated heliomicins adopt a similar scaffold, including the so-called cysteine-stabilized alphabeta motif. A comparison of their structures with other defensin-type molecules indicates that common hydrophobic characteristics can be assigned to all the antifungal proteins. A comparative analysis of various structural features of heliomicin mutant and of antibacterial defensins enables common properties to be assessed, which will help to design new mutants with increased antibacterial activity.

Eurocin, a New Fungal Defensin

“…Many invertebrate (11) and fungal (12) defensins have a cysteine-stabilized ␣-helix/␤-sheet structure similar to the structure of ␤-defensins from vertebrates. Among others, structures have been solved for Phormia defensin (13), Sarcophaga defensin (14), drosomycin (15), heliomycin (16), terramycin (17), MGD-1 (18), lucifensin (19), and plectasin (20). They all form an ␣-helix and two anti-parallel ␤-strands (␣␤␤-scaffold) with the ␣-helix stabilized by two disulfide bridges to one strand of the ␤-sheet.…”

“…The cDNA clone encoded a putative defensin-like peptide that was named eurocin. The gene encodes 90 amino acids (signal peptide (amino acids (aa) [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20], propeptide (aa , and defensin peptide (aa 49 -90)). The mature peptide consists of 42 amino acids ( Fig.…”

Defensins from Insects and Plants Interact with Fungal Glucosylceramides

“…Insect defensins combine an ␣-helix and a double-stranded ␤-sheet stabilized by three disulfide bridges, organized in a cysteine-stabilized ␣␤ motif as found in plant defensins (2,3). An even higher homology to plant defensins is found for the insect defensin-like peptide heliomicin, which carries a triplestranded ␤-sheet in a ␤␣␤␤ fold forming a cysteine-stabilized ␣␤ motif (4). In addition to similarities in their global folds, heliomicin and the radish plant defensin RsAFP2 exhibit very similar distributions of hydrophobic residues (4) and display similar biological activities: both peptides are antifungal rather than antibacterial (5,6).…”

Nematode-derived drosomycin-type antifungal peptides provide evidence for plant-to-ecdysozoan horizontal transfer of a disease resistance gene

“…In addition to the hits described above with significant E-values, some hits with low sequence similarity (E-values of 2.6-9 Â 10 À 4 ) should also be considered as remote homologues of DTAFPs due to the similarity in structure and function, according to the criteria of Russell et al 21 All these hits are heliomicin-like antifungal peptides with a CSab-fold 22 . Their evolutionary relationship with DTAFPs is strengthened by the discovery that heliomicin and plant defensins interact with the same target in the fungal plasma membrane 23 .…”