Solution structures of the antifungal heliomicin and a selected variant with both antibacterial and antifungal activities

In response to an experimental infection, the lepidopteran Heliothis viresc ens produces an antifungal protein named heliomicin. Heliomicin displays se quence similarities with antifungal plant defensins and antibacterial or an tifungal insect defensins. To gain information about the structural element s 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 he liomicin with the sequences of members of the insect defensin family, were analyzed using antibacterial and antifungal assays. One of the variants sho ws significant activity against Gram-positive bacteria while remaining effi cient against fungi. The three-dimensional structures of this variant and o f the wild-type protein were determined by two-dimensional H-1 NMR to estab lish a correlation between structure and antibacterial or antifungal activi ty. Wild-type and mutated heliomicins adopt a similar scaffold, including t he so-called cysteine-stabilized alpha beta motif. A comparison of their st ructures with other defensin-type molecules indicates that common hydrophob ic characteristics can be assigned to all the antifungal proteins. A compar ative analysis of various structural features of heliomicin mutant and of a ntibacterial defensins enables common properties to be assessed, which will help to design new mutants with increased antibacterial activity.