ABSTRACTFungal pathogens kill more people per year globally than malaria or tuberculosis and threaten international food security through crop destruction. New sophisticated strategies to inhibit fungal growth are thus urgently needed. Among the potential candidate molecules that strongly inhibit fungal spore germination are small cationic, cysteine-stabilized proteins of the AFP family secreted by a group of filamentous Ascomycetes. Its founding member, AFP fromAspergillus giganteus, is of particular interest since it selectively inhibits the growth of filamentous fungi without affecting the viability of mammalian, plant, or bacterial cells. AFPs are also characterized by their high efficacy and stability. Thus, AFP can serve as a lead compound for the development of novel antifungals. Notably, all members of the AFP family comprise a γ-core motif which is conserved in all antimicrobial proteins from pro- and eukaryotes and known to interfere with the integrity of cytoplasmic plasma membranes. In this study, we used classical molecular dynamics simulations combined with wet laboratory experiments and nuclear magnetic resonance (NMR) spectroscopy to characterize the structure and dynamical behavior of AFP isomers in solution and their interaction with fungal model membranes. We demonstrate that the γ-core motif of structurally conserved AFP is the key for its membrane interaction, thus verifying for the first time that the conserved γ-core motif of antimicrobial proteins is directly involved in protein-membrane interactions. Furthermore, molecular dynamic simulations suggested that AFP does not destroy the fungal membrane by pore formation but covers its surface in a well-defined manner, using a multistep mechanism to destroy the membranes integrity.IMPORTANCEFungal pathogens pose a serious danger to human welfare since they kill more people per year than malaria or tuberculosis and are responsible for crop losses worldwide. The treatment of fungal infections is becoming more complicated as fungi develop resistances against commonly used fungicides. Therefore, discovery and development of novel antifungal agents are of utmost importance.
The Evolutionary Conserved γ-Core Motif Influences the Anti-Candida Activity of the Penicillium chrysogenum Antifungal Protein PAF
