Molecular recognition of fungal methylated glucosylceramides by ETD151 defensin

The growing resistance to antifungal drugs and the limited number of antifungals currently available highlight the need for new antifungal strategies. In this context, there is significant potential for plant and insect antifungal defensins, which target fungal glucosylceramides (GlcCer), a growth and virulence determinant. The ETD151 peptide, optimized from the insect defensin Heliomicin, binds to fungal GlcCer as a crucial step in its activity. Nevertheless, further investigation is necessary to elucidate the mechanisms by which ETD151 targets fungal GlcCer at the molecular and atomic scales. The binding affinity was experimentally measured with isothermal titration calorimetry and microscale thermophoresis. The results collectively revealed affinity with a membrane-bound methylated GlcCer in the micromolar range. NMR has been employed to identify the area of ETD151 that would monopolize methylated GlcCer binding, including the two adjacent hydrophobic loops. Furthermore, our findings indicate that ETD151 specifically inserts into and disorders phosphatidylcholine lipid vesicles containing methylated GlcCer. Finally, it is worth noting that the C9-methyl in the sphingoid base plays a pivotal role in the ETD151-GlcCer interaction. Its presence increases the binding affinity between the two partners, resulting in stronger structural changes in ETD151 and deeper insertion into the hydrophobic core of model membranes. This study reveals key biochemical and structural elements of the ETD151-GlcCer interaction, provides a basis for elucidating the structure and dynamics of other GlcCer-targeting defensins in relation to their function, and facilitates the development of defensin-mimetic antifungals.