Structural basis for tRNA-dependent sterol aminoacylation underlying cell membrane integrity

Ergosteryl-3β-O-L-aspartate synthase (ErdS) catalyzes tRNA-dependent aspartylation of ergosterol, a lipid essential for fungal cell membrane integrity. However, the functional significance of ergosteryl-aspartate and the molecular mechanisms underlying its synthesis remain unclear. Here, we show that ErdS localization is highly dynamic and provide evidence that Erg-Asp is required for proper hyphal growth, sporulation, and spore germination, and likely influences stress tolerance. The cryo-electron microscopy structure of ErdS revealed an unprecedented sterol-binding pocket. In addition, the structures in complex with non-hydrolyzable Asp-N-tRNA ^Asp uncovered a tRNA-guided intramolecular aminoacyl transfer mechanism between two functional domains of the enzyme. The CCA end of tRNA ^Asp undergoes a large displacement to reach the aa-tRNA transfer active site, while the tRNA elbow is clamped by a long extension of the N-terminal α-helix. The present structural and mutational analyses demonstrate that domain fusion, dynamic repositioning, and tRNA-mediated substrate handover underlie the multifunctional catalytic efficiency of ErdS and facilitate Erg-Asp synthesis independently from protein synthesis. These findings elucidate the unique regulatory mechanism of tRNA-dependent sterol modification and provide insights into fungal membrane dynamics, highlighting potential novel targets for antifungal therapies.