Crystal Structure of Autophagy-Associated Protein 8 at 1.36 Å Resolution and Its Inhibitory Interactions with Indole Analogs

Autophagy-associated protein 8 (ATG8) is essential for autophagy and organismal growth and development. In this study, we successfully resolved the crystal structure of Drosophila melanogaster (D. melanogaster) ATG8a (DmATG8a) at 1.36 Å resolution. Being distinct from previously characterized ATG8 homologues, DmATG8a (121 residues) adopts a unique fold comprising five α-helices and four β-folding strands, in contrast to the canonical four α-helices and four β-folding strands observed in other ATG8 proteins. DmATG8a features two active cavities: hydrophobic pocket 1 (HP1) and hydrophobic pocket 2 (HP2), which are essential for the normal physiological function of ATG8. Indole and its analogs can bind specifically with HP1. Microscale thermophoresis results demonstrated a strong affinity of 6-fluoroindole with DmATG8a (3.54 μmol/L), but no affinity with the DmATG8a^K48A mutant, suggesting that Lys48 is critical in binding 6-fluoroindole probably via a hydrogen bond interaction. The half-maximum lethal concentration (LC₅₀) of 6-fluoroindole against D. melanogaster adult flies was 169 μg/mL. Our findings establish DmATG8a as a promising target for developing indole-based insecticides.