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

Abstract

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.

Keywords: 6-fluoroindole; ATG8a; DmATG8a; Drosophila melanogaster; insecticide.

Figure 1

Involvement of autophagy-associated protein 8 (ATG8) family proteins in the macroautophagy pathway. PE, phosphatidylethanolamine; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; ATG8-I, a form of ATG8 formed after cleavage of the amino acid residues at the C-terminal glycine (116G) of ATG8; ATG8-II, a form of ATG8 after the C-terminal glycine residue of ATG8-I is esterified with phosphatidylethanolamine (PE).

Figure 2

Structures of indole analogs (a–n).

Figure 3

(A) SDS-PAGE gel image of purified DmATG8a in lanes 1–3 (10 μg). M: protein markers. The loading bufferws 0.25 M Tris-HCl (pH 8.8), 2% SDS, 10% β-mercaptoethanol, and 20% glycerol. The electrophoresis conditions are 100 V for the stacking gel and 120 V for the separating gel. (B) SDS-PAGE gel image of DmATG8a with His tag in lanes 1–2 (10 μg), of DmATG8a without His tag in lanes 3–4 (5 μg). M: protein markers. The loading buffer is 0.25 M Tris-HCl (pH 8.8), 2% SDS, 10% β-mercaptoethanol, and 20% glycerol. The electrophoresis conditions are 100 V for the stacking gel and 120 V for the separating gel.

Figure 4

(A) Overall structure of DmATG8a. α-Helices depicted in purple and β-folding strands shown in blue. (B) Interactions between the ubiquitin fold and the N-terminal helices are depicted, with the N-terminal helices shown in white and the ubiquitin fold in purple. The residues involved in these interactions are marked. (C) Interactions occurring within the ubiquitin fold are illustrated, with the relevant residues also labeled. (D) The two active cavities of DmATG8a are presented. With residues in the HP1 cavity are shown in blue and those in the HP2 cavity are shown in white.

Figure 5

Comparison of five similar structures. (A) Structure-based sequence alignment of GABARAP (88.73% identity), GABARAPL1 (77.95% identity), GABARAPL2 (55.37% identity), and LC3 (28.68% identity) with DmATG8a. Alignments were performed with the program ESPript. (B) A superposition of the overall structures shows DmATG8a (pink) alongside GABARAP (green), GABARAPL1 (blue), GABARAPL2 (purple), and LC3 (yellow).

Figure 6

MST binding curves of (A) 6-fluoroindole with DmATG8a at 25 °C. (B) Tryptophan with DmATG8a at 25 °C.

Figure 7

Molecular docking result of 6-fluoroindole and DmATG8a. (A) Cavity binding sites of DmATG8a and 6-fluoroindole. (B) Binding residue sites of DmATG8a and 6-fluoroindole.

Figure 8

(A) SDS-PAGE gel image of purified DmATG8A^K48A in lanes 1–5 (10 μg). M: protein markers. The loading buffer is 0.25 M Tris-HCl (pH 8.8), 2% SDS, 10% β-mercaptoethanol, and 20% glycerol. The electrophoresis conditions are 100 V for the stacking gel and 120 V for the separating gel; and (B) MST results of 6-fluoroindole with DmATG8A^K48A.