Characterization of the molecular mechanism of the autophagy-related Atg8–Atg3 protein interaction in Toxoplasma gondii

Toxoplasmosis is caused by an obligate intracellular parasite, the protozoan Toxoplasma gondii. Discovery of novel drugs against T. gondii infection could circumvent the toxicity of existing drugs and T. gondii resistance to current treatments. The autophagy-related protein 8 (Atg8)-Atg3 interaction in T. gondii is a promising drug target because of its importance for regulating Atg8 lipidation. We reported previously that TgAtg8 and TgAtg3 interact directly. Here we validated that substitutions of conserved residues of TgAtg8 interacting with the Atg8 family-interacting motif (AIM) in Atg3 disrupt the TgAtg8-TgAtg3 interaction and reduce TgAtg8 lipidation and autophagosome formation. These findings were consistent with results reported previously for Plasmodium Atg8, suggesting functional conservation of Atg8 in Toxoplasma and Plasmodium. Moreover, using peptide and AlphaScreen assays, we identified the AIM sequence in TgAtg3 that binds TgAtg8. We determined that the core TgAtg3 AIM contains a Phe(239)-Ala(240)-Asp(241)-Ile(242) ((239)FADI(242)) signature distinct from the (WLLP108)-W-105 signature in the AIM of Plasmodium Atg3. Furthermore, an alanine-scanning assay revealed that the TgAtg8-TgAtg3 interaction in T. gondii also depends strongly on several residues surrounding the core TgAtg3 AIM, such as Asn(238), Asp(243), and Cys(244). These results indicate that distinct AIMs in Atg3 contribute to differences between Toxoplasma and Plasmodium Atg8-Atg3 interactions. By elucidating critical residues involved in the TgAtg8-TgAtg3 interaction, our work paves the way for the discovery of potential anti-toxoplasmosis drugs. The quantitative and straightforward AlphaScreen assay developed here may enable high-throughput screening for small molecules disrupting the TgAtg8-TgAtg3 interaction.