Identification and characterization of the linear region of ATG3 that interacts with ATG7 in higher eukaryotes.

Transfer of GABARAP thioester from the E1 ATG7 to the E2 ATG3 requires the interaction between the N-terminal domain of ATG7 and the flexible region (FR) of ATG3. This interaction has been visualized in the yeast Atg7-Atg3 complex crystal structure, but remains to be defined in higher eukaryotes. Here, our NMR data precisely define the region of the FR of human ATG3 that interacts with ATG7 (RIA7) and demonstrate RIA7 partially overlaps with the E3-interacting region, explaining how the E1-E2 and E2-E3 interactions are mutually exclusive. Mutational analyses identify critical residues of the RIA7 for the E1 interaction and GABARAP transfer, advancing our understanding of a molecular mechanism of the autophagic conjugation cascade in higher eukaryotes.