Autophagy is an intracellular degradation system regulating cellular homeostasis. The two ubiquitin-like modification systems named the Atg
8
system and the Atg12 system are essential for autophagy. Atg
8
and Atg12 are ubiquitin-like proteins covalently conjugated with a phosphatidylethanolamine (PE) and Atg5, respectively,
via enzymatic reactions. The Atg
8
–PE conjugate binds to autophagic membranes and recruits various proteins through direct interaction, whereas the Atg12–Atg5 conjugate recognizes Atg
3
, the
E
2
enzyme for Atg
8
, and facilitates Atg
8
–PE conjugation by functioning as the
E
3
enzyme. Although structural and biochemical analyses have well established the Atg
8
-family interacting motif (AIM), studies on the interacting sequence for Atg12 are rare (only one example for human ATG12–ATG
3
), thereby making it challenging to define a binding motif. Here we determined the crystal structure of the plant ATG12
b
as a complex with the ATG12
b
-binding region of ATG
3
and revealed that ATG12
b
recognizes the aspartic acid (Asp)–methionine (Met) motif in ATG
3
via a hydrophobic pocket and a basic residue, which we confirmed critical for the complex formation by mutational analysis. This recognition mode is similar to that reported between human ATG12 and ATG
3
, suggesting that the Asp–Met sequence is a conserved Atg12-interacting motif (AIM12). These data suggest that AIM12 mediates
E
2
-
E
3
interaction during Atg
8
lipidation and provide structural basis for developing chemicals that regulate autophagy by targeting Atg12-family proteins.
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