Structural basis of ATG3 recognition by the autophagic ubiquitin-like protein ATG12

The autophagic ubiquitin-like protein (ublp) autophagy-related (ATG)12 is a component of the ATG12∼ATG5–ATG16L1 E3 complex that promotes lipid conjugation of members of the LC3 ublp family. A role of ATG12 in the E3 complex is to recruit the E2 enzyme ATG3. Here we report the identification of the ATG12 binding sequence in the flexible region of human ATG3 and the crystal structure of the minimal E3 complexed with the identified binding fragment of ATG3. The structure shows that 13 residues of the ATG3 fragment form a short β-strand followed by an α-helix on a surface area that is exclusive to ATG12. Mutational analyses of ATG3 confirm that four residues whose side chains make contacts with ATG12 are important for E3 interaction as well as LC3 lipidation. Conservation of these four critical residues is high in metazoan organisms and plants but lower in fungi. A structural comparison reveals that the ATG3 binding surface on ATG12 contains a hydrophobic pocket corresponding to the binding pocket of LC3 that accommodates the leucine of the LC3-interacting region motif. These findings establish the mechanism of ATG3 recruitment by ATG12 in higher eukaryotes and place ATG12 among the members of signaling ublps that bind liner sequences.Autophagy is a bulk degradation/recycling process essential for quality control in eukaryotic cells (1, 2). During autophagy, portions of cytoplasm are sequestered into membrane-bound vesicles called autophagosomes and transported into lysosomes for degradation. Autophagosome formation is mediated by a concerted action of a number of conserved autophagy-related (ATG) proteins (2, 3). The two classes of ubiquitin-like proteins (ublps) among these are members of the LC3 family in mammals or Atg8 in yeast and the conserved ATG12, which share some sequence similarities (4). LC3 and ATG12 are activated by the same E1 enzyme, ATG7, and then transferred to different E2 enzymes, ATG3 and ATG10, respectively. ATG3 conjugates LC3 to a lipid molecule, phosphatidylethanolamine (PE), on autophagosomal membranes. PE-conjugated LC3 plays crucial roles in control of membrane dynamics during autophagosome formation as well as in recruitment of cargos, such as aberrant proteins and damaged organelles, through binding to receptor proteins carrying an LC3-interacting region (LIR) motif (5). On the other hand, ATG10 attaches ATG12 to a structural protein, ATG5, and the resulting conjugate ATG12∼ATG5 acts like an E3 factor by stimulating the transfer of LC3 from ATG3 to PE (6). ATG12∼ATG5 exists as a complex with the coiled-coil protein ATG16L1 that localizes on autophagosome precursor membranes, resulting in LC3 lipidation for autophagosome formation (7). We previously showed that E3 activity requires the native covalent linkage between ATG12 and ATG5 and a composite surface patch formed by the residues of both of these proteins, and that the interaction with the E2 ATG3 is mostly mediated by ATG12 (8). However, it remains unclear how E3 communicates with or recruits E2.Here we describe atomic details of the ATG3–ATG12 interaction based on our crystal structure of a human E2–E3 complex containing this interaction. The results from accompanying interaction analyses and cellular LC3 lipidation assays support the importance of this interaction in LC3 lipidation and autophagy. We also show that the ATG3–ATG12 and LIR–LC3 interactions share some structural features. This unexpected finding together with our experimental data suggests that ATG12 is a protein recruitment apparatus in autophagy.