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

Abstract

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 | protein-peptide interaction | X-ray crystallography A utophagy 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 com...

Posttranslational modification of autophagy-related proteins in macroautophagy

“…This interaction is critical for E2-E3 complex formation. 169,170 In addition, the N-terminal 20 amino acids of mouse and human ATG3 form a membrane curvaturesensing amphipathic helix that allows LC3/GABARAP lipidation to proceed efficiently. 171 Finally, the N-terminal domain of ATG16L1 is responsible for the localization of the ATG12-ATG5 conjugate to membranes during autophagosome formation.…”

“…171 Finally, the N-terminal domain of ATG16L1 is responsible for the localization of the ATG12-ATG5 conjugate to membranes during autophagosome formation. 168,170 Proteolysis of Atg/ATG Proteins Some proteins are activated by proteolysis following translation because they are synthesized as inactive precursors, and proteolysis is involved in several different stages of autophagy. In the initiation phase of autophagy, Atg8 and its orthologs are synthesized as inactive precursors, which are then activated through removal of a C-terminal polypeptide segment.…”

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

“…We previously showed that the FR is important for the interaction with E3 in humans and demonstrated that a short region of human ATG3 FR interacts with ATG12 of E3 (termed the region that interacts with ATG12; RIA12) [18]. On the basis of our structural and biochemical characterizations of this interaction, we proposed that the RIA12 is a novel binding motif for ATG12, another ubiquitin-like protein in autophagy, and is required for recruitment of ATG3 by E3 [18]. Another previous study showed that binding of ATG3 to the E1 and E3 are mutually exclusive due to the overlapping E1 and E3 binding sites within the FR [19].…”

Human ATG3 Binding to Lipid Bilayers. Role of Lipid Geometry and Electric Charge

“…The details of Atg3 interaction with the membrane lipid matrix are, to our knowledge, virtually unexplored. Due to the ability of Atg3 to interact with Atg12 17,18 , Atg12-Atg5-Atg16L1 would promote a more efficient recruitment of Atg3 to membranes in vivo 19,20 . Atg12-Atg5 induces a reorientation of Atg3 catalytic cysteine toward a threonine residue so that Atg3 conjugase activity is stimulated 13,21 .…”