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. 2013 Feb;14(2):206-11.
doi: 10.1038/embor.2012.208. Epub 2012 Dec 14.

Structure of the Atg12-Atg5 conjugate reveals a platform for stimulating Atg8-PE conjugation

Nobuo N Noda  1 , Yuko Fujioka, Takao Hanada, Yoshinori Ohsumi, Fuyuhiko Inagaki
Affiliations

Structure of the Atg12-Atg5 conjugate reveals a platform for stimulating Atg8-PE conjugation

Nobuo N Noda et al. EMBO Rep. 2013 Feb.

Abstract

Atg12 is conjugated to Atg5 through enzymatic reactions similar to ubiquitination. The Atg12-Atg5 conjugate functions as an E3-like enzyme to promote lipidation of Atg8, whereas lipidated Atg8 has essential roles in both autophagosome formation and selective cargo recognition during autophagy. However, the molecular role of Atg12 modification in these processes has remained elusive. Here, we report the crystal structure of the Atg12-Atg5 conjugate. In addition to the isopeptide linkage, Atg12 forms hydrophobic and hydrophilic interactions with Atg5, thereby fixing its position on Atg5. Structural comparison with unmodified Atg5 and mutational analyses showed that Atg12 modification neither induces a conformational change in Atg5 nor creates a functionally important architecture. Rather, Atg12 functions as a binding module for Atg3, the E2 enzyme for Atg8, thus endowing Atg5 with the ability to interact with Atg3 to facilitate Atg8 lipidation.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Crystal structure of the Atg12–Atg5 conjugate bound to Atg16N. (A) Ribbon representation of the Atg12–Atg5 conjugate bound to Atg16N. Atg12, Atg5 and Atg6 are coloured salmon pink, green and orange, respectively. Atg12 Phe 185 and Gly 186 and Atg5 Lys 149 are shown with a stick model. The disordered C-terminal region of Atg12 (residues 182–184) is indicated with a broken line. The left figure was obtained by a 90° rotation of the right figure along the vertical axis. The amino- and carboxy-termini are denoted as N and C, respectively. All of the figures representing the molecular structures were generated with PyMOL [26]. (B) Stereo view of the detailed interactions between Atg12 and Atg5. The side chains of the residues involved in the Atg12–Atg5 interaction are shown with a stick model, in which nitrogen and oxygen atoms are coloured red and blue, respectively. (C) Electron density map of the isopeptide linkage between Atg12 Gly 186 and Atg5 Lys 149. The simulated annealing Fo–Fc difference Fourier map was calculated by omitting Atg12 Phe 185 and Gly 186 and Atg5 Lys 149, and is shown with black meshes at 5.0σ. (D) Superimposition of the Atg5-Atg16N complex structure (PDB ID 2DYM) on that of the Atg12–Atg5-Atg16N complex. The Atg12–Atg5-Atg16N complex is coloured red, while the Atg5-Atg16N complex is coloured grey. Atg12 Phe 185 and Gly 186 and the side chain of Atg5 Lys 149 are shown with a stick model. (E) In vivo analyses of the Atg12 and Atg5 mutants for studying the functional significance of the non-covalent Atg12-Atg5 interactions. Yeast cells with or without starvation were lysed and subjected to urea SDS–polyacrylamide gel electrophoresis (PAGE) followed by western blotting. Ape1, aminopeptidase I; mApe1, mature form of Ape1; PE, phosphatidylethanolamine; prApe1, preform of Ape1; WT, wild-type.
Figure 2
Figure 2
Atg12 functions as a binding module for Atg3. (A) In vitro pull-down assay using recombinant plant Atg homologues. (B) Mapping of the mutated residues on the Atg12–Atg5-Atg16N complex structure. Atg5 and Atg16N are shown with a ribbon model, whereas Atg12 is shown with both surface and ribbon models. The side chains of Tyr 147, Tyr 149 and Phe 154 of Atg12 are shown with a stick model and coloured grey (Tyr 147) or blue (Tyr 149 and Phe 154). Corresponding AtAtg12b residues are indicated in parentheses. Atg12 Phe 185 and Gly 186 and the side chain of Atg5 Lys 149 are shown with a stick model. (C) Proposed model of Atg8–PE conjugate formation mediated by the Atg12–Atg5-Atg16 complex. CBB, Coomassie Brilliant Blue staining; GST, glutathione S-transferase; HA, haemagglutinin; PE, phosphatidylethanolamine; WT, wild-type.
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