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Structure of the human ATG12~ATG5 conjugate required for LC3 lipidation in autophagy

Abstract

The autophagy factor ATG12~ATG5 conjugate exhibits E3 ligase–like activity which facilitates the lipidation of members of the LC3 family. The crystal structure of the human ATG12~ATG5 conjugate bound to the N-terminal region of ATG16L1, the factor that recruits the conjugate to autophagosomal membranes, reveals an integrated architecture in which ATG12 docks onto ATG5 through conserved residues. ATG12 and ATG5 are oriented such that other conserved residues on each molecule, including the conjugation junction, form a continuous surface patch. Mutagenesis data support the importance of both the interface between ATG12 and ATG5 and the continuous patch for E3 activity. The ATG12~ATG5 conjugate interacts with the E2 enzyme ATG3 with high affinity through another surface location that is exclusive to ATG12, suggesting a different role of the continuous patch in E3 activity. These findings provide a foundation for understanding the mechanism of LC3 lipidation.

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Figure 1: Artificial mimics of ATG12~ATG5 do not promote LC3 lipidation.
Figure 2: The crystal structure of human ATG12~ATG5–ATG16N.
Figure 3: Identification of potential functional patches on ATG12~ATG5 by conservation mapping.
Figure 4: Conserved residues of ATG12~ATG5 are important for E3 activity.
Figure 5: Residues of ATG12~ATG5 important for E3 activity in vitro are also important in cells.
Figure 6: Point mutations in the binding patch in ATG12 reduce the affinity for ATG3.
Figure 7: Point mutations in the binding patch on ATG12 impair ATG3 interaction in MEFs.

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Protein Data Bank

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Acknowledgements

We thank N. Mizushima (Tokyo Medical and Dental University) for Atg5 knockout MEFs; T. Kitamura (University of Tokyo) for pMXs-puro vector and Plat-E retrovirus packaging cells; P.E. Wright for his critical reading of the manuscript; K. Baker, O. Pornillos, K. Ihara, M. Machius and the beamline staff for assistance and advice on crystallographic data collection; A. Ward and G. Amarasinghe for technical advice regarding the ITC experiments. This work was supported by US National Institutes of Health (NIH) grant GM092740 to T.O. and by funds from Japan Science and Technology Agency through Keio Kanrinmaru Project to G.T. The synchrotron beamlines the Advanced Photon Source 22-BM operated by the Southeast Regional Collaborative Access Team at the Argonne National Laboratory and the Advanced Light Source 5.0.2 and 8.2.1 operated by the Berkeley Center for Structural Biology (BCSB) at the Lawrence Berkeley National Laboratory are supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under contracts W-31-109-Eng-38 and DE-AC02-05Ch11231, respectively. The BCSB is also supported in part by the NIH, National Institute of General Medical Sciences and the Howard Hughes Medical Institute.

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C.O. performed protein preparation, crystallization and cell biology, and assisted with biochemistry; Z.M. assisted with structure refinement, biochemical analyses and manuscript preparation; G.T. managed cell biological assays; T.O. conceived the project and performed structure determination, in vitro biochemistry and ITC analyses, and wrote the paper.

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Correspondence to Takanori Otomo.

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Otomo, C., Metlagel, Z., Takaesu, G. et al. Structure of the human ATG12~ATG5 conjugate required for LC3 lipidation in autophagy. Nat Struct Mol Biol 20, 59–66 (2013). https://doi.org/10.1038/nsmb.2431

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