Abstract
Core functions of autophagy are mediated by ubiquitin-like protein (UBL) cascades, in which a homodimeric E1 enzyme, Atg7, directs the UBLs Atg8 and Atg12 to their respective E2 enzymes, Atg3 and Atg10. Crystallographic and mutational analyses of yeast (Atg7–Atg3)2 and (Atg7–Atg10)2 complexes reveal noncanonical, multisite E1-E2 recognition in autophagy. Atg7's unique N-terminal domain recruits distinctive elements from the Atg3 and Atg10 'backsides'. This, along with E1 and E2 conformational variability, allows presentation of 'frontside' Atg3 and Atg10 active sites to the catalytic cysteine in the C-terminal domain from the opposite Atg7 protomer in the homodimer. Despite different modes of binding, the data suggest that common principles underlie conjugation in both noncanonical and canonical UBL cascades, whereby flexibly tethered E1 domains recruit E2s through surfaces remote from their active sites to juxtapose the E1 and E2 catalytic cysteines.
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Acknowledgements
This work was supported by American Lebanese Syrian Associated Charities (ALSAC), the Howard Hughes Medical Institute, St. Jude Cancer Center grant 5P30CA021765 and US National Institutes of Health (NIH) grant R01GM077053 to B.A.S. and NIH grant R01GM053396 to D.J.K. B.A.S. is an Investigator of the Howard Hughes Medical Institute. We are grateful to C. Regni and A. Williams for help in early stages of this project, M. Frank and C. Rock (St. Jude Children's Research Hospital, Memphis, Tennessee, USA) for providing liposomes and D.W. Miller, S. Bozeman, D.J. Miller, Y. Qiu and J. Bollinger for administrative, computational or technical support. The Northeastern Collaborative Access Team is supported by NIH National Center for Research Resources RR-15301, Advanced Photon Source (APS) by US Department of Energy (US DOE) W-31-109-ENG-38, Advanced Light Source (ALS) by US DOE DE-AC02-05CH11231. Anti-Pgk1 antibody was provided by J. Thorner (University of California, Berkeley, California, USA).
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S.E.K., J.L.O., D.M.D., I.K. and T.D.F. performed the X-ray crystallography experiments. S.E.K., A.M.T., S.Y., J.L.O. and A.D. purified wild-type and mutant proteins and performed in vitro biochemical assays. K.M. performed all yeast experiments. B.A.S., along with S.E.K. and D.J.K., oversaw experiments and prepared the manuscript with input from all authors.
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Kaiser, S., Mao, K., Taherbhoy, A. et al. Noncanonical E2 recruitment by the autophagy E1 revealed by Atg7–Atg3 and Atg7–Atg10 structures. Nat Struct Mol Biol 19, 1242–1249 (2012). https://doi.org/10.1038/nsmb.2415
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DOI: https://doi.org/10.1038/nsmb.2415
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