Abstract
Ubiquitin-like proteins (UBLs) are conjugated by dynamic E1–E2–E3 enzyme cascades. E1 enzymes activate UBLs by catalysing UBL carboxy-terminal adenylation, forming a covalent E1˜UBL thioester intermediate, and generating a thioester-linked E2˜UBL product, which must be released for subsequent reactions. Here we report the structural analysis of a trapped UBL activation complex for the human NEDD8 pathway, containing NEDD8’s heterodimeric E1 (APPBP1–UBA3), two NEDD8s (one thioester-linked to E1, one noncovalently associated for adenylation), a catalytically inactive E2 (Ubc12), and MgATP. The results suggest that a thioester switch toggles E1–E2 affinities. Two E2 binding sites depend on NEDD8 being thioester-linked to E1. One is unmasked by a striking E1 conformational change. The other comes directly from the thioester-bound NEDD8. After NEDD8 transfer to E2, reversion to an alternate E1 conformation would facilitate release of the E2˜NEDD8 thioester product. Thus, transferring the UBL’s thioester linkage between successive conjugation enzymes can induce conformational changes and alter interaction networks to drive consecutive steps in UBL cascades.
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Acknowledgements
We dedicate this manuscript to the memory of C. Pickart for her contributions to our understanding of ubiquitin and UBL pathways, and for advice and encouragement throughout the course of these studies. We thank C. Ralston, B. Sankaran and A. Howard for assistance with data collection at the 8.2.2 beamline at ALS and at the SERCAT beamline at APS. We thank P. Murray, D. Minor, B. Dye and D. Scott for critical reading of the manuscript, members of the Schulman laboratory for discussions, and C. Ross for X-ray support. This work was supported in part by ALSAC and grants from the NIH (B.A.S.) and the Charles A. King Medical Foundation (M.D.O.). B.A.S. is an Investigator of the Howard Hughes Medical Institute.
Author Contributions D.T.H. and B.A.S. designed the experiments. D.T.H., H.W.H., M.Z., J.M.H. and B.A.S. performed the experiments. M.D.O. assessed the quality of crystallization samples and made conceptual contributions. D.T.H. and B.A.S. wrote the manuscript.
Coordinates and structure factors are deposited in the Protein Data Bank under accession code 2NVU.
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Huang, D., Hunt, H., Zhuang, M. et al. Basis for a ubiquitin-like protein thioester switch toggling E1–E2 affinity. Nature 445, 394–398 (2007). https://doi.org/10.1038/nature05490
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DOI: https://doi.org/10.1038/nature05490
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