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Insights into the ubiquitin transfer cascade from the structure of the activating enzyme for NEDD8

Abstract

Post-translational modification by ubiquitin-like proteins (Ublps) is an essential cellular regulatory mechanism1,2,3. The Ublp NEDD8 regulates cell division, signalling and embryogenesis4,5,6. Ublps are conjugated to their targets by the sequential action of E1, E2 and often E3 enzymes3. Each Ublp has a dedicated E1, or activating enzyme, that initiates its conjugation cascade1,3,7,8,9,10. First, E1 associates with the Ublp and catalyses adenylation of the carboxy terminus of the Ublp. Second, E1 forms a thioester between its catalytic cysteine and the Ublp. Next, E1 is loaded with a second Ublp molecule, adenylating the C terminus of this second Ublp while still carrying the first thioester-bound Ublp. Last, E1 binds E2 and promotes Ublp transfer to the catalytic cysteine of E2. We report here the structure and mutational analysis of human APPBP1–UBA3, the heterodimeric E1 enzyme for NEDD8 (ref. 11). Each E1 activity is specified by a domain: an adenylation domain resembling bacterial adenylating enzymes12, an E1-specific domain organized around the catalytic cysteine, and a domain involved in E2 recognition resembling ubiquitin. The domains are arranged around two clefts that coordinate protein and nucleotide binding so that each of E1's reactions drives the next, in an assembly-line fashion.

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Figure 1: E1 reaction scheme1,3,7–10, with noncovalent complexes indicated with a dot, and covalent complexes indicated with a hyphen.
Figure 2: Structure of the APPBP1–UBA3 complex.
Figure 3: Close-up views of the adenylation, catalytic cysteine and ubiquitin-like domains.
Figure 4: Structure-based model for E1 reactions.

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Acknowledgements

We thank P. Jeffrey, D. Minor, J. Berger, J. Huibregste and C. Ross for advice and assistance; M. Becker and staff at National Synchrotron Light Source Beamline X25, and T. Earnest, G. McDermott and staff at Advanced Light Source Beamline 5.0.2 for help with data collection; and P. Murray, T. Izard and L. Hendershot for comments on the manuscript. This work was supported by ALSAC, the NCI Cancer Center (CORE), and a Pew Scholar in Biomedical Sciences Award to B.A.S.

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Correspondence to Brenda A. Schulman.

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Walden, H., Podgorski, M. & Schulman, B. Insights into the ubiquitin transfer cascade from the structure of the activating enzyme for NEDD8. Nature 422, 330–334 (2003). https://doi.org/10.1038/nature01456

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