Abstract
Ubiquitin-like proteins (UBLs) such as NEDD8 are transferred to their targets by distinct, parallel, multienzyme cascades that involve the sequential action of E1, E2 and E3 enzymes. How do enzymes within a particular UBL conjugation cascade interact with each other? We report here that the unique N-terminal sequence of NEDD8's E2, Ubc12, selectively recruits NEDD8's E1 to promote thioester formation between Ubc12 and NEDD8. A peptide corresponding to Ubc12's N terminus (Ubc12N26) specifically binds and inhibits NEDD8's E1, the heterodimeric APPBP1–UBA3 complex. The structure of APPBP1–UBA3– Ubc12N26 reveals conserved Ubc12 residues docking in a groove generated by loops conserved in UBA3s but not other E1s. These data explain why the Ubc12-UBA3 interaction is unique to the NEDD8 pathway. These studies define a novel mechanism for E1-E2 interaction and show how enzymes within a particular UBL conjugation cascade can be tethered together by unique protein-protein interactions emanating from their common structural scaffolds.
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Acknowledgements
We are grateful to M.S. Podgorski for initial characterization of the Ubc12ΔN mutant, to P.D. Jeffrey, N.P. Pavletich, M. Pagano, L. Hendershot, H. Walden and other members of the Schulman lab for many helpful discussions, to D.L. Minor for critical reading of the manuscript, to P.J. Murray for assistance with Figure 8, to C. Ross for crystallography support, to G. Hannon for the MaRX library and initial experiments with Ubc12 in cell proliferation assays, to S. Olsen and K. Rakestraw for expert DNA synthesis and sequencing, and to J. Tanamachi and staff at the 8.3.1 beamline at Advanced Light Source, M. Becker and staff at the X25 beamline at National Synchrotron Light Source, and staff at the SERCAT beamline at Advanced Photon Source for synchrotron support. This work was supported by American Lebanese Syrian Associated Charities, the US National Institutes of Health (P30CA21765 National Cancer Institute Cancer Center Core grant to St. Jude, R01GM69530 to B.A.S., P01CA071907 to M.F.R.), the Department of Defense (DAMD17-03-1-0420), a grant from Phillip and Elizabeth Gross, a Pew Scholar in Biomedical Sciences award to B.A.S., and a St. Jude Special Fellowship to D.H.
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Supplementary Table 1
Kinetic parameters for Ubc12-NEDD8 thioester formation. (PDF 206 kb)
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Huang, D., Miller, D., Mathew, R. et al. A unique E1-E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8. Nat Struct Mol Biol 11, 927–935 (2004). https://doi.org/10.1038/nsmb826
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DOI: https://doi.org/10.1038/nsmb826
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