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Identification of conjugation specificity determinants unmasks vestigial preference for ubiquitin within the NEDD8 E2

Nature Structural & Molecular Biology volume 15, pages 280–287 (2008)Cite this article

Abstract

Ubiquitin-like proteins (UBLs) modify targets via related E1-E2-E3 cascades. How is UBL conjugation fidelity established? Here we report the basis for UBL selection by UBL conjugating enzyme 12 (Ubc12), which is specific for the neural precursor cell expressed, developmentally down-regulated protein 8 (NEDD8), and does not form a thioester-linked conjugate with ubiquitin. We systematically identified Ubc12 surfaces impeding Ubc12∼ubiquitin conjugate formation and found that several structurally dispersed E1 binding elements, rather than UBL-interacting surfaces, determine E2∼UBL specificity. In addition to roles for conserved E1 and E2 domains, unique structures contribute UBL specificity to the NEDD8 and ubiquitin pathways. By removing surface elements, without substituting corresponding sequences from ubiquitin E2s, we unmasked Ubc12's vestigial preference for ubiquitin over NEDD8 by ∼1010-fold. This has implications for the evolution of specific functions among ubiquitin E2s. We also find that Ubc12 sequences dictating UBL selection map to the E3 binding site, thus providing a molecular mechanism preventing inappropriate modification of targets.

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Figure 1: Systematic identification of Ubc12 surfaces selecting against an incorrect Ubc12∼ubiquitin thioester conjugate.
Figure 2: Ubc12 surface elements select against forming an E2∼ubiquitin conjugate.
Figure 3: Mapping Ubc12's ubiquitin-like protein (UBL) selection determinants onto a crystal structure of a trapped complex with APPBP1-UBA3∼NEDD8(T)-NEDD8(A)-MgATP24.
Figure 4: Unmasking Ubc12's vestigial function as a ubiquitin-conjugating enzyme.
Figure 5: A key residue for forming a thioester conjugate with ubiquitin (Ub) conserved between Ubc12 and ubiquitin E2s.
Figure 6: Impaired Cul1 modification by Ubc12core-8ala_2.

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Acknowledgements

We thank A. Varshavsky (Caltech) for the construct for expressing Ubc2p, D. Duda, B. Dye, H. Kamadurai and D. Scott for helpful discussions and critical reading of the manuscript, D. Miller for laboratory support and S. Bozeman for administrative support. This work was supported in part by ALSAC (American Syrian Lebanese Associated Charities), a Beckman Young Investigator Award, a Pew Scholar Award and grants from the US National Institutes of Health (R01GM069530 and R01GM077053) to B.A.S. B.A.S. is an Investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Howard Hughes Medical Institute, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, 38105, Tennessee, USA

    Danny T Huang & Brenda A Schulman

  2. Department of Structural Biology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, 38105, Tennessee, USA

    Danny T Huang, Min Zhuang & Brenda A Schulman

  3. Department of Genetics and Tumor Cell Biology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, 38105, Tennessee, USA

    Danny T Huang, Min Zhuang, Olivier Ayrault & Brenda A Schulman

  4. Integrated Program in Biomedical Sciences, University of Tennessee Health Science Center, 62 South Dunlap, Memphis, 38163, Tennessee, USA

    Min Zhuang & Brenda A Schulman

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D.T.H. and B.A.S. designed and supervised the experiments. D.T.H., M.Z., and O.A. performed the experiments. D.T.H. and B.A.S. wrote the paper.

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Correspondence to Brenda A Schulman.

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Huang, D., Zhuang, M., Ayrault, O. et al. Identification of conjugation specificity determinants unmasks vestigial preference for ubiquitin within the NEDD8 E2. Nat Struct Mol Biol 15, 280–287 (2008). https://doi.org/10.1038/nsmb.1387

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