Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis
Open accessAbstract
Although ubiquitination plays a critical role in virtually all cellular processes, mechanistic details of ubiquitin (Ub) transfer are still being defined. To identify the molecular determinants within E3 ligases that modulate activity, we scored each member of a library of nearly 100,000 protein variants of the murine ubiquitination factor E4B (Ube4b) U-box domain for auto-ubiquitination activity in the presence of the E2 UbcH5c. This assay identified mutations that enhance activity both in vitro and in cellular p53 degradation assays. The activity-enhancing mutations fall into two distinct mechanistic classes: One increases the U-box:E2-binding affinity, and the other allosterically stimulates the formation of catalytically active conformations of the E2∼Ub conjugate. The same mutations enhance E3 activity in the presence of another E2, Ube2w, implying a common allosteric mechanism, and therefore the general applicability of our observations to other E3s. A comparison of the E3 activity with the two different E2s identified an additional variant that exhibits E3:E2 specificity. Our results highlight the general utility of high-throughput mutagenesis in delineating the molecular basis of enzyme activity.
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Self-citation
- Authors
- Starita, L. M., Pruneda, J. N., Lo, R. S., Fowler, D. M., Kim, H. J., Hiatt, J. B., Shendure, J., Brzovic, P. S., Fields, S., & Klevit, R. E.
- Affiliations
- Howard Hughes Medical Institute (US)
- University of Washington (US)
- Department of Biochemistry, and
- ORCID
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2013-03-18
- Volume
- 110
- Issue
- 14
- Pages
- E1263-72
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC3619334
- OpenAlex
- W2010053735
- Cited by
- 183
- References
- 32
- Citations by year
- 2026: 7 · 2025: 18 · 2024: 21 · 2023: 13 · 2022: 17 · 2021: 16 · 2020: 17 · 2019: 13 · 2018: 9 · 2017: 8 · 2016: 15 · 2015: 10 · 2014: 16 · 2013: 3
- FWCI
- 6.56
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Howard Hughes Medical Institute
- National Institute of General Medical Sciences (R01GM088055, P41GM103533, T32GM007270, R01GM098503)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
- Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
- Keywords
- E3 ubiquitin ligase, UBE4B, U-box domain, autoubiquitination, UbcH5c, allosteric regulation, p53 degradation, high-throughput mutagenesis, protein variants
- MeSH terms
- Allosteric Regulation, Amino Acid Sequence, Animals, Models, Molecular, Molecular Sequence Data, Oligonucleotides, Blotting, Western, Mutagenesis, Sequence Alignment, Nuclear Magnetic Resonance, Biomolecular, Ubiquitin, Ubiquitin-Protein Ligases, Immunoprecipitation, Mice, High-Throughput Screening Assays, Cell Surface Display Techniques
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