ArticleProceedings of the National Academy of Sciences of the United States of America2013
Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 115 papers.
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Who cites it
115 citing papers in PubMed, 204 citations in OpenAlex.
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- Deep structure-function analysis of the endonuclease Mus81 with dominant mutational scanning.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
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- Biophysics-based protein language models for protein engineering.bioRxiv : the preprint server for biology · 2025Article
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- DeepPFP: a multi-task-aware architecture for protein function prediction.Briefings in bioinformatics · 2024Article
- Multiplexed profiling of intracellular protein abundance, activity, interactions and druggability with LABEL-seq.Nature methods · 2024Article
- Design of linked-domain protein inhibitors of UBE2D as tools to study cellular ubiquitination.bioRxiv : the preprint server for biology · 2024Article
55 more citing papers are in PubMed but not listed here.
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Authors and funding
10 authors at 3 institutions in 1 country.
Funding
Abstract
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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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.