Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis

Lea M. Starita, Jonathan N. Pruneda, Russell S. Lo, Douglas M. Fowler, Helen J. Kim, Joseph B. Hiatt, Jay Shendure, Peter S. Brzovic, Stanley Fields, Rachel E. Klevit

Research output: Contribution to journalArticle

71 Scopus citations

Abstract

Although ubiquitination plays a critical role in virtually all cellular processes, mechanistic details of ubiquitin (Ub) transfer are still being defined. To identify themolecular 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 activityenhancing 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.

Original languageEnglish (US)
Pages (from-to)E1263-E1272
JournalProceedings of the National Academy of Sciences of the United States of America
Volume110
Issue number14
DOIs
StatePublished - Apr 2 2013

Keywords

  • Deep mutational scanning
  • NMR
  • Phage display
  • Protein stability
  • Ubiquitin E3 ligase

ASJC Scopus subject areas

  • General

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    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. (2013). Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis. Proceedings of the National Academy of Sciences of the United States of America, 110(14), E1263-E1272. https://doi.org/10.1073/pnas.1303309110