title: Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis.
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.
authors.affiliation: Howard Hughes Medical Institute, Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.
authors.forename: Lea M
authors.initials: LM
authors.lastname: Starita
authors.forename: Jonathan N
authors.initials: JN
authors.lastname: Pruneda
authors.forename: Russell S
authors.initials: RS
authors.lastname: Lo
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Fowler
authors.forename: Helen J
authors.initials: HJ
authors.lastname: Kim
authors.forename: Joseph B
authors.initials: JB
authors.lastname: Hiatt
authors.forename: Jay
authors.initials: J
authors.lastname: Shendure
authors.forename: Peter S
authors.initials: PS
authors.lastname: Brzovic
authors.forename: Stanley
authors.initials: S
authors.lastname: Fields
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Klevit
mesh_terms: D000494:Allosteric Regulation / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D061505:Cell Surface Display Techniques; D057166:High-Throughput Screening Assays / Q000379:methods*; D047468:Immunoprecipitation; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D019906:Nuclear Magnetic Resonance, Biomolecular; D009841:Oligonucleotides / Q000235:genetics; D016415:Sequence Alignment; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics* / Q000378:metabolism
chemical_list: D009841:Oligonucleotides; D025801:Ubiquitin; C471292:Ube4b protein, mouse; D044767:Ubiquitin-Protein Ligases