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.

Submission Details

ID: NWeCj5VZ

Submitter: Shu-Ching Ou

Submission Date: Aug. 13, 2018, 1:01 p.m.

Version: 1

Publication Details
Starita LM;Pruneda JN;Lo RS;Fowler DM;Kim HJ;Hiatt JB;Shendure J;Brzovic PS;Fields S;Klevit RE,Proc Natl Acad Sci U S A (2013) Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis. PMID:23509263
Additional Information

Study Summary

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2KR4 2010-01-26 U-box domain of the E3 Ubiquitin Ligase E4B

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Ubiquitin conjugation factor E4 B Q9ES00 UBE4B_MOUSE