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In Vitro Analysis of E3 Ubiquitin Ligase Function
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Structural basis for E4 enzyme Ufd2-catalyzed K48/K29 branched ubiquitin chains
Zebin Tong1,2,3, Xiangwei Wu1,2, Hongyi Cai1,2
1Institute of Translational Medicine, School of Pharmaceutical Sciences, School of Chemistry and Chemical Engineering, National Center for Translational Medicine (Shanghai), Shanghai Key Laboratory for Antibody-Drug Conjugates with Innovative Target, Shanghai Jiao Tong University, Shanghai, China.
Nature Chemical Biology
|August 15, 2025
Summary
The E4 enzyme Ufd2 builds K48/K29 branched ubiquitin chains using a novel binding domain. Structural studies reveal how Ufd2 and Ubc4 cooperate to assemble these complex ubiquitin signals.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- E4 enzymes extend ubiquitin chains beyond the E1-E2-E3 pathway.
- Ufd2 is the first identified E4 enzyme, known for K48/K29 branched ubiquitin chains.
- The structural mechanism of Ufd2-mediated branching was previously unknown.
Purpose of the Study:
- To elucidate the structural mechanism of Ufd2 in catalyzing K48/K29 branched ubiquitin chains.
- To understand the spatial cooperation between ubiquitin-modifying enzymes.
Main Methods:
- Chemical biology techniques
- Cryo-electron microscopy (cryo-EM)
- Visualization of stable enzyme-ubiquitin intermediates
Main Results:
- Identified a novel K29 diubiquitin binding domain in Ufd2's core region.
- Revealed Ufd2's interaction with proximal and distal ubiquitin, orienting substrates for Ubc4.
- Discovered a dimeric conformation of Ufd2 and Ubc4 stabilizing interactions during branching.
Conclusions:
- Provided mechanistic insights into K48/K29 branched ubiquitin chain assembly by Ufd2.
- Highlighted the importance of spatial arrangement and enzyme cooperation in ubiquitin signaling.
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