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Updated: May 12, 2026
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In Vitro Analysis of E3 Ubiquitin Ligase Function
Published on: May 14, 2021
Photocross-linking activity-based probes to capture the dynamics of ubiquitin RING E3 ligase interactions
Sarah F Chandler1, Michael H Tatham1, Emma Branigan1
1Division of Molecular, Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
The Biochemical Journal
|May 11, 2026
Summary
Researchers developed a novel activity-based probe (ABP) using a photoactivatable crosslinker attached to ubiquitin. This tool monitors interactions between E2 enzymes and RING E3 ligases, revealing crucial insights into ubiquitination processes.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Ubiquitination is crucial for cellular processes, with RING E3 ligases providing specificity.
- RING E3 ligases mediate ubiquitin transfer by stabilizing the E2-ubiquitin linkage for substrate attack.
Purpose of the Study:
- To develop a novel method for monitoring E2-E3 ligase interactions.
- To investigate the structural dynamics and interaction interfaces of RING E3 ligase complexes.
Main Methods:
- Utilized an N-Maleimido Diazirine (NMD) photoactivatable crosslinker attached to ubiquitin (Ub).
- Created an activity-based probe (ABP) by linking NMD-Ub to an E2 enzyme.
- Employed crosslinking mass spectrometry to identify interaction regions between ubiquitin and RING E3s.
Main Results:
- Identified specific interaction regions between ubiquitin and various RING E3 ligases.
- Validated existing crystal structures and evaluated in silico models of E2-E3 complexes.
- Provided insights into conformational flexibility within E2-E3 complexes, difficult to study by other methods.
Conclusions:
- NMD-Ub-E2 ABPs are effective tools for studying E2-E3 ligase interactions and complex conformations.
- This methodology offers potential for broader applications within and beyond the ubiquitin system.
