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In Vitro Analysis of E3 Ubiquitin Ligase Function
Published on: May 14, 2021
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Multisite phosphorylation dictates selective E2-E3 pairing as revealed by Ubc8/UBE2H-GID/CTLH assemblies
Jakub Chrustowicz1, Dawafuti Sherpa1, Jerry Li2
1Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried 82152, Germany.
Molecular Cell
|December 19, 2023
Summary
Phosphorylation guides E3-E2 enzyme partnerships in ubiquitylation. This study reveals how multisite phosphorylation of Ubc8/UBE2H E2 enzymes anchors them to GID/CTLH E3 ligases, ensuring specific ubiquitylation.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Ubiquitylation is a crucial post-translational modification regulated by E3 and E2 enzymes.
- The specific mechanisms governing many E3-E2 enzyme interactions, particularly for RING-family GID/CTLH E3 ubiquitin ligases and their cognate E2 enzyme Ubc8/UBE2H, are not fully understood.
- This E3-E2 complex regulates vital cellular processes, including yeast metabolic signaling and human development.
Purpose of the Study:
- To elucidate the molecular principles underlying the specific interaction between GID/CTLH E3 ubiquitin ligases and the Ubc8/UBE2H E2 enzyme.
- To investigate the role of E2 enzyme phosphorylation in mediating this E3-E2 partnership.
- To understand how this specific interaction contributes to the regulation of ubiquitylation.
Main Methods:
- Cryo-electron microscopy (cryo-EM) was employed to determine the structural basis of the E3-E2 complex.
- Biochemical assays were used to characterize the enzymatic activity and interaction interfaces.
- Cell biology techniques were utilized to study the functional consequences in a cellular context.
Main Results:
- The study revealed an unconventional catalytic assembly mechanism for the GID/CTLH-Ubc8/UBE2H complex.
- Multisite phosphorylation of Ubc8/UBE2H, targeted by CK2, specifically anchors the E2 C termini to basic patches on the E3 ligase, distinct from previously observed polyelectrostatic interactions.
- These phospho-dependent interactions occur at a distance from the catalytic sites and correlate evolutionarily, rigidifying the catalytic center and facilitating substrate engagement.
Conclusions:
- Phosphorylation-dependent multivalency is critical for establishing specific E3-E2 partnerships in ubiquitylation.
- This mechanism is antagonistic to dephosphorylation and rigidifies the catalytic machinery.
- The findings provide a detailed molecular understanding of how E3-E2 specificity is achieved, impacting cellular signaling and development.
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