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Elife. 2013 Aug 8;2:e00828. doi: 10.7554/eLife.00828.

Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3.

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1
Department of Structural Biology , St Jude Children's Research Hospital , Memphis , United States.

Abstract

Ubiquitination by HECT E3 enzymes regulates myriad processes, including tumor suppression, transcription, protein trafficking, and degradation. HECT E3s use a two-step mechanism to ligate ubiquitin to target proteins. The first step is guided by interactions between the catalytic HECT domain and the E2∼ubiquitin intermediate, which promote formation of a transient, thioester-bonded HECT∼ubiquitin intermediate. Here we report that the second step of ligation is mediated by a distinct catalytic architecture established by both the HECT E3 and its covalently linked ubiquitin. The structure of a chemically trapped proxy for an E3∼ubiquitin-substrate intermediate reveals three-way interactions between ubiquitin and the bilobal HECT domain orienting the E3∼ubiquitin thioester bond for ligation, and restricting the location of the substrate-binding domain to prioritize target lysines for ubiquitination. The data allow visualization of an E2-to-E3-to-substrate ubiquitin transfer cascade, and show how HECT-specific ubiquitin interactions driving multiple reactions are repurposed by a major E3 conformational change to promote ligation. DOI:http://dx.doi.org/10.7554/eLife.00828.001.

KEYWORDS:

E2 conjugating enzyme; E3 ligase; HECT; NEDD4; Rsp5; S. cerevisiae; ubiquitin

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PMID:
23936628
PMCID:
PMC3738095
DOI:
10.7554/eLife.00828
[Indexed for MEDLINE]
Free PMC Article
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