Products Cited in Publications Worldwide Protein semisynthesis reveals plasticity in HECT E3 ubiquitin ligase mechanisms

Protein semisynthesis reveals plasticity in HECT E3 ubiquitin ligase mechanisms

Jiang, Hanjie; Miller, Bryant D; Viennet, Thibault; Kim, Hyojeon; Lee, Kwangwoon; Arthanari, Haribabu; Cole, Philip A

DOI: PMID:

Abstract

Lys ubiquitination is catalysed by and is central to the regulation of stability and cell signalling in normal and disease states. There are gaps in our understanding of E3 mechanisms, and here we use semisynthesis, chemical rescue, microscale thermophoresis and other biochemical approaches to dissect the role of catalytic base/acid function and conformational interconversion in HECT-domain E3 catalysis. We demonstrate that there is plasticity in the use of the terminal side chain or backbone carboxylate for proton transfer in HECT reactions, with yeast Rsp5 orthologues appearing to be possible evolutionary intermediates. We also show that the HECT-domain covalent intermediate appears to eject the E2 conjugating enzyme, promoting catalytic turnover. These fndings provide key mechanistic insights into how ubiquitination occurs and provide a framework for understanding E3 functions and regulation.

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