Protein semisynthesis reveals plasticity in HECT E3 ubiquitin ligase mechanisms

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

doi:10.1038/s41557-024-01576-z

Protein semisynthesis reveals plasticity in HECT E3 ubiquitin ligase mechanisms

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

^*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

Abstract

Lys ubiquitination is catalysed by E3 ubiquitin ligases and is central to the regulation of protein stability and cell signalling in normal and disease states. There are gaps in our understanding of E3 mechanisms, and here we use protein 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 E3 ubiquitin ligase reactions, with yeast Rsp5 orthologues appearing to be possible evolutionary intermediates. We also show that the HECT-domain ubiquitin covalent intermediate appears to eject the E2 conjugating enzyme, promoting catalytic turnover. These findings provide key mechanistic insights into how protein ubiquitination occurs and provide a framework for understanding E3 functions and regulation. (Figure presented.).

Original language	English
Journal	Nature Chemistry
ISSN	1755-4330
DOIs	https://doi.org/10.1038/s41557-024-01576-z
Publication status	Accepted/In press - 2024
Externally published	Yes

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title = "Protein semisynthesis reveals plasticity in HECT E3 ubiquitin ligase mechanisms",

abstract = "Lys ubiquitination is catalysed by E3 ubiquitin ligases and is central to the regulation of protein stability and cell signalling in normal and disease states. There are gaps in our understanding of E3 mechanisms, and here we use protein 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 E3 ubiquitin ligase reactions, with yeast Rsp5 orthologues appearing to be possible evolutionary intermediates. We also show that the HECT-domain ubiquitin covalent intermediate appears to eject the E2 conjugating enzyme, promoting catalytic turnover. These findings provide key mechanistic insights into how protein ubiquitination occurs and provide a framework for understanding E3 functions and regulation. (Figure presented.).",

author = "Hanjie Jiang and Miller, {Bryant D.} and Thibault Viennet and Hyojeon Kim and Kwangwoon Lee and Haribabu Arthanari and Cole, {Philip A.}",

note = "Publisher Copyright: {\textcopyright} The Author(s), under exclusive licence to Springer Nature Limited 2024.",

year = "2024",

doi = "10.1038/s41557-024-01576-z",

language = "English",

journal = "Nature Chemistry",

issn = "1755-4330",

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T1 - Protein semisynthesis reveals plasticity in HECT E3 ubiquitin ligase mechanisms

AU - Jiang, Hanjie

AU - Miller, Bryant D.

AU - Viennet, Thibault

AU - Kim, Hyojeon

AU - Lee, Kwangwoon

AU - Arthanari, Haribabu

AU - Cole, Philip A.

PY - 2024

Y1 - 2024

N2 - Lys ubiquitination is catalysed by E3 ubiquitin ligases and is central to the regulation of protein stability and cell signalling in normal and disease states. There are gaps in our understanding of E3 mechanisms, and here we use protein 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 E3 ubiquitin ligase reactions, with yeast Rsp5 orthologues appearing to be possible evolutionary intermediates. We also show that the HECT-domain ubiquitin covalent intermediate appears to eject the E2 conjugating enzyme, promoting catalytic turnover. These findings provide key mechanistic insights into how protein ubiquitination occurs and provide a framework for understanding E3 functions and regulation. (Figure presented.).

AB - Lys ubiquitination is catalysed by E3 ubiquitin ligases and is central to the regulation of protein stability and cell signalling in normal and disease states. There are gaps in our understanding of E3 mechanisms, and here we use protein 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 E3 ubiquitin ligase reactions, with yeast Rsp5 orthologues appearing to be possible evolutionary intermediates. We also show that the HECT-domain ubiquitin covalent intermediate appears to eject the E2 conjugating enzyme, promoting catalytic turnover. These findings provide key mechanistic insights into how protein ubiquitination occurs and provide a framework for understanding E3 functions and regulation. (Figure presented.).

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U2 - 10.1038/s41557-024-01576-z

DO - 10.1038/s41557-024-01576-z

M3 - Journal article

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SN - 1755-4330

JO - Nature Chemistry

JF - Nature Chemistry

ER -

Protein semisynthesis reveals plasticity in HECT E3 ubiquitin ligase mechanisms

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