Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3

Hari B. Kamadurai; Yu Qiu; Alan Deng; Joseph S. Harrison; Chris MacDonald; Marcelo Actis; Patrick Rodrigues; Darcie J. Miller; Judith Souphron; Steven M. Lewis; Igor Kurinov; Naoaki Fujii; Michal Hammel; Robert Piper; Brian Kuhlman; Brenda A. Schulman

doi:10.7554/eLife.00828

Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3

Hari B. Kamadurai, Yu Qiu, Alan Deng, Joseph S. Harrison, Chris MacDonald, Marcelo Actis, Patrick Rodrigues, Darcie J. Miller, Judith Souphron, Steven M. Lewis, Igor Kurinov, Naoaki Fujii, Michal Hammel, Robert Piper, Brian Kuhlman, Brenda A. Schulman

Biology

Research output: Contribution to journal › Article › peer-review

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.

Original language	English
Pages (from-to)	e00828
Journal	eLife
Volume	2
DOIs	https://doi.org/10.7554/eLife.00828
Publication status	Published - 2013

Keywords

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

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10.7554/eLife.00828

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title = "Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3",

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",

author = "Kamadurai, {Hari B.} and Yu Qiu and Alan Deng and Harrison, {Joseph S.} and Chris MacDonald and Marcelo Actis and Patrick Rodrigues and Miller, {Darcie J.} and Judith Souphron and Lewis, {Steven M.} and Igor Kurinov and Naoaki Fujii and Michal Hammel and Robert Piper and Brian Kuhlman and Schulman, {Brenda A.}",

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doi = "10.7554/eLife.00828",

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pages = "e00828",

journal = "eLife",

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T1 - Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3

AU - Kamadurai, Hari B.

AU - Qiu, Yu

AU - Deng, Alan

AU - Harrison, Joseph S.

AU - MacDonald, Chris

AU - Actis, Marcelo

AU - Rodrigues, Patrick

AU - Miller, Darcie J.

AU - Souphron, Judith

AU - Lewis, Steven M.

AU - Kurinov, Igor

AU - Fujii, Naoaki

AU - Hammel, Michal

AU - Piper, Robert

AU - Kuhlman, Brian

AU - Schulman, Brenda A.

PY - 2013

Y1 - 2013

N2 - 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.

AB - 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.

KW - E2 conjugating enzyme

KW - E3 ligase

KW - HECT

KW - NEDD4

KW - Rsp5

KW - S. cerevisiae

KW - ubiquitin

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U2 - 10.7554/eLife.00828

DO - 10.7554/eLife.00828

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SN - 2050-084X

VL - 2

SP - e00828

JO - eLife

JF - eLife

ER -

Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3

Abstract

Keywords

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