Activity-based E3 ligase profiling uncovers an E3 ligase with esterification activity

Kuan-Chuan Pao, Nicola T. Wood, Axel Knebel, Karim Rafie, Mathew Stanley, Peter D. Mabbitt, Ramasubramanian Sundaramoorthy, Kay Hofmann, Daan M. F. van Aalten, Satpal Virdee (Lead / Corresponding author)

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Abstract

Ubiquitination is initiated by transfer of ubiquitin (Ub) from a ubiquitin-activating enzyme (E1) to a ubiquitin-conjugating enzyme (E2), producing a covalently linked intermediate (E2-Ub) 1 . Ubiquitin ligases (E3s) of the 'really interesting new gene' (RING) class recruit E2-Ub via their RING domain and then mediate direct transfer of ubiquitin to substrates 2 . By contrast, 'homologous to E6-AP carboxy terminus' (HECT) E3 ligases undergo a catalytic cysteine-dependent transthiolation reaction with E2-Ub, forming a covalent E3-Ub intermediate3,4. Additionally, RING-between-RING (RBR) E3 ligases have a canonical RING domain that is linked to an ancillary domain. This ancillary domain contains a catalytic cysteine that enables a hybrid RING-HECT mechanism 5 . Ubiquitination is typically considered a post-translational modification of lysine residues, as there are no known human E3 ligases with non-lysine activity. Here we perform activity-based protein profiling of HECT or RBR-like E3 ligases and identify the neuron-associated E3 ligase MYCBP2 (also known as PHR1) as the apparent single member of a class of RING-linked E3 ligase with esterification activity and intrinsic selectivity for threonine over serine. MYCBP2 contains two essential catalytic cysteine residues that relay ubiquitin to its substrate via thioester intermediates. Crystallographic characterization of this class of E3 ligase, which we designate RING-Cys-relay (RCR), provides insights into its mechanism and threonine selectivity. These findings implicate non-lysine ubiquitination in cellular regulation of higher eukaryotes and suggest that E3 enzymes have an unappreciated mechanistic diversity.

LanguageEnglish
Pages381-385
Number of pages5
JournalNature
Volume556
DOIs
StatePublished - 11 Apr 2018

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Ubiquitin-Protein Ligases
Esterification
Ubiquitin
Genes
Ubiquitination
Cysteine
Threonine
Ubiquitin-Activating Enzymes
Ubiquitin-Conjugating Enzymes
Post Translational Protein Processing
Ligases
Eukaryota
Serine
Lysine
Neurons
Enzymes

Keywords

  • Journal Article
  • Chemical tools
  • Enzymemechanisms
  • Ubiquitylation
  • X-ray crystallography

Cite this

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title = "Activity-based E3 ligase profiling uncovers an E3 ligase with esterification activity",
abstract = "Ubiquitination is initiated by transfer of ubiquitin (Ub) from a ubiquitin-activating enzyme (E1) to a ubiquitin-conjugating enzyme (E2), producing a covalently linked intermediate (E2-Ub) 1 . Ubiquitin ligases (E3s) of the 'really interesting new gene' (RING) class recruit E2-Ub via their RING domain and then mediate direct transfer of ubiquitin to substrates 2 . By contrast, 'homologous to E6-AP carboxy terminus' (HECT) E3 ligases undergo a catalytic cysteine-dependent transthiolation reaction with E2-Ub, forming a covalent E3-Ub intermediate3,4. Additionally, RING-between-RING (RBR) E3 ligases have a canonical RING domain that is linked to an ancillary domain. This ancillary domain contains a catalytic cysteine that enables a hybrid RING-HECT mechanism 5 . Ubiquitination is typically considered a post-translational modification of lysine residues, as there are no known human E3 ligases with non-lysine activity. Here we perform activity-based protein profiling of HECT or RBR-like E3 ligases and identify the neuron-associated E3 ligase MYCBP2 (also known as PHR1) as the apparent single member of a class of RING-linked E3 ligase with esterification activity and intrinsic selectivity for threonine over serine. MYCBP2 contains two essential catalytic cysteine residues that relay ubiquitin to its substrate via thioester intermediates. Crystallographic characterization of this class of E3 ligase, which we designate RING-Cys-relay (RCR), provides insights into its mechanism and threonine selectivity. These findings implicate non-lysine ubiquitination in cellular regulation of higher eukaryotes and suggest that E3 enzymes have an unappreciated mechanistic diversity.",
keywords = "Journal Article, Chemical tools, Enzymemechanisms, Ubiquitylation, X-ray crystallography",
author = "Kuan-Chuan Pao and Wood, {Nicola T.} and Axel Knebel and Karim Rafie and Mathew Stanley and Mabbitt, {Peter D.} and Ramasubramanian Sundaramoorthy and Kay Hofmann and {van Aalten}, {Daan M. F.} and Satpal Virdee",
note = "This work was funded by the Scottish Funding Council, the UK Medical Research Council (MC_UU_12016/8), BBSRC (BB/P003982/1), and pharmaceutical companies supporting the Division of Signal Transduction Therapy (Boehringer-Ingelheim, GlaxoSmithKline and Merck KGaA). D.M.F.v.A. is funded by a Wellcome Trust Investigator Award (110061).",
year = "2018",
month = "4",
day = "11",
doi = "10.1038/s41586-018-0026-1",
language = "English",
volume = "556",
pages = "381--385",
journal = "Nature",
issn = "0028-0836",
publisher = "Nature Publishing Group",

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TY - JOUR

T1 - Activity-based E3 ligase profiling uncovers an E3 ligase with esterification activity

AU - Pao,Kuan-Chuan

AU - Wood,Nicola T.

AU - Knebel,Axel

AU - Rafie,Karim

AU - Stanley,Mathew

AU - Mabbitt,Peter D.

AU - Sundaramoorthy,Ramasubramanian

AU - Hofmann,Kay

AU - van Aalten,Daan M. F.

AU - Virdee,Satpal

N1 - This work was funded by the Scottish Funding Council, the UK Medical Research Council (MC_UU_12016/8), BBSRC (BB/P003982/1), and pharmaceutical companies supporting the Division of Signal Transduction Therapy (Boehringer-Ingelheim, GlaxoSmithKline and Merck KGaA). D.M.F.v.A. is funded by a Wellcome Trust Investigator Award (110061).

PY - 2018/4/11

Y1 - 2018/4/11

N2 - Ubiquitination is initiated by transfer of ubiquitin (Ub) from a ubiquitin-activating enzyme (E1) to a ubiquitin-conjugating enzyme (E2), producing a covalently linked intermediate (E2-Ub) 1 . Ubiquitin ligases (E3s) of the 'really interesting new gene' (RING) class recruit E2-Ub via their RING domain and then mediate direct transfer of ubiquitin to substrates 2 . By contrast, 'homologous to E6-AP carboxy terminus' (HECT) E3 ligases undergo a catalytic cysteine-dependent transthiolation reaction with E2-Ub, forming a covalent E3-Ub intermediate3,4. Additionally, RING-between-RING (RBR) E3 ligases have a canonical RING domain that is linked to an ancillary domain. This ancillary domain contains a catalytic cysteine that enables a hybrid RING-HECT mechanism 5 . Ubiquitination is typically considered a post-translational modification of lysine residues, as there are no known human E3 ligases with non-lysine activity. Here we perform activity-based protein profiling of HECT or RBR-like E3 ligases and identify the neuron-associated E3 ligase MYCBP2 (also known as PHR1) as the apparent single member of a class of RING-linked E3 ligase with esterification activity and intrinsic selectivity for threonine over serine. MYCBP2 contains two essential catalytic cysteine residues that relay ubiquitin to its substrate via thioester intermediates. Crystallographic characterization of this class of E3 ligase, which we designate RING-Cys-relay (RCR), provides insights into its mechanism and threonine selectivity. These findings implicate non-lysine ubiquitination in cellular regulation of higher eukaryotes and suggest that E3 enzymes have an unappreciated mechanistic diversity.

AB - Ubiquitination is initiated by transfer of ubiquitin (Ub) from a ubiquitin-activating enzyme (E1) to a ubiquitin-conjugating enzyme (E2), producing a covalently linked intermediate (E2-Ub) 1 . Ubiquitin ligases (E3s) of the 'really interesting new gene' (RING) class recruit E2-Ub via their RING domain and then mediate direct transfer of ubiquitin to substrates 2 . By contrast, 'homologous to E6-AP carboxy terminus' (HECT) E3 ligases undergo a catalytic cysteine-dependent transthiolation reaction with E2-Ub, forming a covalent E3-Ub intermediate3,4. Additionally, RING-between-RING (RBR) E3 ligases have a canonical RING domain that is linked to an ancillary domain. This ancillary domain contains a catalytic cysteine that enables a hybrid RING-HECT mechanism 5 . Ubiquitination is typically considered a post-translational modification of lysine residues, as there are no known human E3 ligases with non-lysine activity. Here we perform activity-based protein profiling of HECT or RBR-like E3 ligases and identify the neuron-associated E3 ligase MYCBP2 (also known as PHR1) as the apparent single member of a class of RING-linked E3 ligase with esterification activity and intrinsic selectivity for threonine over serine. MYCBP2 contains two essential catalytic cysteine residues that relay ubiquitin to its substrate via thioester intermediates. Crystallographic characterization of this class of E3 ligase, which we designate RING-Cys-relay (RCR), provides insights into its mechanism and threonine selectivity. These findings implicate non-lysine ubiquitination in cellular regulation of higher eukaryotes and suggest that E3 enzymes have an unappreciated mechanistic diversity.

KW - Journal Article

KW - Chemical tools

KW - Enzymemechanisms

KW - Ubiquitylation

KW - X-ray crystallography

U2 - 10.1038/s41586-018-0026-1

DO - 10.1038/s41586-018-0026-1

M3 - Article

VL - 556

SP - 381

EP - 385

JO - Nature

T2 - Nature

JF - Nature

SN - 0028-0836

ER -