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  NleG Type 3 Effectors from Enterohaemorrhagic Escherichia coli Are U-Box E3 Ubiquitin Ligases

Wu, B., Skarina, T., Yee, A., Jobin, M.-C., DiLeo, R., Semesi, A., et al. (2010). NleG Type 3 Effectors from Enterohaemorrhagic Escherichia coli Are U-Box E3 Ubiquitin Ligases. PLoS Pathogens, 6(6): e1000960. doi:10.1371/journal.ppat.1000960.

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Wu, Bin1, 2, Author
Skarina, Tatiana3, Author
Yee, Adelinda1, 2, Author
Jobin, Marie-Claude2, 3, Author
DiLeo, Rosa3, Author
Semesi, Anthony1, 2, Author
Farès, Christophe1, 2, Author           
Lemak, Alexander1, 2, Author
Coombes, Brian K.4, Author
Arrowsmith, Cheryl H.1, 2, 3, Author
Singer, Alexander U.2, 3, Author
Savchenko, Alexei2, 3, Author
1Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, Toronto, Ontario, Canada, ou_persistent22              
2Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada , ou_persistent22              
3 Banting and Best Department for Medical Research, University of Toronto, C.H. Best Institute, Toronto, Ontario, Canada , ou_persistent22              
4 Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada, ou_persistent22              


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 Abstract: NleG homologues constitute the largest family of type 3 effectors delivered by pathogenic E. coli, with fourteen members in the enterohaemorrhagic (EHEC) O157:H7 strain alone. Identified recently as part of the non-LEE-encoded (Nle) effector set, this family remained uncharacterised and shared no sequence homology to other proteins including those of known function. The C-terminal domain of NleG2-3 (residues 90 to 191) is the most conserved region in NleG proteins and was solved by NMR. Structural analysis of this structure revealed the presence of a RING finger/U-box motif. Functional assays demonstrated that NleG2-3 as well as NleG5-1, NleG6-2 and NleG9′ family members exhibited a strong autoubiquitination activity in vitro; a characteristic usually expressed by eukaryotic ubiquitin E3 ligases. When screened for activity against a panel of 30 human E2 enzymes, the NleG2-3 and NleG5-1 homologues showed an identical profile with only UBE2E2, UBE2E3 and UBE2D2 enzymes supporting NleG activity. Fluorescence polarization analysis yielded a binding affinity constant of 56±2 µM for the UBE2D2/NleG5-1 interaction, a value comparable with previous studies on E2/E3 affinities. The UBE2D2 interaction interface on NleG2-3 defined by NMR chemical shift perturbation and mutagenesis was shown to be generally similar to that characterised for human RING finger ubiquitin ligases. The alanine substitutions of UBE2D2 residues Arg5 and Lys63, critical for activation of eukaryotic E3 ligases, also significantly decreased both NleG binding and autoubiquitination activity. These results demonstrate that bacteria-encoded NleG effectors are E3 ubiquitin ligases analogous to RING finger and U-box enzymes in eukaryotes.


Language(s): eng - English
 Dates: 2010-06-24
 Publication Status: Published online
 Pages: 17
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1371/journal.ppat.1000960
 Degree: -



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Title: PLoS Pathogens
  Other : PLoS Pathog.
Source Genre: Journal
Publ. Info: San Francisco, CA : Public Library of Science
Pages: - Volume / Issue: 6 (6) Sequence Number: e1000960 Start / End Page: - Identifier: ISSN: 1553-7366
CoNE: https://pure.mpg.de/cone/journals/resource/1000000000018830