title: Covalent Inhibition of Ubc13 Affects Ubiquitin Signaling and Reveals Active Site Elements Important for Targeting.
abstract: Ubc13 is an E2 ubiquitin conjugating enzyme that functions in nuclear DNA damage signaling and cytoplasmic NF-κB signaling. Here, we present the structures of complexes of Ubc13 with two inhibitors, NSC697923 and BAY 11-7082, which inhibit DNA damage and NF-κB signaling in human cells. NSC697923 and BAY 11-7082 both inhibit Ubc13 by covalent adduct formation through a Michael addition at the Ubc13 active site cysteine. The resulting adducts of both compounds exploit a binding groove unique to Ubc13. We developed a Ubc13 mutant which resists NSC697923 inhibition and, using this mutant, we show that the inhibition of cellular DNA damage and NF-κB signaling by NSC697923 is largely due to specific Ubc13 inhibition. We propose that unique structural features near the Ubc13 active site could provide a basis for the rational development and design of specific Ubc13 inhibitors.
authors.affiliation: †Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: Curtis D
authors.initials: CD
authors.lastname: Hodge
authors.affiliation: †Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: Ross A
authors.initials: RA
authors.lastname: Edwards
authors.affiliation: †Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: Craig J
authors.initials: CJ
authors.lastname: Markin
authors.affiliation: ‡Department of Oncology, University of Alberta, Edmonton, Alberta, Canada T6G 1Z2.
authors.forename: Darin
authors.initials: D
authors.lastname: McDonald
authors.affiliation: ∥Department of Biomedical Genetics, University of Rochester, Rochester, New York 14642, United States.
authors.forename: Mary
authors.initials: M
authors.lastname: Pulvino
authors.affiliation: §Department of Anatomy and Centre for Cancer Research, The University of Hong Kong, Hong Kong, China.
authors.forename: Michael S Y
authors.initials: MS
authors.lastname: Huen
authors.affiliation: ∥Department of Biomedical Genetics, University of Rochester, Rochester, New York 14642, United States.
authors.forename: Jiyong
authors.initials: J
authors.lastname: Zhao
authors.affiliation: †Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: Leo
authors.initials: L
authors.lastname: Spyracopoulos
authors.affiliation: ‡Department of Oncology, University of Alberta, Edmonton, Alberta, Canada T6G 1Z2.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Hendzel
authors.affiliation: †Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: J N Mark
authors.initials: JN
authors.lastname: Glover
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D009570:Nitriles / Q000494:pharmacology*; D009581:Nitrofurans / Q000494:pharmacology*; D016415:Sequence Alignment; D015398:Signal Transduction / Q000187:drug effects; D013450:Sulfones / Q000494:pharmacology*; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects*
chemical_list: C000604167:2-((4-methylphenyl)sulfonyl)-5-nitrofuran; C434003:3-(4-methylphenylsulfonyl)-2-propenenitrile; D016328:NF-kappa B; D009570:Nitriles; D009581:Nitrofurans; D013450:Sulfones; D025801:Ubiquitin; C471607:UBE2N protein, human; D044763:Ubiquitin-Conjugating Enzymes