title: Ubiquitin binding to A20 ZnF4 is required for modulation of NF-κB signaling.
abstract: Inactivating mutations in the ubiquitin (Ub) editing protein A20 promote persistent nuclear factor (NF)-κB signaling and are genetically linked to inflammatory diseases and hematologic cancers. A20 tightly regulates NF-κB signaling by acting as an Ub editor, removing K63-linked Ub chains and mediating addition of Ub chains that target substrates for degradation. However, a precise molecular understanding of how A20 modulates this pathway remains elusive. Here, using structural analysis, domain mapping, and functional assays, we show that A20 zinc finger 4 (ZnF4) does not directly interact with E2 enzymes but instead can bind mono-Ub and K63-linked poly-Ub. Mutations to the A20 ZnF4 Ub-binding surface result in decreased A20-mediated ubiquitination and impaired regulation of NF-κB signaling. Collectively, our studies illuminate the mechanistically distinct but biologically interdependent activities of the A20 ZnF and ovarian tumor (OTU) domains that are inherent to the Ub editing process and, ultimately, to regulation of NF-κB signaling.
authors.affiliation: Department of Structural Biology, Genentech, Inc, 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Ivan
authors.initials: I
authors.lastname: Bosanac
authors.forename: Ingrid E
authors.initials: IE
authors.lastname: Wertz
authors.forename: Borlan
authors.initials: B
authors.lastname: Pan
authors.forename: Christine
authors.initials: C
authors.lastname: Yu
authors.forename: Saritha
authors.initials: S
authors.lastname: Kusam
authors.forename: Cynthia
authors.initials: C
authors.lastname: Lam
authors.forename: Lilian
authors.initials: L
authors.lastname: Phu
authors.forename: Qui
authors.initials: Q
authors.lastname: Phung
authors.forename: Brigitte
authors.initials: B
authors.lastname: Maurer
authors.forename: David
authors.initials: D
authors.lastname: Arnott
authors.forename: Donald S
authors.initials: DS
authors.lastname: Kirkpatrick
authors.forename: Vishva M
authors.initials: VM
authors.lastname: Dixit
authors.forename: Sarah G
authors.initials: SG
authors.lastname: Hymowitz
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D016328:NF-kappa B / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D025821:Polyubiquitin / Q000378:metabolism; D011485:Protein Binding; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D013379:Substrate Specificity; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: D016328:NF-kappa B; D009687:Nuclear Proteins; D025801:Ubiquitin; D025821:Polyubiquitin; C097308:UBE2D1 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D008239:Lysine