Ubiquitin Binding to A20 ZnF4 Is Required for Modulation of NF-κB Signaling

Department of Structural Biology, Genentech, Inc, 1 DNA Way, South San Francisco, CA 94080, USA.
Molecular cell (Impact Factor: 14.02). 11/2010; 40(4):548-57. DOI: 10.1016/j.molcel.2010.10.009
Source: PubMed


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.

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Available from: Vishva Dixit, Sep 25, 2015
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    • "A different approach set up for the production of di-, tri-, and tetraUb was reported independently [53] and, in more details, by researchers at Genentech [43]. Cdc34 and Ubc13-Uev1A are used for K48-and K63-linkages, respectively [43]. "
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    • "Our structural analysis based on the PRE revealed that the C1 and C2 closed states utilize different binding interfaces. Importantly, the ligand-free structures of K63-Ub 2 are similar to the respective ligandbound structures (Kulathu et al., 2009; Sato et al., 2009a; Bosanac et al., 2010) (Figure 4B,C). This suggests a conformational selection mechanism for K63-Ub 2 target recognition. "
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    • "Although this binding interaction was not definitively observed in our prior NMR experiments, it was not definitively excluded either, given the low signal to noise of experiments performed in the range of low binding affinities expected for this interaction. Bosanac et al [41] reported a crystal structure of the zinc-finger domain of the protein A20 bound to three separate mono-Ubs with each site having progressively lower binding affinities. Our structure may represent even further progression to beyond the limit of detection by NMR or traditional pull-downs, where the majority of the binding events in solution represent the dominant interaction previously identified. "
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