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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    • "An MTS paramagnetic probe was conjugated at N25C or K48C site. Each of the conjugation sites was designed so that the paramagnetic probe was away from the binding partners of K63-Ub 2 , which include Rap80 tUIM domain (Sekiyama et al., 2012), TAB2 NZF domain (Kulathu et al., 2009; Sato et al., 2009b), and A20 ZnF4 domain (Bosanac et al., 2010), and therefore the conjugation does not interfere with ligand binding (Figure 1—figure supplement 2). Indeed, the binding affinities between K63-Ub 2 and tUIM or NZF remain unchanged for the paramagnetically tagged K63-Ub 2 proteins (Figure 1—figure supplement 3). "
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    ABSTRACT: A polyubiquitin comprises multiple covalently linked ubiquitins and recognizes myriad targets. Free or bound to ligands, a polyubiquitin is found in different arrangements of ubiquitin subunits. To understand the structural basis for polyubiquitin quaternary plasticity and to explore the target recognition mechanism, we characterize the conformational space of Lys63-linked diubiquitin (K63-Ub2). Refining against inter-subunit paramagnetic NMR data, we show that free K63-Ub2 exists as a dynamic ensemble comprising multiple closed and open quaternary states. The quaternary dynamics enables K63-Ub2 to be specifically recognized in a variety of signaling pathways. When binding to a target protein, one of the preexisting quaternary states is selected and stabilized. A point mutation that shifts the equilibrium between the different states modulates the binding affinities towards K63-Ub2 ligands. This conformational selection mechanism at quaternary level may be used by polyubiquitins of different lengths and linkages for target recognition.
    eLife Sciences 06/2015; 4. DOI:10.7554/eLife.05767 · 9.32 Impact Factor
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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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    ABSTRACT: The Caspase Recruitment Domain (CARD) from the innate immune receptor NOD1 was crystallized with Ubiquitin (Ub). NOD1 CARD was present as a helix-swapped homodimer similar to other structures of NOD1 CARD, and Ub monomers formed a homodimer similar in conformation to Lys48-linked di-Ub. The interaction between NOD1 CARD and Ub in the crystal was mediated by novel binding sites on each molecule. Comparisons of these sites to previously identified interaction surfaces on both molecules were made along with discussion of their potential functional significance.
    PLoS ONE 08/2014; 9(8):e104017. DOI:10.1371/journal.pone.0104017 · 3.23 Impact Factor
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    • "Additional UB-binding domain(s), such as the multiple NZFs of TRABID, are involved in targeting to substrate sites and enhance cleavage activity with longer UB chains (Licchesi et al., 2012). Furthermore, the different C-terminal linkage-specific UB chain-binding motifs (ZnF1- 7) in H. sapiens A20 are crucial for its recruitment by distinct signaling proteins and complexes (Bosanac et al., 2010; Skaug et al., 2011; Tokunaga et al., 2012; Verhelst et al., 2012; Lu et al., 2013). "
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    ABSTRACT: The reverse reaction of ubiquitylation is catalyzed by different classes of deubiquitylation enzymes (DUBs), including ovarian tumor domain (OTU)-containing DUBs; experiments using Homo sapiens proteins have demonstrated that OTU DUBs modulate various cellular processes. With the exception of OTLD1, plant OTU DUBs have not been characterized. We identified 12 Arabidopsis thaliana OTU loci and analyzed 11 of the encoded proteins in vitro to determine their preferences for the ubiquitin (UB) chains of M1, K48, and K63 linkages as well as the UB-/RUB-/SUMO-GST fusions. The A. thaliana OTU DUBs were shown to be cysteine proteases and classified into four groups with distinct linkage preferences: OTU1 (M1=K48>K63), OTU3/4/7/10 (K63>K48>M1), OTU2/9 (K48=K63), and OTU5/11/12/OTLD1 (inactive). Five active OTU DUBs (OTU3/4/7/9/10) also cleaved RUB fusion. OTU1/3/4 cleaved M1 UB chains, suggesting a possible role for M1 chains in plant cellular signaling. The different substrate specificities of the various A. thaliana OTU DUBs indicate the involvement of distinct structural elements; for example, the OTU1 oxyanion residue D89 is essential for cleaving isopeptide bond-linked chains but dispensable for M1 chains. UB-binding activities were detected only for OTU2 and OTLD1, with distinct linkage preferences. These differences in biochemical properties support the involvement of A. thaliana OTU DUBs in different functions. Moreover, based on the established phylogenetic tree, plant- and H. sapiens-specific clades exist, which suggests that the encoded proteins have taxa-specific functions. We also detected five OTU clades that are conserved across species, which suggests that the orthologs in different species within each clade are involved in conserved cellular processes, such as ERAD and DNA damage responses. However, different linkage preferences have been detected among potential cross-species OTU orthologs, indicating functional and mechanistic differentiation.
    Frontiers in Plant Science 03/2014; 5:84. DOI:10.3389/fpls.2014.00084 · 3.95 Impact Factor
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