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. 2008 Feb 15;376(2):526-40.
doi: 10.1016/j.jmb.2007.11.092. Epub 2007 Dec 4.

Molecular basis for the unique deubiquitinating activity of the NF-kappaB inhibitor A20

Su-Chang Lin  1 , Jee Y Chung, Betty Lamothe, Kanagalaghatta Rajashankar, Miao Lu, Yu-Chih Lo, Amy Y Lam, Bryant G Darnay, Hao Wu
Affiliations

Molecular basis for the unique deubiquitinating activity of the NF-kappaB inhibitor A20

Su-Chang Lin et al. J Mol Biol. .

Abstract

Nuclear factor kappaB (NF-kappaB) activation in tumor necrosis factor, interleukin-1, and Toll-like receptor pathways requires Lys63-linked nondegradative polyubiquitination. A20 is a specific feedback inhibitor of NF-kappaB activation in these pathways that possesses dual ubiquitin-editing functions. While the N-terminal domain of A20 is a deubiquitinating enzyme (DUB) for Lys63-linked polyubiquitinated signaling mediators such as TRAF6 and RIP, its C-terminal domain is a ubiquitin ligase (E3) for Lys48-linked degradative polyubiquitination of the same substrates. To elucidate the molecular basis for the DUB activity of A20, we determined its crystal structure and performed a series of biochemical and cell biological studies. The structure reveals the potential catalytic mechanism of A20, which may be significantly different from papain-like cysteine proteases. Ubiquitin can be docked onto a conserved A20 surface; this interaction exhibits charge complementarity and no steric clash. Surprisingly, A20 does not have specificity for Lys63-linked polyubiquitin chains. Instead, it effectively removes Lys63-linked polyubiquitin chains from TRAF6 without dissembling the chains themselves. Our studies suggest that A20 does not act as a general DUB but has the specificity for particular polyubiquitinated substrates to assure its fidelity in regulating NF-kappaB activation in the tumor necrosis factor, interleukin-1, and Toll-like receptor pathways.

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Figures

Figure 1
Figure 1. Overall view of the A20 structure
(a) Experimental map from Se-MAD phasing in the small unit cell and the P3221 space group. (b) Final 2Fo−Fc map of the same region in the P32 space group. Both maps are contoured at 1.0σ. (c) Ribbon diagram of A20. Secondary structures, active site residues and regions of the teakettle-shaped structure are shown. (d) Surface representation of A20 with conserved residues colored. The active site, the predicted ubiquitin-binding site and the elevated β1-α4 and the α7–α8 loops are shown.
Figure 2
Figure 2. Structure-based sequence alignment of A20
Every 10th residue in human A20 is indicated by a dot above the sequence. Regions of otubain 2 that are structurally aligned with A20 are also shown. Residues identical with human A20 are highlighted in yellow. Active site residues are highlighted in magenta. Residues subjected to mutagenesis are highlighted in green. Residues at the predicted ubiquitin-binding site are marked with *.
Figure 3
Figure 3. The A20 Active Site
(a) Active site of A20 shown in stereo. The observed distances between the proposed catalytic residues Cys103, His256 and Thr97 are labeled. (b) Superposition of the A20 active site with other cysteine proteases including papain, otubain 2, Yuh1 and HAUSP. The unusual β1–α4 loop in A20 and the corresponding loop in otubain 2 are shown. (c) Superposition of the active sites of A20 and otubain 2 in stereo. In addition to Cys103, His256 and Thr97, the proposed third catalytic residue for otubain 2, Asn226, is also shown. (d) Superposition of A20 with otubain 2, with conserved secondary structural elements labeled. (e) Superposition of the A20 active site with those of otubain 2, HAUSP and Yuh1. Note that the third catalytic residues for otubain 2, HAUSP and Yuh1 are Asn226, Asp481 and Asp181, respectively.
Figure 4
Figure 4. The predicted interaction of A20 with ubiquitin
(a) and (b) Mapping of conserved residues on the A20 surface. The location of the active site is circled. (c) Ribbon diagram of A20 with modeled ubiquitins. The locations of the ubiquitin based on the HAUSP-ubiquitin and the Yuh1-ubiquitin complexes are shown in gray and orange, respectively. The location of the ubiquitin after adjustment to avoid steric clash is shown in yellow. (d) Surface presentation of A20 shown in complex with the modeled ubiquitin in a ribbon diagram. The active site is circled. (e) Electrostatic surface presentation of A20 shown in complex with the modeled ubiquitin in a ribbon diagram. The ubiquitin-binding site is mostly negatively charged. (f) and (g) The model of the A20-ubiquitin complex shown with A20 in a ribbon diagram and the ubiquitin in a electrostatic surface representation. The positive electrostatic potential of the side of ubiquitin in contact with A20 is shown. (h) The proposed oxyanion hole construction of A20 by the main chain amides of Cys103, Gly101 and Asp100. The corresponding regions in Yuh1 and HAUSP are superimposed and shown.
Figure 5
Figure 5. A20-mediated de-ubiquitination
(a) Cleavage by A20 of Ub-AMC in a series of concentrations. The cleavage of Ub-AMC by the same concentration of Yuh1 is for the range between approximately 200 to 600 seconds to show complete conversion of Ub-AMC. (b) Time-dependent cleavage of Lys48- (left panel) and Lys63-linked (right panel) tetraubiquitin by A20. (c) De-ubiquitination of Lys63-linked polyubiquitinated GST-TRAF6 by A20 at different concentrations. The active site mutant C103A was used as a negative control. (d) A time course for de-ubiquitination of Lys63-linked polyubiquitinated GST-TRAF6 by A20. De-ubiquitination is exemplified by the appearance of ubiquitin chains in the supernatant of the glutathione beads. (e) Co-immunoprecipitation of Flag-tagged TRAF6 and A20 (left) and de-ubiquitination of Flag-tagged TRAF6 by A20 in HEK293 cells (right).
Figure 6
Figure 6. Structure-based mutational analyses
(a) Cleavage of Lys48-linked polyubiquitin chains by wildtype and mutant A20. (b) Cleavage of Lys48-linked diubiquitin by wildtype and mutant A20. (c) De-ubiquitination of Lys63-linked polyubiquitinated GST-TRAF6 by wildtype and mutant A20. The left panel shows the appearance of ubiquitin chains in the supernatant of the glutathione beads. The right panel shows the decrease in polyubiquitination of GST-TRAF6 bound to the glutathione beads.
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References

    1. Jaattela M, Mouritzen H, Elling F, Bastholm L. A20 zinc finger protein inhibits TNF and IL-1 signaling. J Immunol. 1996;156:1166–1173. - PubMed
    1. Cooper JT, Stroka DM, Brostjan C, Palmetshofer A, Bach FH, Ferran C. A20 blocks endothelial cell activation through a NF-kappaB-dependent mechanism. J Biol Chem. 1996;271:18068–18073. - PubMed
    1. Ferran C, Stroka DM, Badrichani AZ, Cooper JT, Bach FH. Adenovirus-mediated gene transfer of A20 renders endothelial cells resistant to activation: a means of evaluating the role of endothelial cell activation in xenograft rejection. Transplant Proc. 1997;29:879–880. - PubMed
    1. Grey ST, Arvelo MB, Hasenkamp W, Bach FH, Ferran C. A20 inhibits cytokine-induced apoptosis and nuclear factor kappaB-dependent gene activation in islets. J Exp Med. 1999;190:1135–1146. - PMC - PubMed
    1. Beyaert R, Heyninck K, Van Huffel S. A20 and A20-binding proteins as cellular inhibitors of nuclear factor-kappa B-dependent gene expression and apoptosis. Biochem Pharmacol. 2000;60:1143–1151. - PubMed

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