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Structural basis for specific recognition of Lys 63-linked polyubiquitin chains by NZF domains of TAB2 and TAB3 - PubMed

  • ️Thu Jan 01 2009

Structural basis for specific recognition of Lys 63-linked polyubiquitin chains by NZF domains of TAB2 and TAB3

Yusuke Sato et al. EMBO J. 2009.

Abstract

TAB2 and TAB3 activate the Jun N-terminal kinase and nuclear factor-kappaB pathways through the specific recognition of Lys 63-linked polyubiquitin chains by its Npl4 zinc-finger (NZF) domain. Here we report crystal structures of the TAB2 and TAB3 NZF domains in complex with Lys 63-linked diubiquitin at 1.18 and 1.40 A resolutions, respectively. Both NZF domains bind to the distal ubiquitin through a conserved Thr-Phe dipeptide that has been shown to be important for the interaction of the NZF domain of Npl4 with monoubiquitin. In contrast, a surface specific to TAB2 and TAB3 binds the proximal ubiquitin. Both the distal and proximal binding sites of the TAB2 and TAB3 NZF domains recognize the Ile 44-centred hydrophobic patch on ubiquitin but do not interact with the Lys 63-linked isopeptide bond. Mutagenesis experiments show that both binding sites are required to enable binding of Lys 63-linked diubiquitin. We therefore propose a mechanism for the recognition of Lys 63-linked polyubiquitin chains by TAB2 and TAB3 NZF domains in which diubiquitin units are specifically recognized by a single NZF domain.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1

Overall structures of the TAB2- and TAB3-NZFs in complex with K63-Ub2. (A) Pull-down assays using GST-fused TAB2-, TAB3-, Npl4- and Vps36-NZFs to assess their interaction with K63-, K48- or linear Ub2. The bound proteins were analysed by SDS–PAGE and stained with Coomassie Brilliant Blue. (B) The TAB2-NZF·K63-Ub2 complex. The NZF is coloured grey. The proximal and distal ubiquitin moieties are coloured pink and cyan, respectively. Ile 44 in each ubiquitin moiety is shown as green spheres. The Nɛ atoms of lysine residues and the nitrogen atom of the N-terminal Met in the proximal ubiquitin are shown as blue spheres. The K63R mutation in the distal ubiquitin is indicated as sticks. (C) The TAB3-NZF·K63-Ub2 complex. The drawing schemes are the same as in B.

Figure 2
Figure 2

Recognition of the distal and proximal ubiquitin moieties by TAB2. Stereo view of the detailed TAB2-NZF·K63-Ub2 binding interface. The colouring schemes are the same as in Figure 1. Hydrogen bonds are indicated as dashed orange lines. The labels of the NZF and the proximal and distal ubiquitin moieties are coloured black, pink and cyan, respectively. (A) The interface between the proximal ubiquitin and the TAB2-NZF. (B) The interface between the distal ubiquitin and the TAB2-NZF.

Figure 3
Figure 3

Pull-down assays assessing the K63-Ub2 binding by proximal and distal ubiquitin binding site mutants of the TAB2-NZF. The bound K63-Ub2 molecules were analysed by SDS–PAGE and stained with Coomassie Brilliant Blue.

Figure 4
Figure 4

Pull-down assays using NZF variants containing point mutations in the Φ position of the TF/Φ motif to assess their mono- or diubiquitin binding. (A) Pull-down assays using the GST-fused TAB2-, Npl4- and Vps36-NZF mutants to assess their monoubiquitin binding. The bound monoubiquitin molecules were analysed by tricine SDS–PAGE and stained with Coomassie Brilliant Blue. (B) Pull-down assays using GST-fused TAB2-, Npl4- and Vps36-NZF mutants to assess their binding with K63-Ub2, K48-Ub2 or linear Ub2. The bound diubiquitin molecules were analysed by SDS–PAGE and stained with Coomassie Brilliant Blue.

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