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

TAB2 and TAB3 activate the Jun N‐terminal kinase and nuclear factor‐κB 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 Å 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.

[1]  Lan Huang,et al.  Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry. , 2008, Journal of proteome research.

[2]  J. Abrahams,et al.  Methods used in the structure determination of bovine mitochondrial F1 ATPase. , 1996, Acta crystallographica. Section D, Biological crystallography.

[3]  Eric Blanc,et al.  Automated structure solution with autoSHARP. , 2007, Methods in molecular biology.

[4]  M. Bienz,et al.  Trabid, a new positive regulator of Wnt-induced transcription with preference for binding and cleaving K63-linked ubiquitin chains. , 2008, Genes & development.

[5]  Christine Yu,et al.  Ubiquitin Chain Editing Revealed by Polyubiquitin Linkage-Specific Antibodies , 2008, Cell.

[6]  R. Kind,et al.  The Nature of the 660-Kilometer Upper-Mantle Seismic Discontinuity from Precursors to the PP Phase , 1996, Science.

[7]  G. Bricogne,et al.  [27] Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. , 1997, Methods in enzymology.

[8]  O. Nureki,et al.  Structural basis for specific cleavage of Lys 63-linked polyubiquitin chains , 2008, Nature.

[9]  Richard J Morris,et al.  ARP/wARP and automatic interpretation of protein electron density maps. , 2003, Methods in enzymology.

[10]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[11]  Zhijian J. Chen,et al.  Direct Activation of Protein Kinases by Unanchored Polyubiquitin Chains , 2009, Nature.

[12]  S. Akira,et al.  Involvement of linear polyubiquitylation of NEMO in NF-κB activation , 2009, Nature Cell Biology.

[13]  K D Cowtan,et al.  Improvement of macromolecular electron-density maps by the simultaneous application of real and reciprocal space constraints. , 1993, Acta crystallographica. Section D, Biological crystallography.

[14]  Zhijian J. Chen,et al.  Activation of the IκB Kinase Complex by TRAF6 Requires a Dimeric Ubiquitin-Conjugating Enzyme Complex and a Unique Polyubiquitin Chain , 2000, Cell.

[15]  J. Mizukami,et al.  Phosphorylation‐dependent activation of TAK1 mitogen‐activated protein kinase kinase kinase by TAB1 , 2000, FEBS letters.

[16]  J. Tavernier,et al.  TLR-4, IL-1R and TNF-R signaling to NF-κB: variations on a common theme , 2008, Cellular and Molecular Life Sciences.

[17]  Zhijian J. Chen,et al.  Ubiquitin in NF-kappaB signaling. , 2009, Chemical reviews.

[18]  David Komander,et al.  Molecular discrimination of structurally equivalent Lys 63‐linked and linear polyubiquitin chains , 2009, EMBO reports.

[19]  Zhijian J. Chen,et al.  The role of ubiquitin in NF-kappaB regulatory pathways. , 2009, Annual review of biochemistry.

[20]  K. Hofmann.  Ubiquitin-binding domains and their role in the DNA damage response. , 2009, DNA repair.

[21]  J. Ninomiya-Tsuji,et al.  The kinase TAK1 can activate the NIK-IκB as well as the MAP kinase cascade in the IL-1 signalling pathway , 1999, Nature.

[22]  A. Vagin,et al.  MOLREP: an Automated Program for Molecular Replacement , 1997 .

[23]  Michael Assfalg,et al.  Structural determinants for selective recognition of a Lys48-linked polyubiquitin chain by a UBA domain. , 2005, Molecular cell.

[24]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[25]  John Rush,et al.  Quantitative Proteomics Reveals the Function of Unconventional Ubiquitin Chains in Proteasomal Degradation , 2009, Cell.

[26]  P. Cohen,et al.  TAB3, a new binding partner of the protein kinase TAK1. , 2004, The Biochemical journal.

[27]  R. Gaynor,et al.  Role of the TAB2‐related protein TAB3 in IL‐1 and TNF signaling , 2003, The EMBO journal.

[28]  Joshua J. Sims,et al.  Linkage-specific avidity defines the lysine 63-linked polyubiquitin-binding preference of rap80. , 2009, Molecular cell.

[29]  Nobuhiro Suzuki,et al.  Specific Recognition of Linear Ubiquitin Chains by NEMO Is Important for NF-κB Activation , 2009, Cell.

[30]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[31]  A. Sorkin,et al.  Regulation of receptors and transporters by ubiquitination: new insights into surprisingly similar mechanisms. , 2007, Molecular interventions.

[32]  M. Rapé,et al.  The Multiple Layers of Ubiquitin-Dependent Cell Cycle Control , 2009 .

[33]  Zhijian J. Chen,et al.  TAK1 is a ubiquitin-dependent kinase of MKK and IKK , 2001, Nature.

[34]  G. Sheldrick,et al.  SHELXL: high-resolution refinement. , 1997, Methods in enzymology.

[35]  C. Kojima,et al.  pCold-GST vector: a novel cold-shock vector containing GST tag for soluble protein production. , 2008, Protein expression and purification.

[36]  Kazuhiro Iwai,et al.  Linear polyubiquitination: a new regulator of NF‐κB activation , 2009, EMBO reports.

[37]  P. Loll,et al.  Crystal structures of Lys‐63‐linked tri‐ and di‐ubiquitin reveal a highly extended chain architecture , 2009, Proteins.

[38]  Su-Chang Lin,et al.  TAK1-dependent Signaling Requires Functional Interaction with TAB2/TAB3* , 2006, Journal of Biological Chemistry.

[39]  Yusuke Sato,et al.  Structural basis for specific recognition of Lys 63‐linked polyubiquitin chains by tandem UIMs of RAP80 , 2009, The EMBO journal.

[40]  Scott D Emr,et al.  Ubiquitin interactions of NZF zinc fingers , 2004, The EMBO journal.

[41]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[42]  Zhijian J. Chen,et al.  TAB2 and TAB3 activate the NF-kappaB pathway through binding to polyubiquitin chains. , 2004, Molecular cell.

[43]  Zhijian J. Chen,et al.  Nonproteolytic functions of ubiquitin in cell signaling. , 2009, Molecular cell.

[44]  O. Nureki,et al.  Structural basis for specific cleavage of Lys 63-linked polyubiquitin chains , 2008, Nature.

[45]  W. Sundquist,et al.  Structure and Ubiquitin Interactions of the Conserved Zinc Finger Domain of Npl4* , 2003, Journal of Biological Chemistry.

[46]  A. Ashworth,et al.  The structure of the CYLD USP domain explains its specificity for Lys63-linked polyubiquitin and reveals a B box module. , 2008, Molecular cell.

[47]  Merlin Crossley,et al.  Sticky fingers: zinc-fingers as protein-recognition motifs. , 2007, Trends in biochemical sciences.

[48]  A. Ciechanover,et al.  The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. , 2002, Physiological reviews.

[49]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[50]  K. Irie,et al.  TAB1: An Activator of the TAK1 MAPKKK in TGF-β Signal Transduction , 1996, Science.

[51]  G. Warren,et al.  Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and Ufd1–Npl4 , 2002, The EMBO journal.

[52]  Tao Wang,et al.  Evidence for bidentate substrate binding as the basis for the K48 linkage specificity of otubain 1. , 2009, Journal of molecular biology.