Structures of CYLD USP with Met1- or Lys63-linked diubiquitin reveal mechanisms for dual specificity (original) (raw)

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Acknowledgements

We thank S. Kaiser for critically reading and improving this manuscript. We also thank the beamline staff at NW12A, BL-1A, BL-5A and BL17A of the Photon Factory (Tsukuba, Japan) and BL32XU and BL41XU of SPring-8 (Hyogo, Japan) for technical help during data collection. This work was supported by Grants-in-Aid for Scientific Research on Innovative Areas 22121003 (S.F.), 22117002 (J.I.), 22117003 (M.T.), 22117006 (F.T.), 25117711 (Y. Sato) and 25112505 (Y. Sato), Grant-in-Aid for Young Scientists (A) 24687012 (Y. Sato), Grant-in-Aid for Scientific Research (A) 24247014 (S.F.) and CREST, JST (S.F.).

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Authors and Affiliations

  1. Life Science Division, Synchrotron Radiation Research Organization, University of Tokyo, Tokyo, Japan
    Yusuke Sato, Atsushi Yamagata, Sakurako Goto-Ito, Keiko Kubota & Shuya Fukai
  2. Center for Structural Biology of Challenging Proteins, Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan
    Yusuke Sato, Atsushi Yamagata, Sakurako Goto-Ito, Keiko Kubota & Shuya Fukai
  3. Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan
    Yusuke Sato, Atsushi Yamagata, Sakurako Goto-Ito, Keiko Kubota & Shuya Fukai
  4. Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan
    Eiji Goto & Fuminori Tokunaga
  5. Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan
    Yuri Shibata & Jun-ichiro Inoue
  6. Division of Cell Signaling and Molecular Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan
    Yuji Kubota & Mutsuhiro Takekawa
  7. Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Tokyo, Japan
    Jun-ichiro Inoue
  8. Core Research for Evolutionary Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo, Japan
    Shuya Fukai

Authors

  1. Yusuke Sato
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  2. Eiji Goto
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  3. Yuri Shibata
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  4. Yuji Kubota
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  5. Atsushi Yamagata
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  6. Sakurako Goto-Ito
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  7. Keiko Kubota
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  8. Jun-ichiro Inoue
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  9. Mutsuhiro Takekawa
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  10. Fuminori Tokunaga
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  11. Shuya Fukai
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Contributions

Y. Sato designed and performed all experiments except the cell-based assays with hCYLD and wrote the paper. E.G. prepared the expression vectors of hCYLD and performed the DUB assay of hCYLD and the luciferase assay with LUBAC. Y. Shibata performed the luciferase assay with TRAF6. Y.K. performed the experiment for detection of JNK1 phosphorylation. A.Y., S.G.-I. and K.K. discussed the results. J.I., M.T. and F.T. designed and supervised the cell-based assays. S.F. supervised the work, designed the experiments and wrote the paper.

Corresponding author

Correspondence toShuya Fukai.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Characterization of zCYLDΔBbox.

(a) Linkage specificity of zCYLDΔBbox. Ub2 species (10 μM) were incubated in the presence of 10 μM zCYLDΔBbox at 37 °C for 5 and 90 min and analyzed by tricine SDS-PAGE with Coomassie brilliant blue staining.

(b) Superposition of zCYLDΔBbox in the Met1 catalytic state and hCYLD (containing B-box) in the apo state (PDB:2VHF). The drawing scheme is the same as that in Fig. 1. The USP domain and the B-box domain of hCYLD are colored orange and yellow, respectively. In the Met1 catalytic state, both Ubdist and Ubprox are more than 12 Å from the likely position of the B-box domain, as estimated from the apo structure of the hCYLD USP domain. This is consistent with the B-box domain being dispensable for Met1- and Lys63-linkage-specific DUB activities of CYLD.

Supplementary Figure 2 Kinetic analyses of zCYLDΔBbox, Met1- and Lys63-Ub2 mutants.

Plots of initial velocity as a function of Met1- or Lys63-Ub2 concentration. Error bars represent standard deviations in three independent experiments. The data were fitted to the Michaelis-Menten kinetic model.

(a) Cleavage of Met1-Ub2 by mutant zCYLDΔBbox

(b) Cleavage of mutant Met1-Ub2 by zCYLDΔBbox

(c) Cleavage of Lys63-Ub2 by mutant zCYLDΔBbox

(d) Cleavage of mutant Lys63-Ub2 by zCYLDΔBbox

(e) DUB activity (_k_cat/_K_M) of mutant zCYLDΔBbox and Met1- and Lys63-Ub2. The experiments were carried out three times for each mutant except the Q711K mutant of zCYLDΔBbox (twice). Data are presented as mean ± SD. N.D. indicates non-detectable level.

Supplementary Figure 3 Structural details of zCYLDΔBbox in catalytic and precatalytic states.

(a) Comparison between the Met1 and Lys63 catalytic states. The structures in both states are superposed using zCYLDΔBbox as the reference. The drawing scheme is the same as that in Fig. 1, except that Ubprox in the Lys63 catalytic state is colored yellow.

(b) Comparison between the Met1 precatalytic states I and II. The structures of zCYLDΔBbox in these two states are superposed. Ubdist and zCYLDΔBbox are colored cyan and gray, respectively. Ubprox moieties in the Met1 precatalytic states I and II are colored green and magenta, respectively.

(c) Close-up views around Glu64 of Ubprox in the Met1 and Lys63 catalytic states. The drawing scheme is the same as that in Fig. 1.

(d) Cleavage of 10 μM Lys11- and Lys48-Ub2 by 40 μM wild-type or mutant zCYLDΔBbox at 37 °C for 90 min. Samples were analyzed by SDS-PAGE using SuperSepTM Ace, 5-20% (Wako) with Coomassie brilliant blue staining.

(e) Close-up views around catalytic triad of CYLD in the Met1 precatalytic states I and II. The drawing scheme is the same as that in Fig. 1.

Supplementary Figure 4 Sequence alignment of CYLD USP domains from representative organisms.

100% and more than 75% identical residues are highlighted with red backgrounds and red characters, respectively. Residues that interact with the Ubdist core, Ubdist tail and Ubprox are indicated by cyan squares, cyan triangles and pink squares, respectively. Filled squares and triangles indicate that the side chains are involved in the interaction, whereas open symbols indicate that the main chains but not side chains are involved in the interaction. Residues that form the catalytic triad are indicated by yellow stars. The residue number and secondary structure of zCYLDΔBbox are shown above the alignment.

Supplementary Figure 5 DUB activity of full-length hCYLD.

The full-length hCYLD was overexpressed in HEK293T cells.

(a) Linkage specificity of full-length hCYLD. All possible linkage types of Ub2 were cleaved by the full-length hCYLD.

(b) Cleavage of Met1 and Lys63 chains by full-length wild-type or mutant hCYLD

Supplementary Figure 6 Sequence and structure alignments of USP domains of CYLD, USP2, USP7 and USP21.

Structure alignment is based on superposition of the USP2, USP7 or USP21 complex on the zCYLD complex in the Met1 catalytic state using the PDBeFold server with the zCYLD USP domain as the reference (Cα r.m.s.d. values are 2.64, 2.70 and 2.72 Å over 219 residues in total, respectively).

(a) Structure-based sequence alignment of USP domains of zCYLD, human USP2 (PDB:2HD5), human USP7 (PDB:1NBF) and human USP21 (PDB:2Y5B). The drawing scheme is the same as that in Supplementary Fig. 4. Blue bars above the alignment correspond to the CYLD-specific truncations and insertion. The secondary structures of zCYLD and hUSP7 are shown above the alignment.

(b) Structure alignment of Ub molecules bound to USP2, USP7 and USP21 (colored yellow, green and blue, respectively). The USP domain of USP7 (colored red) is also shown as a representative USP domain.

(c) Structure alignment of Ub(dist) molecules bound to zCYLDΔBbox and USP7 (colored cyan and green, respectively). Ubprox and zCYLDΔBbox (colored pink and grey, respectively) are also shown.

(d) Structure alignment of zCYLDΔBbox in the Met1 catalytic state and USP7. The coloring scheme is the same as that in Supplementary Fig. 6b,c. Truncations (Fingers subdomain, the β-sheet intervening between β4 and β5 and the loop connecting β6 and β7) and an insertion (β9-β10 sheet) are indicated by blue circles.

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Sato, Y., Goto, E., Shibata, Y. et al. Structures of CYLD USP with Met1- or Lys63-linked diubiquitin reveal mechanisms for dual specificity.Nat Struct Mol Biol 22, 222–229 (2015). https://doi.org/10.1038/nsmb.2970

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