Structural basis for specific cleavage of Lys6-linked polyubiquitin chains by USP30


Parkin ubiquitin (Ub) ligase (also known as PARK2) ubiquitinates damaged mitochondria for their clearance and quality control. USP30 deubiquitinase opposes parkin-mediated Ub-chain formation on mitochondria by preferentially cleaving Lys6-linked Ub chains. Here, we report the crystal structure of zebrafish USP30 in complex with a Lys6-linked diubiquitin (diUb or Ub2) at 1.87-Å resolution. The distal Ub-recognition mechanism of USP30 is similar to those of other USP family members, whereas Phe4 and Thr12 of the proximal Ub are recognized by a USP30-specific surface. Structure-based mutagenesis showed that the interface with the proximal Ub is critical for the specific cleavage of Lys6-linked Ub chains, together with the noncanonical catalytic triad composed of Cys-His-Ser. The structural findings presented here reveal a mechanism for Lys6-linkage-specific deubiquitination.

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Figure 1: Characterization of zUSP30.
Figure 2: Crystal structure of zUSP30 in complex with Lys6-Ub2.
Figure 3: Noncanonical catalytic triad of zUSP30.
Figure 4: Deubiquitination of mitochondria by hUSP30.
Figure 5: Cleavage of Ub3 and phosphorylated Ub chains by zUSP30.

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NCBI Reference Sequence

Protein Data Bank


  1. 1

    Nunnari, J. & Suomalainen, A. Mitochondria: in sickness and in health. Cell 148, 1145–1159 (2012).

  2. 2

    Turrens, J.F. Mitochondrial formation of reactive oxygen species. J. Physiol. (Lond.) 552, 335–344 (2003).

  3. 3

    Kubli, D.A. & Gustafsson, A.B. Mitochondria and mitophagy: the yin and yang of cell death control. Circ. Res. 111, 1208–1221 (2012).

  4. 4

    Saiki, S., Sato, S. & Hattori, N. Molecular pathogenesis of Parkinson's disease: update. J. Neurol. Neurosurg. Psychiatry 83, 430–436 (2012).

  5. 5

    Yamano, K., Matsuda, N. & Tanaka, K. The ubiquitin signal and autophagy: an orchestrated dance leading to mitochondrial degradation. EMBO Rep. 17, 300–316 (2016).

  6. 6

    Vincow, E.S. et al. The PINK1-Parkin pathway promotes both mitophagy and selective respiratory chain turnover in vivo. Proc. Natl. Acad. Sci. USA 110, 6400–6405 (2013).

  7. 7

    Narendra, D., Walker, J.E. & Youle, R. Mitochondrial quality control mediated by PINK1 and Parkin: links to parkinsonism. Cold Spring Harb. Perspect. Biol. 4, a011338 (2012).

  8. 8

    Jin, S.M. et al. Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL. J. Cell Biol. 191, 933–942 (2010).

  9. 9

    Matsuda, N. et al. PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy. J. Cell Biol. 189, 211–221 (2010).

  10. 10

    Koyano, F. et al. Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature 510, 162–166 (2014).

  11. 11

    Yamano, K. et al. Site-specific interaction mapping of phosphorylated ubiquitin to uncover parkin activation. J. Biol. Chem. 290, 25199–25211 (2015).

  12. 12

    Wauer, T., Simicek, M., Schubert, A. & Komander, D. Mechanism of phospho-ubiquitin-induced PARKIN activation. Nature 524, 370–374 (2015).

  13. 13

    Kumar, A. et al. Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis. EMBO J. 34, 2506–2521 (2015).

  14. 14

    Sauvé, V. et al. A Ubl/ubiquitin switch in the activation of Parkin. EMBO J. 34, 2492–2505 (2015).

  15. 15

    Ordureau, A. et al. Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis. Mol. Cell 56, 360–375 (2014).

  16. 16

    Cunningham, C.N. et al. USP30 and parkin homeostatically regulate atypical ubiquitin chains on mitochondria. Nat. Cell Biol. 17, 160–169 (2015).

  17. 17

    Ordureau, A. et al. Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy. Proc. Natl. Acad. Sci. USA 112, 6637–6642 (2015).

  18. 18

    Pickrell, A.M. & Youle, R.J. The roles of PINK1, parkin, and mitochondrial fidelity in Parkinson's disease. Neuron 85, 257–273 (2015).

  19. 19

    Kitada, T. et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392, 605–608 (1998).

  20. 20

    Valente, E.M. et al. Hereditary early-onset Parkinson's disease caused by mutations in PINK1. Science 304, 1158–1160 (2004).

  21. 21

    Bingol, B. et al. The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy. Nature 510, 370–375 (2014).

  22. 22

    Nakamura, N. & Hirose, S. Regulation of mitochondrial morphology by USP30, a deubiquitinating enzyme present in the mitochondrial outer membrane. Mol. Biol. Cell 19, 1903–1911 (2008).

  23. 23

    Clague, M.J. et al. Deubiquitylases from genes to organism. Physiol. Rev. 93, 1289–1315 (2013).

  24. 24

    Wang, Y. et al. Deubiquitinating enzymes regulate PARK2-mediated mitophagy. Autophagy 11, 595–606 (2015).

  25. 25

    Liang, J.R. et al. USP30 deubiquitylates mitochondrial Parkin substrates and restricts apoptotic cell death. EMBO Rep. 16, 618–627 (2015).

  26. 26

    Wauer, T. et al. Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis. EMBO J. 34, 307–325 (2015).

  27. 27

    Faesen, A.C. et al. The differential modulation of USP activity by internal regulatory domains, interactors and eight ubiquitin chain types. Chem. Biol. 18, 1550–1561 (2011).

  28. 28

    Komander, D. et al. Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains. EMBO Rep. 10, 466–473 (2009).

  29. 29

    Sato, 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).

  30. 30

    Ernst, A. et al. A strategy for modulation of enzymes in the ubiquitin system. Science 339, 590–595 (2013).

  31. 31

    Vijay-Kumar, S., Bugg, C.E. & Cook, W.J. Structure of ubiquitin refined at 1.8 A resolution. J. Mol. Biol. 194, 531–544 (1987).

  32. 32

    Virdee, S., Ye, Y., Nguyen, D.P., Komander, D. & Chin, J.W. Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase. Nat. Chem. Biol. 6, 750–757 (2010).

  33. 33

    Hospenthal, M.K., Freund, S.M. & Komander, D. Assembly, analysis and architecture of atypical ubiquitin chains. Nat. Struct. Mol. Biol. 20, 555–565 (2013).

  34. 34

    Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D Biol. Crystallogr. 60, 2256–2268 (2004).

  35. 35

    Hu, M. et al. Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde. Cell 111, 1041–1054 (2002).

  36. 36

    Renatus, M. et al. Structural basis of ubiquitin recognition by the deubiquitinating protease USP2. Structure 14, 1293–1302 (2006).

  37. 37

    Ye, Y., Scheel, H., Hofmann, K. & Komander, D. Dissection of USP catalytic domains reveals five common insertion points. Mol. Biosyst. 5, 1797–1808 (2009).

  38. 38

    Zhang, W. et al. Contribution of active site residues to substrate hydrolysis by USP2: insights into catalysis by ubiquitin specific proteases. Biochemistry 50, 4775–4785 (2011).

  39. 39

    Bondalapati, S. et al. Chemical synthesis of phosphorylated ubiquitin and diubiquitin exposes positional sensitivities of e1-e2 enzymes and deubiquitinases. Chemistry 21, 7360–7364 (2015).

  40. 40

    Huguenin-Dezot, N. et al. Synthesis of isomeric phosphoubiquitin chains reveals that phosphorylation controls deubiquitinase activity and specificity. Cell Rep. 16, 1180–1193 (2016).

  41. 41

    Ye, Y. et al. Polyubiquitin binding and cross-reactivity in the USP domain deubiquitinase USP21. EMBO Rep. 12, 350–357 (2011).

  42. 42

    Sato, Y. et al. Structural basis for specific cleavage of Lys 63-linked polyubiquitin chains. Nature 455, 358–362 (2008).

  43. 43

    Bremm, A., Freund, S.M. & Komander, D. Lys11-linked ubiquitin chains adopt compact conformations and are preferentially hydrolyzed by the deubiquitinase Cezanne. Nat. Struct. Mol. Biol. 17, 939–947 (2010).

  44. 44

    Michel, M.A. et al. Assembly and specific recognition of k29- and k33-linked polyubiquitin. Mol. Cell 58, 95–109 (2015).

  45. 45

    Kristariyanto, Y.A. et al. Assembly and structure of Lys33-linked polyubiquitin reveals distinct conformations. Biochem. J. 467, 345–352 (2015).

  46. 46

    Pickart, C.M. & Raasi, S. Controlled synthesis of polyubiquitin chains. Methods Enzymol. 399, 21–36 (2005).

  47. 47

    Keusekotten, K. et al. OTULIN antagonizes LUBAC signaling by specifically hydrolyzing Met1-linked polyubiquitin. Cell 153, 1312–1326 (2013).

  48. 48

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

  49. 49

    Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

  50. 50

    Vagin, A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Cryst. 30, 1022–1025 (1997).

  51. 51

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

  52. 52

    Zwart, P.H. et al. Automated structure solution with the PHENIX suite. Methods Mol. Biol. 426, 419–435 (2008).

  53. 53

    Ohtake, F., Saeki, Y., Ishido, S., Kanno, J. & Tanaka, K. The K48–K63 branched ubiquitin chain regulates NF-κB signaling. Mol. Cell 64, 251–266 (2016).

  54. 54

    Tsuchiya, H., Tanaka, K. & Saeki, Y. The parallel reaction monitoring method contributes to a highly sensitive polyubiquitin chain quantification. Biochem. Biophys. Res. Commun. 436, 223–229 (2013).

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We thank J. Chen (Rockefeller University) for providing the NleL plasmid. We thank K. Iwai (Kyoto University) for providing the Ub and E2-25K plasmids. We thank the beamline staff of the biological crystallography beamlines of Photon Factory (Tsukuba, Japan) and BL41XU of SPring-8 (Hyogo, Japan) for technical help during data collection. This work was supported by JSPS/MEXT KAKENHI (JP24687012, JP15H01175 and JP16H04750 to Y. Sato, JP15J10559 to K.O., JP24112008 to Y. Saeki, JP16K18545 to K.Y., JP26000014 to K.T. and JP24247014 to S.F.), JST PRESTO (to N.M.), Takeda Science Foundation (to N.M. and K.T.), the Chieko Iwanaga Fund for Parkinson's Disease Research (to N.M.) and JST CREST (JPMJCR12M5 to S.F.).

Author information

Y. Sato designed and performed all experiments except the MS-based assays using hUSP30, and wrote the paper. K.O., Y. Saeki and A.K. performed the MS-based assays. K.Y. and N.M. established the hUSP30 KO cell line. K.T. supervised the MS-based assays and the establishment of the hUSP30 KO cell line. M.I. and Y.H. prepared reagents for DUB assays. A.Y. and S.G.-I. assisted with structure determination. All authors discussed the results. S.F. supervised the work, designed the experiments and wrote the paper.

Correspondence to Shuya Fukai.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Amino-acid sequence alignment of the USP domains of zUSP30 and hUSP21.

The Ubdist- and Ubprox-interacting residues of zUSP30 are indicated by cyan and pink squares, respectively. Residues interacting with both Ub moieties are indicated by blue squares. The catalytic triad and the Zn2+-coordinating residues are indicated by yellow and green stars, respectively. The residue number and secondary structure of zUSP30 are shown above the alignment. Secondary structure elements of the thumb, palm and fingers subdomains and insertions of zUSP30 are colored green, gray, orange and yellow, respectively. The disordered regions of zUSP30 and hUSP21 (PDBID 3I3T)30 are highlighted in purple.

Supplementary Figure 2 Fluorescence anisotropy-based affinity measurements of zUSP30 or Lys6-Ub2 mutants.

Error bars represent the standard deviations from the mean values of measurements performed in triplicate. The data were fitted to a one-site binding model to derive dissociation constants (Kd). (a) Fluorescence anisotropy-based affinity measurements of zUSP30 (C73A) mutants with Lys6-Ub2. Fluorescence polarization values are plotted as a function of the concentration of each zUSP30 (C73A) mutant. (b) Fluorescence anisotropy-based affinity measurements of zUSP30 (C73A) with Lys6-Ub2 mutants. Fluorescence polarization values are plotted as a function of the concentration of zUSP30 (C73A). (c) Summary of affinity measurements. Data are presented as mean ± standard deviation. Asterisks indicate that the actual Kd is above half of the upper limit of the substrate concentration (more than 20 μM).

Supplementary Figure 3 Fluorescence anisotropy-based kinetic analyses of zUSP30 or Ub2 mutants.

Error bars represent standard deviations from the mean values of measurements performed in triplicate. The initial rates of the DUB reaction were fitted to the Michaelis–Menten kinetic model. (a) Fluorescence anisotropy-based kinetic analyses of zUSP30 mutants for Lys6- or Lys11-Ub2. (b) Fluorescence anisotropy-based kinetic analyses of zUSP30 for Lys6-Ub2 mutants.

Supplementary Figure 4 Amino-acid sequence alignment of USP30 from representative organisms.

The drawing scheme is the same as that in Supplementary Fig. 1. 100% and more than 80% identical residues are highlighted with red backgrounds and red characters, respectively. The transmembrane domain is indicated by an orange bar.

Supplementary Figure 5 K6R mutation of Ubdist in Lys6-Ub2.

(a) Positions of all lysine residues of Ubdist in the zUSP30–Lys6-Ub2 structure. The lysine and N-terminal methionine residues of Ubdist are shown as ball-and-stick models. Lys6-Ub2 and zUSP30 are shown as cartoon and surface models, respectively. The coloring scheme is the same as that in Fig. 2. (b) Close-up view of the area around Lys6* of Ubdist (*; replaced by Arg in the present structure). The drawing scheme is the same as that in Fig. 2. Water molecules are shown as green spheres. (c) Fluorescence anisotropy-based kinetic analysis of zUSP30 for the wild-type or K6Rdist Lys6-Ub2. The drawing scheme is the same as that in Supplementary Fig. 3. (d) Fluorescence anisotropy-based affinity measurement of zUSP30 (C73A) for the wild-type or K6Rdist Lys6-Ub2. The drawing scheme is the same as that in Supplementary Fig. 2.

Supplementary Figure 6 His-Lys6-Ub3 preparation.

(a) Schematic drawings of His-Lys6-Ub3 preparation. His-Lys6-Ub3 and His-Lys6-Ub3 (R74Adist) were enzymatically synthesized by E1, UbcH7 and NleL. Lys48 of all the Ub molecules was mutated to Arg to prevent the elongation of Lys48-linked chains. Lys6 of His-Ub and His-Ub (R74A) was mutated to Arg to prevent the elongation of Lys6-linked chains on the distal end of His-Ub. See also online methods. (b) SDS-PAGE of individual reaction steps in His-Lys6-Ub3 preparation. The lanes (i)–(iv) correspond to the steps (i)–(iv) shown in (a).

Supplementary Figure 7 Ub phosphorylation at Ser65.

(a) Schematic drawings of phosphorylated Lys6-Ub2 preparation. pSer65-containing Ub (produced by Tribolium castaneum PINK1) and non-phosphorylated (native) Ub were enzymatically linked by E1, NleL and UbcH7. Lys48 of all the Ub molecules was mutated to Arg to avoid elongation of the Lys48-linked chains. For efficient production of Ub2, Lys6 of Ubdist was mutated to Arg, and Asp was attached to the C-terminus of Ubprox. See also online methods. (b) SDS-PAGE of the purified Ub and Lys6-Ub2 with or without phosphorylation. The left panel shows the result in the absence of Phos-tag, whereas the right panel shows that in the presence of Phos-tag. These data guarantee the purities of the phosphorylated samples.

Supplementary Figure 8 Difference in interface between USP and Ub.

USP and Ub are shown as surface and cartoon models, respectively. (a) zUSP30 and Lys6-Ub2. The coloring scheme is the same as that in Fig. 2. (b) hUSP21 and Ub (PDBID 3I3T)30. The coloring scheme is the same as that in Fig. 3a. (c) Zebrafish CYLD and Met1-Ub2 (PDBID 3WXF)29. CYLD, Ubdist and Ubprox are colored green cyan, blue and red, respectively. (d) Zebrafish CYLD and Lys63-Ub2 (PDBID 3WXG)29. The coloring scheme is the same as that in (c).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Table 1. (PDF 1864 kb)

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Supplementary Table 2

Excel source for Table 2. (XLSX 23 kb)

Supplementary Table 3

Excel source for Fig. 4. (XLSX 15 kb)

Supplementary Data Set 1

Uncropped SDS-PAGE gel images for Fig. 1b, c and e. (PDF 760 kb)

Supplementary Data Set 2

Uncropped western blot for Fig. 4c. (PDF 164 kb)

Supplementary Data Set 3

Uncropped SDS-PAGE gel images for Fig. 5a and d. (PDF 429 kb)

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Sato, Y., Okatsu, K., Saeki, Y. et al. Structural basis for specific cleavage of Lys6-linked polyubiquitin chains by USP30. Nat Struct Mol Biol 24, 911–919 (2017).

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