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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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.).
The authors declare no competing financial interests.
Integrated supplementary information
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).
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.
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.
(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.
(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).
(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.
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 Figures 1–8 and Supplementary Table 1. (PDF 1864 kb)
Excel source for Table 2. (XLSX 23 kb)
Excel source for Fig. 4. (XLSX 15 kb)
Uncropped SDS-PAGE gel images for Fig. 1b, c and e. (PDF 760 kb)
Uncropped western blot for Fig. 4c. (PDF 164 kb)
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). https://doi.org/10.1038/nsmb.3469
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