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Fig. 1 | Design, characterization and inhibition of chimeric <t>USP30.</t> a, USP30 antagonizes PINK1–parkin-mediated mitophagy. MOMP, mitochondrial outer membrane protein. b, Chemical structures of a covalent and a non-covalent USP30 inhibitor. Inhibition of USP30 to enhance mitochondrial quality control is explored as a therapeutic strategy for Parkinson’s and kidney diseases. ABPP, activity-based protein profiling. c, Crystal structure of human USP30 obtained with a previously engineered construct as the Ub-PA complex (PDB 5OHK). USP subdomains are shown in different colors. d, Architecture of full-length human USP30, the previously used c1 (named c13 in ref. 37) and the USP30 ch3 described here. See Extended Data Fig. 2 for other chimeras. e, AlphaFold2-predicted model of USP30c1 (top left), crystal structures of the catalytic domains of USP14 (PDB 2AYN, top right) and USP35 (PDB 5TXK, bottom left) and AlphaFold2-predicted model of USP30 ch3 (bottom right). Regions used for grafting are shown in corresponding colors. f, Catalytic efficiencies of the indicated USP30 constructs, determined from Ub–RhoG cleavage assays. See Extended Data Fig. 3 for raw
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Fig. 1 | Design, characterization and inhibition of chimeric USP30. a, USP30 antagonizes PINK1–parkin-mediated mitophagy. MOMP, mitochondrial outer membrane protein. b, Chemical structures of a covalent and a non-covalent USP30 inhibitor. Inhibition of USP30 to enhance mitochondrial quality control is explored as a therapeutic strategy for Parkinson’s and kidney diseases. ABPP, activity-based protein profiling. c, Crystal structure of human USP30 obtained with a previously engineered construct as the Ub-PA complex (PDB 5OHK). USP subdomains are shown in different colors. d, Architecture of full-length human USP30, the previously used c1 (named c13 in ref. 37) and the USP30 ch3 described here. See Extended Data Fig. 2 for other chimeras. e, AlphaFold2-predicted model of USP30c1 (top left), crystal structures of the catalytic domains of USP14 (PDB 2AYN, top right) and USP35 (PDB 5TXK, bottom left) and AlphaFold2-predicted model of USP30 ch3 (bottom right). Regions used for grafting are shown in corresponding colors. f, Catalytic efficiencies of the indicated USP30 constructs, determined from Ub–RhoG cleavage assays. See Extended Data Fig. 3 for raw

Journal: Nature structural & molecular biology

Article Title: Chimeric deubiquitinase engineering reveals structural basis for specific inhibition of the mitophagy regulator USP30.

doi: 10.1038/s41594-025-01534-4

Figure Lengend Snippet: Fig. 1 | Design, characterization and inhibition of chimeric USP30. a, USP30 antagonizes PINK1–parkin-mediated mitophagy. MOMP, mitochondrial outer membrane protein. b, Chemical structures of a covalent and a non-covalent USP30 inhibitor. Inhibition of USP30 to enhance mitochondrial quality control is explored as a therapeutic strategy for Parkinson’s and kidney diseases. ABPP, activity-based protein profiling. c, Crystal structure of human USP30 obtained with a previously engineered construct as the Ub-PA complex (PDB 5OHK). USP subdomains are shown in different colors. d, Architecture of full-length human USP30, the previously used c1 (named c13 in ref. 37) and the USP30 ch3 described here. See Extended Data Fig. 2 for other chimeras. e, AlphaFold2-predicted model of USP30c1 (top left), crystal structures of the catalytic domains of USP14 (PDB 2AYN, top right) and USP35 (PDB 5TXK, bottom left) and AlphaFold2-predicted model of USP30 ch3 (bottom right). Regions used for grafting are shown in corresponding colors. f, Catalytic efficiencies of the indicated USP30 constructs, determined from Ub–RhoG cleavage assays. See Extended Data Fig. 3 for raw

Article Snippet: The following sequences were used: codon-optimized human USP30 catalytic domain, Addgene 110746, UniProt Q70CQ3 with modifications described previously37 and in Supplementary Information; human USP7, UniProt Q93009; human USP14, UniProt P54578; human USP35, UniProt Q9P2H5; human CYLD, UniProt Q9NQC7.

Techniques: Inhibition, Membrane, Control, Activity Assay, Construct

Fig. 2 | Structure of USP30 in complex with NK036. a, Cartoon representation of the crystal structure of USP30ch3 bound to NK036. The compound is shown under an orange surface. b, Composite omit electron density map of NK036 in chain A (2mFo – DFc, contoured at 1σ, covering all atoms of the compound, created with simulated annealing from the final coordinates). See Extended Data Fig. 5e,f for unbiased mFo – DFc maps. c, Structure as in a with surface representation of USP30. d, Compound binding site highlighting typical USP regions involved in binding to NK036. These include the switching loop (yellow), blocking loop 1

Journal: Nature structural & molecular biology

Article Title: Chimeric deubiquitinase engineering reveals structural basis for specific inhibition of the mitophagy regulator USP30.

doi: 10.1038/s41594-025-01534-4

Figure Lengend Snippet: Fig. 2 | Structure of USP30 in complex with NK036. a, Cartoon representation of the crystal structure of USP30ch3 bound to NK036. The compound is shown under an orange surface. b, Composite omit electron density map of NK036 in chain A (2mFo – DFc, contoured at 1σ, covering all atoms of the compound, created with simulated annealing from the final coordinates). See Extended Data Fig. 5e,f for unbiased mFo – DFc maps. c, Structure as in a with surface representation of USP30. d, Compound binding site highlighting typical USP regions involved in binding to NK036. These include the switching loop (yellow), blocking loop 1

Article Snippet: The following sequences were used: codon-optimized human USP30 catalytic domain, Addgene 110746, UniProt Q70CQ3 with modifications described previously37 and in Supplementary Information; human USP7, UniProt Q93009; human USP14, UniProt P54578; human USP35, UniProt Q9P2H5; human CYLD, UniProt Q9NQC7.

Techniques: Binding Assay, Blocking Assay

Fig. 3 | Engagement of the Leu73 pocket and conformational plasticity of the switching loop underlie ligand engagement by USP30. a, Cartoon representation of the crystal structure of USP30 bound to NK036. The compound is shown under an orange surface. b, Structure of the USP30~Ub-PA complex (PDB 5OHK). Ubiquitin is shown under a yellow surface. c, Superposition of a and b. d, Close-up view of the compound binding site. Catalytic triad residues of USP30 and Leu73 of ubiquitin are labeled. The conformational change of the USP30 switching loop is indicated. e, Close-up view on the engagement of the ubiquitin Leu73 side chain by USP30, with residues forming the hydrophobic

Journal: Nature structural & molecular biology

Article Title: Chimeric deubiquitinase engineering reveals structural basis for specific inhibition of the mitophagy regulator USP30.

doi: 10.1038/s41594-025-01534-4

Figure Lengend Snippet: Fig. 3 | Engagement of the Leu73 pocket and conformational plasticity of the switching loop underlie ligand engagement by USP30. a, Cartoon representation of the crystal structure of USP30 bound to NK036. The compound is shown under an orange surface. b, Structure of the USP30~Ub-PA complex (PDB 5OHK). Ubiquitin is shown under a yellow surface. c, Superposition of a and b. d, Close-up view of the compound binding site. Catalytic triad residues of USP30 and Leu73 of ubiquitin are labeled. The conformational change of the USP30 switching loop is indicated. e, Close-up view on the engagement of the ubiquitin Leu73 side chain by USP30, with residues forming the hydrophobic

Article Snippet: The following sequences were used: codon-optimized human USP30 catalytic domain, Addgene 110746, UniProt Q70CQ3 with modifications described previously37 and in Supplementary Information; human USP7, UniProt Q93009; human USP14, UniProt P54578; human USP35, UniProt Q9P2H5; human CYLD, UniProt Q9NQC7.

Techniques: Ubiquitin Proteomics, Binding Assay, Labeling

Fig. 4 | Molecular basis of inhibitor specificity for USP30. a, Sequence alignment of the indicated human USP DUBs. Arrows indicate the unique Leu328 and Phe453 residues in USP30. b, Close-up view of the compound binding site. c, Superposition with indicated USP DUB structures in complex with inhibitors (PDB 5N9R, 6IIN, 6GH9), highlighting how equivalent Phe and Tyr residues in other human USP DUBs interfere with compound binding. d, Catalytic activities of the indicated wild-type (WT) and mutant USP30 proteins, assessed by Ub–RhoG cleavage. Mean ± s.e.m. (derived from curve fitting; Extended Data Fig. 7). e, Inhibitory potencies of NK036, pre-incubated with the indicated

Journal: Nature structural & molecular biology

Article Title: Chimeric deubiquitinase engineering reveals structural basis for specific inhibition of the mitophagy regulator USP30.

doi: 10.1038/s41594-025-01534-4

Figure Lengend Snippet: Fig. 4 | Molecular basis of inhibitor specificity for USP30. a, Sequence alignment of the indicated human USP DUBs. Arrows indicate the unique Leu328 and Phe453 residues in USP30. b, Close-up view of the compound binding site. c, Superposition with indicated USP DUB structures in complex with inhibitors (PDB 5N9R, 6IIN, 6GH9), highlighting how equivalent Phe and Tyr residues in other human USP DUBs interfere with compound binding. d, Catalytic activities of the indicated wild-type (WT) and mutant USP30 proteins, assessed by Ub–RhoG cleavage. Mean ± s.e.m. (derived from curve fitting; Extended Data Fig. 7). e, Inhibitory potencies of NK036, pre-incubated with the indicated

Article Snippet: The following sequences were used: codon-optimized human USP30 catalytic domain, Addgene 110746, UniProt Q70CQ3 with modifications described previously37 and in Supplementary Information; human USP7, UniProt Q93009; human USP14, UniProt P54578; human USP35, UniProt Q9P2H5; human CYLD, UniProt Q9NQC7.

Techniques: Sequencing, Binding Assay, Mutagenesis, Derivative Assay, Incubation

Fig. 5 | Cellular evaluation of compound-resistant USP30 mutations. a, Mitochondrial ubiquitination analysis. HeLa cells expressing YFP–parkin were treated with USP30 inhibitors for 19 h where indicated. Mitophagy was induced with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 1 h. Ubiquitinated proteins were enriched through pulldowns with OtUBD, and samples were analyzed by western blot. Cmpd, compound; IB, immunoblot. b, Target engagement assay with endogenous USP30. HEK293 cells were treated with the indicated compounds. Lysates were then incubated with ubiquitin probe where indicated and analyzed by western blot for USP30. The asterisk denotes

Journal: Nature structural & molecular biology

Article Title: Chimeric deubiquitinase engineering reveals structural basis for specific inhibition of the mitophagy regulator USP30.

doi: 10.1038/s41594-025-01534-4

Figure Lengend Snippet: Fig. 5 | Cellular evaluation of compound-resistant USP30 mutations. a, Mitochondrial ubiquitination analysis. HeLa cells expressing YFP–parkin were treated with USP30 inhibitors for 19 h where indicated. Mitophagy was induced with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 1 h. Ubiquitinated proteins were enriched through pulldowns with OtUBD, and samples were analyzed by western blot. Cmpd, compound; IB, immunoblot. b, Target engagement assay with endogenous USP30. HEK293 cells were treated with the indicated compounds. Lysates were then incubated with ubiquitin probe where indicated and analyzed by western blot for USP30. The asterisk denotes

Article Snippet: The following sequences were used: codon-optimized human USP30 catalytic domain, Addgene 110746, UniProt Q70CQ3 with modifications described previously37 and in Supplementary Information; human USP7, UniProt Q93009; human USP14, UniProt P54578; human USP35, UniProt Q9P2H5; human CYLD, UniProt Q9NQC7.

Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Drug discovery, Incubation

Fig. 6 | Inhibition of USP30 by NK036 uses a ligandability hotspot and a cryptic pocket, distinct from other USP DUB inhibitors. a–g, Cartoon representations of human USP family DUB catalytic domains in complex with the indicated small-molecule inhibitors (USP7 and compound 2 (PDB 5N9R; a), USP7 and GNE6776 (PDB 5UQX; b), USP14 and IU1-206 (PDB 6IIM; c), USP28 and FT206 (PDB 8P1Q; d), USP1 and ML323 (PDB 7ZH4; e), USP7 and compound 23 (PDB 6VN3; f), USP30 and NK036 (g)). Compounds are shown as both sticks and transparent

Journal: Nature structural & molecular biology

Article Title: Chimeric deubiquitinase engineering reveals structural basis for specific inhibition of the mitophagy regulator USP30.

doi: 10.1038/s41594-025-01534-4

Figure Lengend Snippet: Fig. 6 | Inhibition of USP30 by NK036 uses a ligandability hotspot and a cryptic pocket, distinct from other USP DUB inhibitors. a–g, Cartoon representations of human USP family DUB catalytic domains in complex with the indicated small-molecule inhibitors (USP7 and compound 2 (PDB 5N9R; a), USP7 and GNE6776 (PDB 5UQX; b), USP14 and IU1-206 (PDB 6IIM; c), USP28 and FT206 (PDB 8P1Q; d), USP1 and ML323 (PDB 7ZH4; e), USP7 and compound 23 (PDB 6VN3; f), USP30 and NK036 (g)). Compounds are shown as both sticks and transparent

Article Snippet: The following sequences were used: codon-optimized human USP30 catalytic domain, Addgene 110746, UniProt Q70CQ3 with modifications described previously37 and in Supplementary Information; human USP7, UniProt Q93009; human USP14, UniProt P54578; human USP35, UniProt Q9P2H5; human CYLD, UniProt Q9NQC7.

Techniques: Inhibition