codon-optimized human usp30 catalytic domain  (Addgene inc)

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: 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. ) and the USP30 ch3 described here. See Extended Data Fig. for other chimeras. e , AlphaFold2-predicted model of USP30 c1 (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. for raw data. Mean ± s.e.m. (derived from curve fitting). g , Stability assessment of USP30 constructs in their apo states derived from thermal shift assays (TSAs). T m , protein melting temperature. Mean ( n = 3 independent replicates). h , Changes in protein stability upon binding to the ubiquitin probe Ub-PA. Mean ± s.d. ( n = 3 independent replicates). i , Gel-based Ub-PA binding assay. j , Crystal structure of USP30 ch3 ~Ub-PA. Regions within the chimeric USP30 construct derived from different USP DUBs are shown in blue (USP30), orange (USP14) and red (USP35). The Ub-PA probe is shown in yellow. See Table for statistics. k , Structure of NK036, a solubility-enhanced derivative of compound 39. l , Inhibitory potencies of NK036 for the indicated USP30 constructs. IC 50 values are given as mean ± s.e.m. (derived from curve fitting, with activity data for each concentration recorded as n = 3 independent replicates and shown as mean ± s.d.). m , Protein stability of the indicated USP30 constructs in the presence of NK036. Mean values are shown ( n = 3 independent replicates). Panel a created with BioRender.com .

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

Techniques: Membrane, Inhibition, Control, Activity Assay, Construct, Derivative Assay, Binding Assay, Ubiquitin Proteomics, Solubility, Concentration Assay

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: a , AlphaFold2 (AF2)-model of the catalytic domain of human USP30. USP boxes and domain insertions are indicated. b , Statistics of human USP enzymes regarding their characterization by crystallography and regarding the presence of Zn 2+ -coordinating residues in the tip of the fingers subdomain. c , AF2-model of USP30 c1 , previously optimized for structural studies and used as a starting point for this project. d-g , Experimental structures of catalytic domains of USP7 ( d ), USP14 ( e ), USP35 ( f ), and CYLD ( g ). Elements used for USP30 chimeric engineering are highlighted. h-l , AF2-models of USP30 constructs explored in this study ( h : USP30-GS (c2), i : USP30-USP7 (ch1), j : USP30-USP14 (ch2), k : USP30-USP14-USP35 (ch3), l : USP30-CYLD (ch4)). Catalytic residues are shown in pink. m , Architecture of USP30 constructs with close-up view of the boundaries of the chimeric portions.

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

Techniques: Construct

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: a , Protein stability assessment of USP30 constructs with thermal shift assays. Mean (N = 3 independent replicates). b , Ubiquitin probe reactivity assay. Samples were analyzed by SDS-PAGE and Coomassie staining. c , Changes in protein stability upon binding to Ub-PA. Δ T m was calculated as T m (Ub-PA-bound) subtracted from T m (apo protein). Mean ± s.d. (N = 3 independent replicates). d , Quantification of enzyme activity. Varying concentrations of USP30 proteins were incubated with Ubiquitin-RhoG substrate and fluorescence was recorded. e , Observed rate constants derived from plots in d were plotted over enzyme concentrations (upper panel) to derive catalytic efficiencies (lower panel). Mean ± s.e.m. (derived from curve fitting, with data for each concentration recorded as N = 3 independent replicates). f , Inhibitory potencies of Compound 39 and NK036. Compounds were pre-incubated with USP30 constructs for 1.5 h, and remaining activities were determined from Ub-RhoG cleavage assays. Mean ± s.d. (N = 3 independent replicates). g , IC 50 values of assays shown in f. Mean ± s.e.m. (derived from curve fitting). h , Assessment of binding of Compound 39 and NK036 to indicated USP30 constructs by thermal shift assays. Δ T m was calculated as T m of the inhibitor-bound sample subtracted from the T m of the apo protein. Mean ± s.d. (N = 3 independent replicates).

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

Techniques: Construct, Ubiquitin Proteomics, SDS Page, Staining, Binding Assay, Activity Assay, Incubation, Fluorescence, Derivative Assay, Concentration Assay

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: Chemical structures of representative examples of small molecule USP30 inhibitors are given together with characterization data and references. Cyanamides of covalent inhibitors form isothiourea linkages with the active site cysteine of USP30. Non-covalent inhibitors belong either to chemical series which were developed from phenylalanine derivatives (with either a benzenesulfonamide or a naphthylsulfonamide) or are natural product derivatives.

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

Techniques:

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: a , Cartoon representation of crystal structure of USP30 ch3 ~ Ub-PA, with representative 2m F o -D F c electron density (contoured at 1σ) shown for indicated areas. b , USP30 ch3 ~ Ub-PA structure and superpositions with previously reported Ub-PA complexes of USP30 c1 , USP14 and USP35.

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

Techniques:

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: a , Cartoon representation of the crystal structure of USP30 ch3 bound to NK036. The compound is shown under an orange surface. b , Composite omit electron density map of NK036 in chain A (2 mF o – D F c , contoured at 1 σ , covering all atoms of the compound, created with simulated annealing from the final coordinates). See Extended Data Fig. for unbiased mF o – DF c 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 (pink) and blocking loop 2 (red). Residues of the catalytic triad are shown as sticks. e , Close-up view of the compound binding site highlighting key residues involved in hydrogen bonding. f , Close-up view of the USP30 hydrophobic patch engaging the fluorobenzoyl moiety of the compound. g , Close-up view of hydrophobic interactions of the phenylalanine group of the compound. h , Close-up view of the benzenesulfonamide moiety of the compound engaged by USP30 residues.

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

Techniques: Binding Assay, Blocking Assay

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: a , Cartoon representations of the two copies within the asymmetric unit in the crystal structure of USP30 ch3 -NK036. b , Close-up view of the superposition shown in a highlighting the ligand geometries in both chains. c-d , Representative electron density map of the α5 helix in chain A (panel c ) and chain B (panel d ). 2m F o -D F c density, contoured at 1σ, with –30 Å 2 B -factor sharpening, is shown covering all atoms of the region. e-f , Unbiased electron density omit map of NK036 in chain A (panel e ) and chain B (panel f ). A m F o -D F c map, contoured at 1σ, covering all atoms of the compound is shown, which was calculated from the protein geometry before the ligand was modelled. g , Composite omit electron density map of NK036 in chain B (2m F o -D F c , contoured at 1σ, covering all atoms of the compound, created as described for Fig. ). h , Structure as in a, with chimeric elements shown in different colors. Of note, no chimeric residue is near the compound binding site. i , Cartoon representation of crystal structure of USP30 ch3 -NK036, highlighting different USP subdomains. The compound is shown under an orange surface, active site residues are shown in pink. j , Structure as in panel e with surface representation of USP30 highlighting different USP subdomains.

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

Techniques: Residue, Binding Assay

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: 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 pocket highlighted. f , Superposition of the structures, focused on the Leu73 binding pocket, showing its occupation by the fluorobenzoyl group of NK036. g , h , Close-up views of the conformational changes of the switching loop, focusing on entry of the cryptic pocket within the thumb subdomain ( g ) and anchoring of the switching loop on the α 5 helix ( h ). Putative movements of residues are indicated with arrows. See also Supplementary Video , in which the equivalent transition is shown based on the Lys6 diubiquitin-bound structure of USP30 (PDB 5OHP ) .

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

Techniques: Ubiquitin Proteomics, Binding Assay, Labeling

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: a-h , Structural superpositions showing the switching loop positions in different USP DUBs, comparing compound-bound to ubiquitin-bound or apo states ( a : USP30 with NK036 compared to Ubiquitin-bound USP30, b : USP7 with GNE6776 compared to apo USP7, c : USP14 with IU1-206 compared to apo USP14, d : USP28 with FT206 compared to apo USP28, e : USP1 with ML323 compared to Ubiquitin-bound USP1, f : USP7 with compound 2 compared to Ubiquitin-bound USP7, g : USP14 with IU1-206 compared to Ubiquitin-bound USP14, h : USP7 with compound 23 compared to Ubiquitin-bound USP7).

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

Techniques: Ubiquitin Proteomics

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: 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. ). e , Inhibitory potencies of NK036, pre-incubated with the indicated USP30 proteins for 1.5 h, determined from Ub–RhoG cleavage assays. IC 50 values are given as mean ± s.e.m. (derived from curve fitting, with activity data for each concentration recorded as n = 3 independent replicates and shown as mean ± s.d.). f , Protein stability of the indicated USP30 proteins in the presence of 20 µM NK036. Δ T m was calculated as T m of the compound-bound sample subtracted from T m of the respective apo protein. Mean ± s.d. ( n = 3 independent replicates). g , Inhibitory potencies of compound 39, determined as in e . h , Protein stability assessment in the presence of compound 39, determined as in f .

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

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

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: a-b , Quantification of USP30 enzyme activity for WT and indicated mutant proteins ( a : fluorescence over time plots for each protein, b : observed rate constant over enzyme concentration plots), as described in Extended Data Fig. . c , Probe competition assay. USP30 and inhibitors were preincubated, followed by addition of Ub-PA, and analysis of samples by SDS-PAGE and Coomassie staining. d , Sequence alignment of indicated human USP DUBs. Arrows indicate Ile154, Phe157 and Ala162 residues in USP30. e , Inhibitory potencies of Compound 39, pre-incubated with indicated USP30 proteins for 1.5 h, determined from Ub-RhoG cleavage assays. IC 50 values are given as mean ± s.e.m. (derived from curve fitting, with data for each concentration recorded as N = 3 independent replicates). f , Protein stability of indicated USP30 proteins in the presence of 20 µM Compound 39. Δ T m was calculated as T m of the compound-bound sample subtracted from T m of the respective apo protein. Mean ± s.d. (N = 3 independent replicates). g , Probe competition assay performed as in c with mutations characterized in e and f.

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

Techniques: Activity Assay, Mutagenesis, Fluorescence, Concentration Assay, Competitive Binding Assay, SDS Page, Staining, Sequencing, Incubation, Derivative Assay

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: 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 an unspecific band. c , Cellular assessment of the USP30 inhibition mechanism. C-terminally Flag-tagged USP30 (wild type or with the compound-resistant mutation F453Y) was overexpressed in HEK293 cells. Cells were analyzed as described in b , with a western blot for the Flag tag. d , Catalytic activities of additional USP30 mutants, assessed by Ub–RhoG cleavage as described for Fig. . e , Protein stability of the indicated USP30 proteins by NK036 as described for Fig. . f , Cellular probe competition assay as described in c with mutations characterized in d and e . GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: The following sequences were used: codon-optimized human USP30 catalytic domain, Addgene 110746, UniProt Q70CQ3 with modifications described previously and in ; 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, Inhibition, Mutagenesis, FLAG-tag, Competitive Binding Assay

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: a-b , Cartoon representations of the crystal structures of USP30 in complex with non-covalent inhibitor NK036 ( a ) and covalent inhibitor 552 ( b ). The fragment antigen-binding region (Fab) present in the structure 8D1T is not shown. c , Superposition of the structure of USP30 + NK036 on the structure of USP30~inhibitor 552. d , Close-up view of the inhibitor 552 binding site highlighting key residues. The chemical structure of inhibitor 552 is shown in Extended Data Fig. . e-h , Close-up views of the conformational changes of the switching loop in the structures of USP30 + NK036 ( e ), USP30~inhibitor 552 ( f ) and USP30~Ub-PA ( g ). A superposition of these three structures is also shown ( h ). Highlighted are different orientations of the switching loop residue Phe157 in the respective structures. Upon binding of the non-covalent compound, Phe157 moves backwards leading to the formation of the cryptic pocket. In contrast, upon binding of the covalent compounds, Phe157 moves forward which creates a shielded tunnel between the modified catalytic cysteine and the S1 Ubiquitin site.

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

Techniques: Binding Assay, Residue, Modification, Ubiquitin Proteomics

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: 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 surfaces. Structural elements of DUBs are labeled, and PDB codes of structures are given , , , , , . The Leu73 ubiquitin binding site is shown with an arrow when engaged by compounds. h , Comparison of USP30 inhibition by NK036 to other DUB inhibitors. Superposition of the structure of USP30 + NK036 on other structures shown in a – f . Compounds are shown as surfaces and are labeled. All USP cartoons except USP30 are semitransparent.

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

Techniques: Labeling, Ubiquitin Proteomics, Binding Assay, Comparison, Inhibition

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: a-c , Cartoon representations of the crystal structures of USP7 ( a ), SARS-CoV2-PLPro ( b ) and USP30 ( c ) in complex with respective inhibitors. Compounds are shown as surfaces. d , Superposition of the structure of USP30 + NK036 on other structures shown in panels a - c . Compounds are shown as surfaces and are labeled. All protein cartoons except USP30 are semitransparent.

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

Techniques: Labeling

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: a-d , Chemical and structural representations of inhibitors of USP7 ( a , b ), USP14 ( c ) and USP28 ( d ). Shown are the overlays of the inhibitor-bound structures with ubiquitin-bound structures of the respective DUBs (first box) and the inhibitor-bound structures of each DUB overlaid on NK036 bound structure of USP30 (second box). Highlighted is the close-up view of the compound binding site at the ubiquitin Leu73 pocket. Please note the chemically related hotspot anchors in b and c . Structural superpositions were aligned taking only the protein residues into account. The chemical moieties of the inhibitors that occupy the Leu73 pocket are highlighted with orange background.

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

Techniques: Ubiquitin Proteomics, Binding Assay

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: a , Schematic of a ubiquitin-bound USP DUB. Structural elements are labeled. b , Schematic of USP30 in complex with an inhibitor of the benzenesulfonamide scaffold, occupying the ubiquitin Leu73 pocket (sand), the cryptic pocket (green) and the cleft toward the S1 ubiquitin binding site (blue) with shown chemical moieties. c , Schematic of DUB inhibition with compounds being composed of a hotspot anchor element (occupying the ubiquitin Leu73 pocket) and one or two specificity extensions (occupying the other shown USP DUB structural elements). Compounds featuring the respective extensions are named together with their cognate DUBs , , , , , , . See Extended Data Fig. for structural superpositions focused on the Leu73 binding pocket. d , Chemical structures of specific DUB inhibitors. Chemical motifs occupying the distinct binding sites are colored according to c . The hotspot anchor motifs shared by all are highlighted.

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

Techniques: Ubiquitin Proteomics, Labeling, Binding Assay, Inhibition