Allosteric activation of the SPRTN protease by ubiquitin maintains genome stability

Abstract

The DNA-dependent protease SPRTN maintains genome stability by degrading toxic DNA-protein crosslinks (DPCs). To understand how SPRTN's promiscuous protease activity is confined to cleavage of crosslinked proteins, we reconstitute the repair of DPCs including their modification with SUMO and ubiquitin chains in vitro. We discover that DPC ubiquitylation strongly activates SPRTN independently of SPRTN's known ubiquitin-binding domains. Using protein structure prediction, MD simulations and NMR spectroscopy we reveal that ubiquitin binds to SPRTN's protease domain, promoting an open, active conformation. Replacing key interfacial residues prevents allosteric activation of SPRTN by ubiquitin, leading to genomic instability and cell cycle defects in cells expressing truncated SPRTN variants that cause premature aging and liver cancer in Ruijs-Aalfs syndrome patients. Collectively, our results reveal a ubiquitin-dependent regulatory mechanism that ensures SPRTN activity is deployed precisely when and where it is needed.

Fig. 1. Ubiquitylation of DPCs promotes their cleavage by SPRTN.

a Schematic of SPRTN’s domain structure and truncated variants, featuring motif interacting with ubiquitin (MIU), protease domain, zinc-binding domain (ZBD), basic region (BR), SHP box for p97-binding, PCNA-interacting motif (PIP) and ubiquitin-binding zinc finger (UBZ). SPRTN^ΔC is caused by a frameshift mutation resulting in a variant composed of SPRTN’s N-terminal 240 residues followed by eight additional amino acids (X8). b Schematic of HMCES^SRAP ubiquitylation to generate DPCs shown in e, f, Fig. 4 and Supplementary Fig. 5b and 6b. HMCES^SRAP-Ub(G76V)-3C-FKBP was incubated with FRB-E3 + E2 (K48 or K63) in the presence of ubiquitin, rapamycin, ubiquitin-E1 and ATP for 2 h (K63) or 6.5 h (K48) at 30 °C. After cleavage of the FKBP-tag via 3C-protease, ubiquitylated HMCES^SRAP was purified by reverse immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC). c Mass spectrometry analysis of ubiquitin linkages formed by ubiquitylation of HMCES^SRAP as shown in (b). Bar chart shows the mean ± SD of three biological replicates. d Schematic of the generation of HMCES^SRAP-DPCs. HMCES^SRAP was incubated for 30 min at 37 °C with a Cy5-labeled 30nt oligonucleotide containing a dU at position 15 and UDG. After crosslinking a complementary 15nt reverse oligonucleotide was annealed to form a ssDNA-dsDNA junction. e Indicated HMCES^SRAP-DPCs (10 nM) were incubated alone or in the presence of FANCJ (100 nM) and indicated concentrations of SPRTN (1-100 nM) for 1 h at 30 °C. Quantification: bar graphs represent the mean ± SD of three independent experiments. All samples derive from the same experiment and gels were processed in parallel. Values for cleavage of unmodified HMCES^SRAP-DPC are the same as in Supplementary Fig. 1b. Source data are provided as a Source Data file. f Indicated HMCES^SRAP-DPCs (10 nM) were incubated alone or in the presence of FANCJ (100 nM) and indicated concentrations of SPRTN or SprT-BR (1-100 nM) for 1 h at 30 °C. Quantification: bar graphs represent the mean ± SD of three independent experiments. All samples derive from the same experiment and gels were processed in parallel. Source data are provided as a Source Data file.

Fig. 2. Ubiquitin promotes an open SPRTN conformation.

a–c Experimental structure of SPRTN’s SprT domain (SPRTN^aa28-214), PDB: 6mdx (a), ColabFold predicted structure of SprT (b) and ColabFold predicted structure of a SprT-ubiquitin (Ub¹) complex (c). Protease domain is colored in blue, zinc-binding domain (ZBD) in orange and the Ub¹ in grey. Zn²⁺ ions are colored in red. d–f Radius of gyration (Rg) of the indicated structures over 400 ns of molecular dynamics (MD) simulation. Each curve represents an independent MD trajectory (n = 3). Source data are provided as a Source Data file. g–i Main MD-clusters of the indicated structures during MD simulation for 400 ns, generated from three independent trajectories. For SprT (ColabFold predicted) two of three main MD-clusters are depicted. Rg correlating frequencies among all performed simulations are labeled above the structures. j, k Zoom-in to regions i and ii of the SprT-Ub¹ complex (i), showing amino acids of ubiquitin (in grey) surrounding residue Leu38 (j) or L99 (k) of SPRTN (in blue) in the wild-type (WT) protein (left) and upon L38S or L99S replacement, respectively (right). l SprT-Ub¹ binding energy difference (ΔΔG) between SprT-L38S or -L99S and WT protein obtained from alanine scanning. Bar graphs show the mean ± SD of 301 snapshots from PBSA calculations for the central structure of the largest cluster. Source data are provided as a Source Data file.

Fig. 3. DNA- and ubiquitin-binding affect SPRTN’s conformation synergistically.

a–f Comparison of NMR spectra, highlighting Trp ε1 amide signals in ¹H,¹⁵N-HSQC experiments of SprT-BR and SprT-BR-L99S. Trp ε1 region is labeled and boxed (bottom). Resonance assignments corresponding to the Trp ε1’s in the zinc-binding domain (ZBD) are shown in orange and those in the protease domain in blue. Broadened or shifted signals upon dsDNA addition are shown as asterisk. a, b SprT-BR (a) and SprT-BR-L99S (b) alone (= Apo) (black), with mono-ubiquitin (Ub¹) (5x molar excess) (red). Minor changes are boxed in blue to highlight the spectral differences between SprT-BR and SprT-BR-L99S upon adding Ub¹. Zoom-in region in Supplementary Fig. 3e is marked with a black box (b). c, d SprT-BR (c) and SprT-BR-L99S (d) alone (black) (= Apo), with dsDNA (2x molar excess) (red). Some of the ZBD resonances affected by dsDNA are labeled in black while the unchanged are labeled in grey. e, f Superimpositions of SprT-BR (e) and SprT-BR-L99S (f) in the presence of dsDNA (2x molar excess) (black) and of both dsDNA (2x molar excess) and Ub¹ (5x molar excess) (red). Additional resonance changes upon adding Ub¹ to the dsDNA-bound SprT-BR are shown with red boxes.

Fig. 4. The ubiquitin-dependent activation of SPRTN is mediated by the USD.

a–c Indicated HMCES^SRAP-DPCs (10 nM) were incubated alone or in the presence of FANCJ (100 nM) and indicated concentrations (0.1–100 nM) and variants of SPRTN (WT, L38S, L99S) for 1 h at 30 °C. Quantification: bar graphs represent the mean ± SD of three independent experiments. Source data are provided as a Source Data file.

Fig. 5. SUMO-targeted DPC ubiquitylation activates SPRTN.

a Schematic of SUMO-targeted ubiquitylation of HMCES^FL-DPCs used in b-f and h. HMCES^FL-DPCs were incubated alone or in the presence of SUMO2, UBC9 and PIAS4, with or without SAE1/UBA2 for 30 min at 37 °C. Next unmodified or SUMOylated HMCES^FL-DPCs were incubated alone or in the presence of ubiquitin (Ub), RNF4, UBE2D3, with or without UBE1 for 30 min at 37 °C. b SUMO-targeted ubiquitylated HMCES^FL-DPCs generated as described in (a), separated by denaturing SDS-PAGE and immunoblotting. Source data are provided as a Source Data file. c Mass spectrometry analysis of ubiquitin linkages formed by SUMO-targeted ubiquitylation of HMCES^FL-DPCs. Bar chart shows the mean ± SD of four biological replicates. d Mass spectrometry analysis of lysine residues within HMCES or SUMO modified upon SUMO-targeted ubiquitylation. Violin blots show the mean ± SD of four biological replicates. e Indicated HMCES^FL-DPCs (10 nM) were incubated alone or in the presence of FANCJ (100 nM) and SPRTN (100 nM) for 1 h at 30 °C. Quantifications: bar graphs represent the mean ± SD of three independent experiments. Source data are provided as a Source Data file. f Indicated HMCES^FL-DPCs (10 nM) were incubated alone or in the presence of FANCJ (100 nM) and indicated concentrations (1-100 nM) and variants of SPRTN (WT, L38S, L99S) for 1 h at 30 °C. Quantifications: bar graphs represent the mean ± SD of three independent experiments. All samples derive from the same experiment and gels were processed in parallel. Source data are provided as a Source Data file. g HeLa-TREx SPRTN^ΔC Flp-In cells complemented with indicated YFP-SPRTN^FL-Strep-tag variants were treated as depicted (top) with 5-azadC (10 µM) and harvested at indicated time points. DNMT1-DPCs were isolated using PxP (middle, see Methods) and analyzed by immunoblotting (bottom). Shown is a representative of three independent experiments. Source data are provided as a Source Data file. h Indicated HMCES^FL-DPCs (10 nM) were incubated alone or in the presence of FANCJ (100 nM) and indicated concentrations (1–100 nM) and variants of SPRTN (FL-WT/L99S, ΔUBZ-WT/L99S, ΔC-WT/L99S) for 1 h at 30 °C. Quantifications: bar graphs represent the mean ± SD of three independent experiments. All samples derive from the same experiment and gels were processed in parallel. Source data are provided as a Source Data file.

Fig. 6. Ubiquitin-dependent activation of SPRTN maintains genome stability in Ruijs-Aalfs syndrome.

a, b Proliferation of Sprtn^F/- Cre-ER^T2 mouse embryonic fibroblasts (MEFs) complemented with indicated SPRTN variants or empty vector (EV, pMSCV) treated with methanol (MeOH) or (Z)−4-hydroxytamoxifen (4-OHT) (2 µM) for 48 h. After seeding, cell numbers were counted at indicated time points. Values are the mean ± SD of eight technical replicates. Shown is a representative of three independent experiments. Source data are provided as a Source Data file. c Image showing micronuclei (asteriks) and chromatin bridges (arrow) in Sprtn^F/- Cre-ER^T2 MEFs + pMSCV-SPRTN^ΔC-L99S treated with 4-OHT (2 µM) for 48 h. DNA was visualized by DAPI staining. Scale bar corresponds to 15 µm. d, e Quantification of micronuclei and chromatin bridges formation in Sprtn^F/- Cre-ER^T2 MEFs complemented with indicated SPRTN variants or EV (pMSCV) treated with MeOH or 4-OHT (2 µM) for 48 h. DNA was visualized by DAPI staining. Bar graphs show the mean ± SD of three independent experiments. The p values were calculated using a two-way ANOVA with Dunnett’s multiple comparison test. P values: d Micronuclei (left): SPRTN-WT vs. SPRTN-L38S = 0.0002; SPRTN-WT vs. SPRTN-L99S < 0.0001. Chromatin bridges (right): SPRTN-WT vs. SPRTN-L38S = 0.9992; SPRTN-WT vs. SPRTN-L99S = 0.8634. e Micronuclei (left): SPRTN^ΔC-WT vs. SPRTN^ΔC-L38S = 0.1411; SPRTN^ΔC-WT vs. SPRTN^ΔC-L99S < 0.0001. Chromatin bridges (right): SPRTN^ΔC-WT vs. SPRTN^ΔC-L38S = 0.4745; SPRTN^ΔC-WT vs. SPRTN^ΔC-L99S = 0.0005. Source data are provided as a Source Data file.

Fig. 7. ‘Triple-lock’ model for SPRTN activation.

The ubiquitin-binding zinc finger (UBZ) recruits SPRTN to ubiquitylated DPCs. Binding of both DNA-binding domains, zinc-binding domain (ZBD) and the basic region (BR) to activating DNA structures induces an open conformation. This open conformation is stabilized by ubiquitin binding to the ubiquitin binding interface at the SprT domain (USD). Recruitment and DNA structure recognition are compromised in Ruijs-Aalfs syndrome patients, which therefore fully rely on the Ub-dependent activation via the USD to maintain genome stability.