Sequence alignments were generated using the L-INS-I algorithm of the MAFFT package 30. The multiple alignments were used for the generation of generalized profiles using pftools 31, and Hidden-Markov-Models using the HHSEARCH 37. Generalized profile searches were performed using all proteins from the Uniprot database (https://www.uniprot.org). Protein clustering was performed using the CLANS software 56. Structure comparisons were performed using the DALI method 40.
Cloning & Mutagenesis
The DrT-VTDs were amplified from Danio rerio genomic DNA or, in the case of DrT1-VTD2, from Danio rerio cDNA (kind gifts from Sigrun Korsching, University of Cologne), Wc-VTD2 was amplified from Waddlia chondrophila genomic DNA (kind gift from Carole Kebbi Beghdadi, University of Lausanne), Si-VTD was amplified from Serendipita indica cDNA (kind gift from Alga Zuccaro, University of Cologne), and Dm-VTD was amplified from Drosophila melanogaster cDNA (kind gift from Mirka Uhlirova, University of Cologne). All amplifications were done by PCR, using Phusion High Fidelity Kit (New England Biolabs). Wc-VTD1, Nf-VTD and Sl-VTD coding regions were obtained by gene synthesis (IDT). The PCR fragments and gBlocks were cloned into pOPIN-S and pOPIN-K vectors 57 using the In-Fusion HD Cloning Kit (Takara Clontech). Point mutations were introduced using the QuikChange Lightning kit (Agilent Technologies).
Constructs for ubiquitin-PA purification (pTXB1-ubiquitin1–75) were a kind gift of David Komander (WEHI, Melbourne). SUMO11-96, SUMO31-91 and ISG1579-156 were amplified by PCR with an N-terminal 3xFlag tag and cloned into the pTXB1 vector (New England Biolabs) by restriction cloning according to the manufacturers protocol. The UbI44A activity-based probe was created by introducing the mutation using the QuikChange Lightning kit (Agilent Technologies).
Protein expression & purification
All VTD candidates including all truncations and mutants were expressed from the pOPIN-S (in case of the bacterial and eukaryotic proteases) with an N-terminal 6His-Smt3-tag or pOPIN-K vector (in case of the transposon-derived candidates) with an N-terminal 6His-GST-tag. Escherichia coli (Strain: Rosetta (DE3) pLysS) were transformed with constructs expressing DUBs and 2-6 l cultures were grown in LB medium at 37 °C until the OD600 of 0.8 was reached. The cultures were cooled down to 18 °C and protein expression was induced by addition of 0.2 mM isopropyl β-d-1-thiogalactopyranoside (IPTG).
The expression of selenomethionine substituted proteins was carried out as described previously by 58: In brief, the expression cultures were grown in minimal medium until the OD600 of 0.8 was reached. The cultures were cooled down to 18 °C, mixed with feed-back inhibition amino acid mix (0.5 g/l final concentration), metal trace elements (0.1% final concentration) and vitamins (0.01% final concentration) and induced with 0.2 mM IPTG. After 16 h, the cultures were harvested by centrifugation at 5000 × g for 15 min. After freeze thaw, the pellets were resuspended in binding buffer (300 mM NaCl, 20 mM TRIS pH 7.5, 20 mM imidazole, 2 mM β-mercaptoethanol) containing DNase and Lysozyme, and lysed by sonication using 10 s pulses with 50 W for a total time of 10 min. Lysates were clarified by centrifugation at 50,000 × g for 1 h at 4 °C and the supernatant was used for affinity purification on HisTrap FF columns (GE Healthcare) according to the manufacturer's instructions. The 6His-Smt3 tag was removed by incubation with SENP1415-644; the 6His-GST tag was removed by incubation with 3C protease. Si-VTD and Sl-VTD were purified including the N-terminal 6His-Smt3 tag. The proteins were simultaneously dialyzed in binding buffer. The liberated affinity-tag and the His-tagged SENP1 and 3C proteases were removed by a second round of affinity purification with HisTrap FF columns (GE Healthcare). All proteins were purified with a final size exclusion chromatography (HiLoad 16/600 Superdex 75 or 200 pg) in 20 mM TRIS pH 7.5, 150 mM NaCl, 2 mM dithiothreitol (DTT), concentrated using VIVASPIN 20 Columns (Sartorius), flash frozen in liquid nitrogen, and stored at −80 °C. Protein concentrations were determined using the absorption at 280 nm (A280) using the proteins’ extinction coefficients derived from their sequences.
Synthesis of activity-based probes
All activity-based probes used in this study were expressed as C-terminal intein fusion proteins as described previously 59: In brief, the fusion proteins were affinity purified in buffer A (20 mM HEPES, 50 mM sodium acetate pH 6.5, 75 mM NaCl) from clarified lysates using Chitin Resin (New England Biolabs) following the manufacturer's protocol. On-bead cleavage was performed by incubation with cleavage buffer (buffer A containing 100 mM MesNa (sodium 2-mercaptoethanesulfonate)) for 24 h at room temperature (RT). The resin was washed extensively with buffer A and the pooled fractions were concentrated and subjected to size exclusion chromatography (HiLoad 16/600 Superdex 75pg) with buffer A. To synthesize the propargylated probe, 300 µM Ub/Ubl-MesNa were reacted with 600 µM propargylamine hydrochloride (Sigma Aldrich) in buffer A containing 150 mM NaOH for 3 h at RT. Unreacted propargylamine was removed by size exclusion chromatography and the probe was concentrated using VIVASPIN 20 Columns (3 kDa cutoff, Sartorius), flash frozen and stored at −80 °C. The NEDD8-PA was a kind gift from David Pérez Berrocal and Monique Mulder (Department of Cell and Chemical Biology, Leiden University).
Met1-linked di-ubiquitin was expressed as a linear fusion protein and purified by ion exchange chromatography and size exclusion chromatography. K6-, K11-, K48-, and K63-linked ubiquitin chains were enzymatically assembled using UBE2SΔC (K11), CDC34 (K48), and Ubc13/UBE2V1 (K63) as previously described 60,61. In brief, ubiquitin chains were generated by incubation of 1 µM E1, 25 µM of the respective E2, and 2 mM ubiquitin in reaction buffer (10 mM ATP, 40 mM TRIS (pH 7.5), 10 mM MgCl2, 1 mM DTT) for 18 h at RT. K6-linked ubiquitin chains were assembled by incubation of 1 µM E1, 25 µM E2 (UbCH7), 25 µM E3 (Nlel) and 2 mM ubiquitin in reaction buffer. The generated mixture of K6- and K48-linked chains was treated with 10 µM OTUB1. The respective reactions were stopped by 20-fold dilution in 50 mM sodium acetate (pH 4.5) and chains of different lengths were separated by cation exchange using a Resource S column (GE Healthcare). Elution of different chain lengths was achieved with a gradient from 0 to 600 mM NaCl.
DrT1-VTD2 (selenomethionine substituted), Wc-VTD1 (selenomethionine substituted) and Wc-VTD1~Ub were crystallized using sitting drop vapor diffusion with commercially available sparse matrix screens. 96 well iQ crystallization plates containing 30 µl of the respective screening conditions were mixed with 10 mg/ml protein in the ratios 1:2, 1:1 and 2:1 in 300 nl drops. For DrT1-VTD2 and Wc-VTD1, the initial conditions containing the most promising crystals were optimized by gradually changing the chemical components included in the respective screening condition. 80 μl reservoir solution were pipetted into 48-well MRC plates and sitting drop vapour diffusion was performed by mixing 10 mg/ml protein in the ratios 1:2, 1:1 and 2:1 in drops of 3 μl. Initial DrT1-VTD2 crystals were detected in Proplex D12 (0.1 M Tris pH 8.5, 20% w/v PEG 6000) and several other conditions after 3 days at 20°C. Optimization was carried out with 3 µl drops (protein/precipitant ratios: 2:1, 1:1 and 1:2) and precipitant solutions varying in pH or PEG 6000 concentration respectively. Optimized crystals were harvested and cryoprotected with reservoir containing 10% glycerol. For unbound Wc-VTD1, initial crystals were detected in JCSG H8 (0.2 M sodium chloride, 0.1 M BisTris pH 5.5, 25 % w/v PEG 3350) and several other conditions after 3 days at 20°C. Optimization was carried out with 3 µl drops (protein/precipitant ratios: 2:1, 1:1 and 1:2) and precipitant solutions varying in sodium chloride or PEG 3350 concentration respectively. Optimized crystals were harvested and cryoprotected with reservoir containing 20% glycerol. 1.2 mM Wc-VTD1 were incubated with 760 µM ubiquitin-PA for 18 h at 4 °C. Unreacted Wc-VTD1 and Ub-PA were removed by size exclusion chromatography and the complex concentrated to a concentration of 7 mg/ml. The covalent Wc-VTD1~Ub crystals were detected in Salt RX B6 (0.1 M BisTris propane pH 7.0, 2.0 M Ammonium citrate tribasic) after 2 days at 20 °C. Crystals were harvested without further optimization and cryoprotected with the addition of 10% glycerol.
Data collection, phasing, model building, and refinement
Diffraction data for DrT1-VTD2, Wc-VTD1 and the Wc-VTD1~Ub-PA complex was collected at the Deutsches Elektronen-Synchroton (DESY), Hamburg, Germany at beamline P13 at the EMBL outstation 62. All datasets were processed using XDS 63. For DrT1-VTD2 and Wc-VTD1, initial phases were determined using selenomethionine SAD experiments and SHELXC/D/E 64, which were driven by HKL2MAP 65. Both protein structures were subsequently built automatically using the ARP/wARP Web Service 66 or the buccaneer pipeline implemented in CCP4 67. The Wc-VTD1~Ub-PA complex was solved by molecular replacement using PHASER 68 as implemented in the phenix package 69 with a single molecule of Wc-VTD1 and a full-length model of ubiquitin as search models. For further refinement, necessary restraints for the propargyl moiety were calculated using AceDRG implemented in the CCP4 package 70. Initial models were refined using iterative cycles of phenix.refine (DrT1-VTD2, Wc-VTD1) or RefMac (Wc-VTD1~Ub-PA) and manually rebuilt using COOT 71. For structural analysis, the PyMOL (http://www.pymol.org) and ChimeraX Graphics Systems72 were used.
Activity assays of DUBs against AMC-labeled substrates were performed using reaction buffer (150 mM NaCl, 20 mM TRIS pH 7.5, 10 mM DTT), 1 µM DUBs (deviating concentrations are indicated in the respective figures and the corresponding legends), 5 µM Ub-AMC (UbiQ-Bio, The Netherlands) or 5 µM NEDD8-AMC (Enzo Life Science). The reaction was performed in black 96-well plates (Corning) at 30 °C and fluorescence was measured using the Infinite F200 Pro plate reader (Tecan) equipped for excitation wavelength of 360 nm and an emission wavelength of 465 nm. The presented results are means of three independent cleavage assays.
Activity based probe assays
DUBs were prediluted to 2× concentration (10 µM) in reaction buffer (20 mM TRIS pH 7.5, 150 mM NaCl and 10 mM DTT) and 1:1 combined with 100 µM Ub-, UbI44A-, SUMO1, SUMO3, ISG15CTD or NEDD8-PA for 18 hours at 4°C. The reaction was stopped by the addition of 2x Laemmli buffer, resolved by SDS-PAGE, and Coomassie stained.
Ubiquitin chain cleavage
DUBs were preincubated in 150 mM NaCl, 20 mM TRIS pH 7.5 and 10 mM DTT for 10 min. The cleavage was performed for the indicated time points with 25 nM up to 500 nM DUBs (as indicated in the respective figure legends) and 25 µM di-ubiquitin (K11, K48, K63, M1, K6 synthesized as described above, others purchased from Boston Biochem) at RT. The reactions were stopped with 2x Laemmli buffer, resolved by SDS-PAGE, and Coomassie stained.