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PDB ID |
Structure Details |
Compound ID |
PARP14 macrodomain 2 with ligand MnK2-13 |
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PARP14 macrodomain 3 with fragment N13417a |
FM010005a |
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PARP14 macrodomain 3 with fragment N08149b |
FM001707a |
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PARP14 macrodomain 3 with fragment N13681a |
FM001999a |
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PARP14 macrodomain 3 with fragment N13729a |
XS106503b |
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PARP14 macrodomain 3 with fragment N13734a |
FM001875a |
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PARP14 macrodomain 3 with fragment N13856a |
FM001884a |
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PARP14 macrodomain 3 with fragment N13979a |
FM010067a |
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PARP14 macrodomain 3 with fragment N13857a |
FM002036a |
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PARP14 macrodomain 3 with fragment N13848a |
FM002044a |
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PARP14 macrodomain 3 with fragment N13987a |
FM001702a |
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PARP14 macrodomain 3 with fragment N14095a |
FM001958a |
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PARP14 macrodomain 3 with fragment N13660a |
FM001909a |
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PARP14 macrodomain 3 with fragment N13844a |
FM002207a |
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PARP14 macrodomain 3 with fragment N13767a |
FM001942a |
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PARP14 macrodomain 3 with fragment N13462a |
FM002062a |
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PARP14 macrodomain 3 with fragment N14015a |
XS097881b |
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PARP14 macrodomain 3 with fragment N13888a |
FM002205a |
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PARP14 macrodomain 3 with fragment N13605a |
FM010020a |
Protein expression and purification
Macrodomain proteins (PARP14 MD1, MD2, MD2SERmut and MD3) were expressed using constructs that add tobacco etch virus (TEV) protease-cleavable His6-tags. Transformed BL21(DE3)-R3-pRARE cells were grown at 37 °C in LB medium (Miller) supplemented with appropriate antibiotics until OD600 reached 0.5‒0.6, then cooled to 18 °C and supplemented with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at an OD600 of 0.8 to induce protein expression overnight. For purification of in vivo biotinylated macrodomain proteins, the constructs were transformed into BL21(DE3)-R3-BirA cell line (BL21 derivative coexpressing BirA using a pACYC coexpression vector). Cells were grown at 37 °C in LB medium (Miller) until OD600 reached 0.5–0.6, then cooled to 18 °C and supplemented with 0.5 mM D-biotin dissolved in 10 mM bicine (pH 8.3) and 0.5 mM IPTG at an OD600 of 0.8 to induce protein expression overnight. Cell pellets re-suspended in lysis buffer (50 mM HEPES (pH 7.4), 500 mM NaCl, 20 mM imidazole, 5 % glycerol, 0.5 mM tris(2-carboxyethyl)phosphine [TCEP], 1:2,000 Calbiochem protease inhibitor cocktail set III) were quickly thawed on ice and lysed by high pressure homogenisation. Following cell lysis, DNA was precipitated with 0.15 % polyethyleneimine (PEI) and insoluble cell debris was removed by centrifugation (36000×g, 1 h, 4 °C). His6- (-biotin) tagged proteins were purified using Ni-Sepharose resin (GE Healthcare) and eluted stepwise in binding buffer with 40–250 mM imidazole. A high salt wash with 1 M NaCl was combined with the first elution step including 40 mM imidazole. As required, the His6-tag was removed from the macrodomain proteins at 4 °C overnight using recombinant TEV protease (used 1:40 -1:100 (w/w)) before gel filtration (Superdex 75 16/60, GE Healthcare) in GF buffer (25 mM HEPES (pH 7.4), 300 mM NaCl, 5 % glycerol, 0.5 mM TCEP). Proteins with removed His6- tag were additionally passed over Ni-Sepharose resin as a final purification step. In the case of PARP14 MD2 and MD3 purification for crystallisation, TEV His6-tag cleavage after the first Ni-IMAC purification step was combined with overnight dialysis using SnakeSkin® Dialysis Tubing, 3500 MWCO (Thermo Scientific). The dialysed sample was passed over Ni-Sepharose resin before purification by gel filtration (Superdex 75 16/60) and further purification steps. For PARP14 MD3 the GF buffer contained 20 mM HEPES (pH 7.4), 300 mM NaCl, 10 % glycerol and 0.5 mM TCEP. Apart from Ni-IMAC rebinding, all purification steps were carried out at 4 °C.
AlphaScreen assay
Assays were performed with minor modifications from the manufacturer’s protocol (PerkinElmer) [27,28]. All reagents were diluted in buffer containing 25 mM HEPES (pH 7.4), 100 mM NaCl, 0.5 mM TCEP, 0.1 % BSA and 0.05 % CHAPS and allowed to equilibrate to RT before addition to plates. The assays were run in 20 µL volumes in low-volume 384-well plates (ProxiPlate™-384 Plus, PerkinElmer) at RT. To determine ideal assay concentrations of the corresponding macrodomain protein and the macrodomain AlphaScreen peptide (a biotinylated and ADP-ribosylated 11 residue sequence), 4 µL volumes of peptide (0-16 μM; final assay concentration: 0‑3.2 μM) were incubated with 4 µL volumes of His6-tagged macrodomain protein (0-16 μM; final assay concentration: 0-3.2 μM) in 4 µL buffer for 30 min at RT in foil-sealed plates. For compound screening and IC50 characterisation, 12 µL of a solution containing peptide and His6‑tagged macrodomain protein in the pre-determined assay concentrations in assay buffer were incubated for 30 min at RT with 50 nL or 100 nL compound solution (pre-dispensed into the assay plate from 10 mM or 50 mM DMSO stocks using an Echo 550 (Labcyte)). Then, 8 µL of streptavidin-coated donor beads (7 μg/ml) and nickel chelate acceptor beads (7 μg/ml) (Perkin Elmer AlphaScreen™ Histidine (Nickel Chelate) Detection Kit) were added under low light conditions and plates were incubated for 60 min at RT protected from light. Plates were read on a PHERAstar FS plate reader (BMG Labtech) using an AlphaScreen 680 excitation/570 emission filter set. Alternatively for counterscreening of the compounds, 12 µL of 75 nM biotinylated and His6-tagged linker peptide (PerkinElmer) was added to 50 nL or 100 nL of the compounds and plates were processed as described above.
Biolayer Inteferometry
Kinetic ligand-binding measurements were performed using an Octet RED384 BLI instrument (fortéBio) [29]. Superstreptavidin (SSA) biosensors were loaded with biotinylated macrodomain protein and equilibrated for 120 sec in assay buffer (25 mM HEPES (pH 7.4), 100 mM NaCl, 0.01 % Tween 20). Association and dissociation were monitored for 240 sec each in assay buffer at 25 °C. For compound characterisation, compounds were typically prepared as seven 1:1 serial dilutions starting from 10 µM or 50 µM. Binding to the reference sensors (no protein attached) was subtracted before calculations and data was processed using the fortéBio analysis software provided by the manufacturer.
Crystallisation of PARP14 MD2 with MnK2-13
Surface entropy reduction (SER) mutations were introduced into PARP14 MD2 (A994−N1191) by the overlapping PCR method. Several mutants were prepared, of which construct PARP14A-c013 with K1048S, K1154S, K1158S, and K1162S mutation could be crystallized with MnK2-13. For protein crystallization, purified PARP14A-c013 was buffer exchanged into 20 mM HEPES (pH 7.4), 500 mM NaCl, 5% glycerol, and 0.5 mM TCEP, and concentrated to 16 mg/mL, using 10 kDa MWCO centrifugal concentrators (Millipore). MnK2-13 inhibitor dissolved to 50 mM in DMSO was added to a final concentration of 1.0 mM (2 % DMSO) and incubated on ice for approximately 30 min. The sample was centrifuged at 14,000 rpm for 10 min at 4 °C prior to setting up 150 nL volume sitting drops at three ratios (2:1, 1:1, or 1:2 protein−inhibitor complex to crystallization solution). A crystal was obtained with a 1:2 ratio of protein to a crystallization solution consisting of 0.8 M sodium phosphate monobasic, 0.8 M potassium phosphate dibasic, and 0.1 M HEPES at pH 7.5 and was cryoprotected in mother liquor supplemented with 25 % ethylene glycol before flash-freezing in liquid nitrogen for data collection. Diffraction data were collected at the Diamond Light Source (Harwell, UK) beamline I02.
Crystallisation of PARP14 MD3 with fragments
PARP14 MD3 apo was crystallised by mixing on pre-cooled sitting drop crystallisation plates 100 nL of 30 mg/mL protein in 20 mM HEPES (pH 7.4), 300 mM NaCl, 10 % glycerol, 0.5 mM TCEP with 50 nL of reservoir solution containing 80 mM KBr and 30 % PEG2000MME and adding 15 nL of a crystal seed solution obtained from a previous crystallisation experiment, diluted 1:500 from the stock in 90 mM KBr and 30 % PEG2000MME. The seeds were prepared from a single crystal growing in 90 mM KBr and 30 % PEG2000MME which was smashed to seeds using a 3 mm PTFE seed bead (Fisher Scientific) using standard laboratory vortex at full speed in 100 µL reservoir solution. Crystal growth was completed after 24 hours with incubation at 4 °C.
PARP14 MD3 apo crystals were soaked with compounds from the DSPL2.0 library consisting of 776 fragments at 500 mM in DMSO-d6 (and a subset in ethylene glycol). Soaking was performed by acoustically transferring 17.5 nL of compound solution to the crystallisation drop using an Echo 550 (Labcyte) [30] resulting in a final compound concentration of 50 mM with 10 % DMSO, calculated based on the initial drop volume. Crystals were incubated for 4-6 hours at 4 °C and then harvested (without cryoprotection) and cryocooled before X-ray diffraction data collection on the beamline I04-1 at Diamond Light Source (Harwell, UK).
Coordinates and structure factors for all data sets are deposited in the RCSB Protein Data Bank. Data collection and refinement statistics are available from the PDB pages.
Structure determination
PARP14 MD2
The diffraction data collected from a PARP14 MD2-MnK2-13 co-crystal was processed using MOSFLM [31] and AIMLESS [32]. The structure was solved by molecular replacement using PHASER [33] and a published structure of PARP14 MD2 (PDB ID 3Q71) as a search model. There was one molecule of PARP14 MD2 in the asymmetric unit. Coot [34] and REFMAC5 [35] were used for building the model and refinement. MOLPROBITY [36] was used for model validation and analysis.
PARP14 MD3
X-ray diffraction data collected for the PARP14 MD3 fragment screen was processed using the Diamond autoprocessing pipeline, utilising xia2 [37] and DIALS [38], and programs from the CCP4 suite [39]. Electron-density maps were generated using the XChemExplorer [40] via DIMPLE [41]. Ligand restraints were generated with AceDRG [42] and ligand binding was detected with PanDDA [43], with ligands built into PanDDA event maps. Iterative refinement and manual model correction was performed using REFMAC5 [35] and Coot [34], respectively.
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