Expression and purification of recombinant human NUDT7
Human NUDT7 (residues 14-235) was cloned into pNIC28-Bsa4 with a TEV-cleavable N-terminal hexahistidine tag. After transformation into E. coli (BL21(DE3)-R3), expression was performed in TB auto induction medium (FroMedium), supplemented with 20 g/L glycerol, 50 μg/mL kanamycin and 34 μg/mL chloramphenicol. Cultures were grown for four hours at 37 °C, then the temperature was decreased to 20 °C and the cultures were grown for another 20 hours. Cells were spun at 5000 rpm for 10 min, then resuspended in 0.5 mg/mL lysozyme, 1 µg/mL benzonase, 20 mM imidazole and stirred for 2 hours at room temperature. 1% Triton X-100 was added and the cells were frozen at -80°C. On thawing, cells were centrifuged for 1 hour at 4000 × g and the supernatant applied to a His GraviTrap column (GE healthcare) equilibrated with binding buffer (10 mM HEPES, 5 % glycerol, 500 mM NaCl, 0.5 mM TCEP, pH 7.5). After washing with binding buffer supplemented to 20 mM imidazole, NUDT7 was eluted with buffer supplemented to 500 mM imidazole. The eluted protein was applied to a PD-10 desalting column (GE Healthcare) and eluted with binding buffer supplemented to 20 mM imidazole. The N-terminal affinity tag was removed by TEV cleavage overnight and uncleaved protein was removed by applying it again to a His GraviTrap column. The flow-through was concentrated and purified further by size exclusion chromatography using a YARRA SEC-2000 PREP column (Phenomenex) equilibrated with binding buffer. Fractions containing protein were pooled, concentrated and stored at -80°C.
NUDT7 crystals were obtained by mixing 100nl of 30mg/mL protein in 10mM Na-HEPES pH7.5, 500mM NaCl, 5% glycerol with 50 nL of reservoir solution containing 0.1M BisTris pH 5.5, 0.1M ammonium acetate and 6%(w/v) PEG10.000. Compact, hexagon-shaped crystals with typical dimensions between 50 – 100 µm appeared within several days from sitting drop plates at 293K. An ECHO 550 acoustic liquid handler (Labcyte) was used to transfer individual fragments from the DSPL (7) and OxXChem (8) fragment library to crystal drops. Briefly, 38 nL of compound solution was added to each crystallisation drop resulting in a final compound concentration of 100 mM with 20 % DMSO, calculated based on the initial drop volume. Compounds from the 3-chloro-phenylamino series were soaked into NUDT7 crystals by adding a mixture containing 600 nL of 100mM compound in DMSO and 1200 nL of reservoir solution containing 0.1M BisTris pH 5.5, 0.1M ammonium acetate and 10% (w/v) PEG10.000. Crystals were incubated overnight at room temperature and then harvested (without further cryoprotection) and flash cooled in liquid nitrogen. Crystals of NUDT7 with covalent binders were grown by mixing 100 nL of 30mg/mL protein in 10mM Na-HEPES pH7.5, 500mM NaCl, 5% glycerol with 30 nL of 20mM compound in DMSO in sitting-drop crystallization plates containing 0.1M BisTris pH 5.5, 0.1M ammonium acetate and 4 - 16%(w/v) PEG10.000 in the reservoir at 293K. After overnight incubation of protein and compound, 100 nL of reservoir solution and 30nL of a crystal seed solution obtained from a previous crystallisation experiment, diluted 1:4 from the stock in 0.1M BisTris pH 5.5, 0.1 M ammonium acetate and 9%(w/v) PEG10.000 were added to the drop. Hexagon-shaped crystals appeared within several days. Prior to data collection, all crystals were transferred to a solution consisting of the precipitation buffer supplemented with 25% Ethylene glycol and subsequently flash cooled in liquid nitrogen. All X-ray diffraction data were collected on the beamline I04-1 at Diamond Light Source (Harwell, UK) unless stated otherwise.
Diffraction data were automatically processed by software pipelines at the Diamond Light Source (9). Initial refinement and map calculation was carried out with DIMPLE (10). PanDDA (11) was used for hit identification and further refinement and model building was performed with REFMAC (12) and COOT (13), respectively. All structure determination steps were performed within the XChemExplorer (14) data management and workflow tool.
Coordinates and structure factors for all data sets are deposited in the RCSB Protein Data Bank under group deposition ID G_1002045. Data collection and refinement statistics are available from the PDB pages. The complete PanDDA analysis and processed data from all crystals (including information about soaked compounds) that were prepared as part of the NUDT7 project can be accessed via the ZENODO data repository under DOI 10.5281/zenodo.1244111.
NUDT7 activity assay
Mass spectrometry assays monitoring acetyl-CoA hydrolysis by NUDT7 were performed on a Agilent 6530 RapidFire QTOF Mass Spectrometer in a 384-well plate format using polypropylene plates (Greiner, code 781280) and an assay buffer containing 20 mM HEPES pH 7.5, 200 mM NaCl and 5 mM MgCl2. All bulk liquid handling steps were performed using a multidrop combi reagent dispenser (Thermo Scientific, Code 5840300) equipped with a small tube plastic tip dispensing cassette (Thermo Scientific, Code 24073290). For inhibitor IC50 determinations an 11-point and 2-fold serial dilution in was prepared from a 50 mM stock solution in DMSO which was transferred to give four replicates using an ECHO 550 acoustic dispenser (Labcyte). The transferred volume was 400 nL giving a final DMSO concentration of 0.4 %. In addition, a DMSO control (400 nL) was transferred into alternate wells in columns 12 and 24 and 50 mM EDTA (NUDT7 inhibitor) was dispensed into alternate wells of column 24 as the background control. 80 µl assay buffer was added to all wells and NUDT7 was prepared to 500 nM (10 X final concentration in assay buffer) and acetyl-CoA was prepared to 200 uM (10 X final concentration in assay buffer). 10 µl NUDT7 was dispensed into half of the assay plate (for two of the compound replicates) and the plate was incubated at room temperature for 30 minutes. 10 µl NUDT7 was then dispensed into the remaining half of the assay plate (for the remaining two compound replicates). 10 µl acetyl-CoA was immediately dispensed into all wells of the assay plate to initiate the reaction and the enzyme reaction was allowed to proceed for 15 min. The enzyme reaction was stopped by addition of 10 ul of 50 mM EDTA and the plate was transferred to a RapidFire RF360 high throughput sampling robot. Samples were aspirated under vacuum and loaded onto a C4 solid phase extraction (SPE) cartridge equilibrated and washed for 5.5 sec with 1 mM octylammonium acetate in LCMS grade water to remove non-volatile buffer components. After the aqueous wash, analytes of interest were eluted from the C4 SPE onto an Agilent 6530 accurate mass Q-TOF in an organic elution step (85% acetonitrile in LC-MS grade water). Ion data for the acetyl-CoA and hydrolysed product were extracted and peak area data integrated using RapidFire integrator software (Agilent). % conversion of substrate to product was calculated in Excel and IC50 curves generated using Graphpad prism version 7.0. The assay had a Z score of 0.79 with the 30 minute pre-incubation and 0.75 without pre-incubation.