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Horizontal Tabs
PDB ID |
Structure Details |
Uridylylated hGALT bound with glucose-1-phosphate |
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hGALT with epitope mutations A21Y:A22T:T23P:R25L |
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hGALK1 bound with galactose and ADP |
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hGALK1 bound with galactose and Fragment a (N-(Cyclobutylmethyl)-1,5-dimethyl-1H-pyrazole-4-carboxamide, x717) |
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hGALK1 bound with galactose and Fragment b (4-{[2-(Methylsulfonyl)-1H-imidazol-1-yl]methyl}-1,3-thiazole, x843) |
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hGALK1 bound with galactose and Fragment c (Ethyl 1-(2-pyrazinyl)-4-piperidinecarboxylate, x682) |
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hGALK1 bound with galactose and Fragment d (1-(4-Methoxyphenyl)-3-(4-pyridinyl)urea, x747) |
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hGALK1 bound with galactose and Fragment e (5-Chloro-N-isobutyl-2-methoxybenzamide, x841) |
GALT
Protein Expression and Purification
DNA fragment encoding full-length hGALT was amplified from a cDNA clone (IMAGE: 3922902) that harbours the p.Asn314Asp polymorphism (11), and subcloned into the pNIC28-Bsa4 vector that incorporates an N-terminal TEV-cleavable His6-tag. hGALT variants were created by either QuikChange site-directed mutagenesis or primer extension PCR method. hGALT (wt or variants) was cultured in 6 L of Terrific Broth at 37 °C, and induced with 0.1 mM IPTG overnight at 18 °C. Cell pellets were harvested, homogenized in lysis buffer (50 mM sodium phosphate pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP) and centrifuged to remove insoluble material. The supernatant was purified by immobilized metal affinity (Talon resin; GE Healthcare) and size-exclusion chromatography in Superdex 200 Hi-Load 16/60 column (GE Healthcare), pre-equilibrated with buffer 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP. Purified protein was treated with His-tagged TEV protease overnight at 4 °C, and further purified by reverse Nickel affinity and size exclusion chromatography. The final purified protein was concentrated to 20 mg∕ml, flash cooled in liquid nitrogen and stored at −80 °C. To produce apo and uridylylated protein, 0.5 mM tag-removed hGALT was incubated overnight at 4 °C with 41 mM Glc-1-P or UDP-Glc before size-exclusion chromatography (Superdex 200 10/300 GL, GE Healthcare). Samples were analysed by denaturing mass spectrometry.
Crystallization and Structure Determination
For the native hGALT protein, crystals were grown by vapour diffusion at 20 °C, from sitting drops mixing 200 nl of protein (20 mg/ml; pre-incubated with 5 m M UDP-Glc) and 100 nl of reservoir solution containing 0.2 M ammonium sulphate, 30% (w/v) PEG 8000. For the hGALT crystal epitope variant (A21Y:A22T:T23P:R25L), crystals were obtained at 4 oC in reservoir solution containing 0.2M ammonium sulphate and 30% PEG4000. Crystals were cryo-protected with reservoir solution supplemented with 25% (v/v) ethylene glycol and flash-cooled in liquid nitrogen. The structures were solved by molecular replacement with PHASER (15), (15), using the structure of E. coli GALT (1HXP) as template. Modelling and refinement were carried out using Refmac (16) and Coot (17).
Rapid Fire Mass Spectrometry Assay
In a typical experiment, 50 μL out of 540 μL assay buffer containing 0.1 μM hGALT (20 mM Hepes pH 7.5, 150 mM NaCl, 5% glycerol, 0.5 mM TCEP) is injected prior to the addition of UDP-Glc. Next, 10 μL of UDP-Glc stock is added to reach the desired final concentration (0.1, 0.5, 1.0, 2.0 and 4.0 μM) of each experiment. The reactions were transferred to a RapidFire RF360 high-throughput sampling robot connected to an Agilent 6530 Accurate-Mass Quadrupole time-of-flight (Q-TOF) mass spectrometer operated in the positive ion mode (Agilent, Wakefield, MA, USA). The peak-area data of each injection were integrated and the percentage activity was calculated. All values were normalised using the percentage of uridylylation obtained from the first time point of each experiment. The initial slope for enzyme progress curves was taken for each UDP-Glc concentration, and data were fitted to the Michaelis–Menten equation in GraphPad Prism 7.
Single-Chain Fragment Variable Antibody
Three scFv antibody fragments for hGALT have been generated via phage-display approach. The scFvs constructs are encoded in the pHAT6 vector with a LacZ promoter, OmpA leader and C-terminal Triple FLAG + His6 tags. Around 1 mg/L is expected from expression in 1.5 L scale from LB or TB media. The scFvs are isolated from cell lysate by affinity chromatography using HiTrap protein A, and dialyzed to 1xPBS overnight for storage.
|
Kd from SPR Single Cycle Kinetics |
IP-MS Normalized Spectral Abundance Factor |
J-GALTA-2 |
1.88 nM |
21 |
J-GALTA-3 |
2.28 nM |
30 |
J-GALTA-4 |
5.85 nM |
33 |
J-GALTA-2EVQLLESGGGLVQPGGSLRLSCAASGFTFYSSYMYWVRQAPGKGLEWVSYISGGGSSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARESGSVGFMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYYYPSTFGQGTKLEIKRTDYKDHDGDYKDHDIDYKDDDDKAAAHHHHHH*
J-GALTA-3EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSYMGWVRQAPGKGLEWVSSIGSYGSGTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSYTNYSLVSIGIFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYYYPLTFGQGTKLEIKRTDYKDHDGDYKDHDIDYKDDDDKAAAHHHHHH*
J-GALTA-4EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYGMYWVRQAPGKGLEWVSSISSSSSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYAGGYGIDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTYLLSTFGQGTKLEIKRTDYKDHDGDYKDHDIDYKDDDDKAAAHHHHHH*
The three scFvs have been validated to bind endogenous hGALT using IP-MS, and display dissociation constants (Kd) in the range of 1.9 – 5.8 nM for recombinant hGALT as measured by SPR.
GALK1
Protein Expression and Purification
The GALK1 construct encodes full-length protein harbouring the surface entropy mutations K252A:E253A, with an engineered N-terminal His6-tag subcloned into the pET21d vector. hGALK1 was cultured in Terrific Broth with 0.1 mM IPTG induction. Cell pellets were harvested, homogenized in lysis buffer (50 mM sodium phosphate pH 7.4, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP) and centrifuged to remove insoluble material. The supernatant was purified by Nickel affinity (Thermo Fisher Scientific) followed by ion exchange (Resource Q, GE Healthcare) and size exclusion (Superdex 200 Hi-Load 16/60, GE Healthcare) chromatography into crystallisation buffer (50 mM sodium phosphate pH7.4, 500 mM NaCl, 5% glycerol, 30mM Galactose and 0.5 mM TCEP). Protein was concentrated to 20 mg/mL and flash-cooled for storage at −80 °C.
Kinase-Glo Activity Assay
GALK activity in vitro was determined using the Luminescent Kinase Assay (Kinase-Glo Promega), which detects ATP depletion, according to the manufacturer’s protocol. Specifically, ten μL/well of reaction containing 5 nM GALK, 100 μM Galactose and 35 μM ATP in assay buffer (20 mM HEPES pH 7.5, 5 mM MgCl2, 60 mM NaCl, 1 mM DTT, 0.01% BSA final concentration) as well as 100 μM of respective compounds was dispensed into 384-well assay plates (Greiner®). Following a 1 hour room temperature incubation, 10 μL of Kinase-Glo Plus detection reagent was added to provide an ATP-dependent luminescent readout (final assay volume: 20 μL/well). Luminescence was detected using a Pherastar Luminescence plate reader (BMG Labtech) after a10 minute incubation, using a 1 second exposure time and 2x binning. For IC50 determination, compounds were serially diluted from 400 μM to 0.2 μM. Experiments were carried out as determined in the manufacturer protocol. Data were plotted in Graphpad Prism.
Crystallization and Structure Determination
For co-crystallisation with ADP, 20mg/ml of GALK1 was pre-incubated with 10mM of ADP for 15 minutes on ice. Crystals were grown by vapour diffusion at 20 °C in 150 nL sitting drops equilibrated against well solution of 0.1M cacodylate pH 6.3, 17.5% PEG8K and 0.2M ammonium sulphate (optimised from Hampton Crystal Screen condition B3). Crystals were cryo-protected by addition of well solution supplemented with 25% ethylene glycol and flash-cooled before diffraction data collection at beamline I03 of the Diamond Light Source. The hGALK1-galactose-ADP structure was solved by molecular replacement using the published hGALK1 structure as template (PDB code: 1WUU)(10). PHENIX (18) and COOT were employed for refinement via iterative cycles.
Crystallography-based Fragment Screening
To produce crystals for XChem, 24 mg/mL hGALK1 was pre-incubated with 0.75 mM of an ATP-analogue (Interbioscreen STOCK4S-53098; referred to in text as cpd 3 (11)) dissolved in NMP (final NMP concentration of 5%). Crystals were grown by vapour diffusion in 300 nL sitting drops at 20°C, equilibrated against well solutions of 0.1 M MOPS/sodium HEPES pH 7.0-7.5, 40-50 % Morpheus® Precipitant Mix 4 (50% mix = 12.5% MPD, 12.5% PEG1000, 12.5% PEG3350), 0.1 M Morpheus® Carboxylic acids mix (0.02M each of:sodium formate, ammonium acetate, sodium citrate tribasic dehydrate, sodium potassium tartrate tetrahydrate and sodium oxamate).
For soaking, 50 nL of each fragment compound (final concentration of 125 mM) was added to a crystallization drop using an ECHO acoustic liquid handler dispenser at the Diamond light source XChem facility. Crystals were soaked for two hours with fragments from the Diamond-SGC Poised Library before being harvested using XChem SHIFTER technology, cryo-cooled in liquid nitrogen, and data sets collected at the beamline I04-1 in “automated unattended” mode. The XChemXplorer pipeline (19) was used for structure solution with parallel molecular replacement using DIMPLE (20) followed by map averaging and statistical modelling to identify weak electron densities generated from low occupancy fragments using Pandda software (21). Coordinates and structure factors for exemplary data sets with bound fragments are deposited in the RCSB Protein Data Bank with the remainder to follow
Non-SGC Resources
GALT |
GALK1 |
Commercially available CRISPR/Cas9 knockout plasmids |
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SCBT: Cat # sc-420482 |
SCBT: Cat # sc-420578 |
Genscript: Cat # 2592 These sgRNA sequences were validated in Sanjana N.E., Shalem O., Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014, 11(8):783-4. |
Addgene #76703, 76704, 76705 Nat Biotechnol. 2016, (2):184-191 |
Commercially available antibodies |
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SCBT: sc-365577 (monoclonal) |
SCBT: sc-393404 (monoclonal) |
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