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Horizontal Tabs
PDB ID |
Structure Details |
Structure of hsMTHFR in complex with FAD and SAH |
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Structure of scMET12 catalytic domain |
Non-SGC Resources
Commercially available CRISPR/Cas9 knockout plasmids | Commercially available antibodies |
---|---|
SCBT: Cat # sc-406554-KO-2 | Thermofisher: MA5-15844 (monoclonal) |
Genscript: Cat # 4524 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. |
SCBT: x sc-517229 (monoclonal) |
Protein Expression and Purification
hsMTHFR1-656 and hsMTHFR38-644
SGC ID: MTHFRA-c034 (hsMTHFR1-656), MTHFRA-c042 (hsMTHFR38-644)
Vector: pFB-CT10HF-LIC
Entry clone accession: BC053509
Cell line: DH10Bac
Tags and additions: C-terminal, TEV protease cleavable deca-histidine tag
Construct protein sequence of MTHFRA-c034 (hsMTHFR1-656):
MVNEARGNSSLNPCLEGSASSGSESSKDSSRCSTPGLDPERHERLREKMRRRLESGDKWFSLEFFPPRTAEGAVNLISRFDRMAAGGPLYIDVTWHPAGDPGSDKETSSMMIASTAVNYCGLETILHMTCCRQRLEEITGHLHKAKQLGLKNIMALRGDPIGDQWEEEEGGFNYAVDLVKHIRSEFGDYFDICVAGYPKGHPEAGSFEADLKHLKEKVSAGADFIITQLFFEADTFFRFVKACTDMGITCPIVPGIFPIQGYHSLRQLVKLSKLEVPQEIKDVIEPIKDNDAAIRNYGIELAVSLCQELLASGLVPGLHFYTLNREMATTEVLKRLGMWTEDPRRPLPWALSAHPKRREEDVRPIFWASRPKSYIYRTQEWDEFPNGRWGNSSSPAFGELKDYYLFYLKSKSPKEELLKMWGEELTSEASVFEVFVLYLSGEPNRNGHKVTCLPWNDEPLAAETSLLKEELLRVNRQGILTINSQPNINGKPSSDPIVGWGPSGGYVFQKAYLEFFTSRETAEALLQVLKKYELRVNYHLVNVKGENITNAPELQPNAVTWGIFPGREIIQPTVVDPVSFMFWKDEAFALWIEQWGKLYEEESPSRTIIQYIHDNYFLVNLVDNDFPLDNCLWQVVEDTLELLNRPTQNARETEAPAENLYFQ*SHHHHHHHHHHDYKDDDDK
Construct protein sequence of MTHFRA-c042 (hsMTHFR38-644):
MDPERHERLREKMRRRLESGDKWFSLEFFPPRTAEGAVNLISRFDRMAAGGPLYIDVTWHPAGDPGSDKETSSMMIASTAVNYCGLETILHMTCCRQRLEEITGHLHKAKQLGLKNIMALRGDPIGDQWEEEEGGFNYAVDLVKHIRSEFGDYFDICVAGYPKGHPEAGSFEADLKHLKEKVSAGADFIITQLFFEADTFFRFVKACTDMGITCPIVPGIFPIQGYHSLRQLVKLSKLEVPQEIKDVIEPIKDNDAAIRNYGIELAVSLCQELLASGLVPGLHFYTLNREMATTEVLKRLGMWTEDPRRPLPWALSAHPKRREEDVRPIFWASRPKSYIYRTQEWDEFPNGRWGNSSSPAFGELKDYYLFYLKSKSPKEELLKMWGEELTSEASVFEVFVLYLSGEPNRNGHKVTCLPWNDEPLAAETSLLKEELLRVNRQGILTINSQPNINGKPSSDPIVGWGPSGGYVFQKAYLEFFTSRETAEALLQVLKKYELRVNYHLVNVKGENITNAPELQPNAVTWGIFPGREIIQPTVVDPVSFMFWKDEAFALWIEQWGKLYEEESPSRTIIQYIHDNYFLVNLVDNDFPLDNCLWQVVEDTLELLNAENLYFQ*SHHHHHHHHHHDYKDDDDK
(underlined sequence contains vector encoded His-tag and TEV protease cleavage site*)
Bacmid DNA was prepared from DH10Bac cells and using to transfect Sf9 insect cells for the preparation of initial baculovirus. MTHFR protein was expressed from infected Sf9 cells cultivated in InsectXpress medium (Lonza) for 72 hours at 27°C.
Harvested cells were resuspended in lysis buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 20 mM Imidazole pH 7.5, 0.5 mM TCEP, 1 µL per 1 mL protease inhibitor cocktail EDTA-free).
Cell pellet was dissolved in approximately 200 mL lysis buffer and broken by sonication, done at 35% amplitude for 5 minutes with cycles of 5 seconds on and 10 seconds off. The cell debris was pelleted at 35000 x g, 1h and the supernatant used for purification with Nickel resin.
Buffers used:
Binding Buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 20 mM Imidazole pH 7.5, 0.5 mM TCEP
Wash Buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 40 mM Imidazole pH 7.5, 0.5 mM TCEP
Elution Buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 250 mM Imidazole pH 7.5, 0.5 mM TCEP
The clarified cell extract was added to 5 ml of Ni-NTA resin pre-equilibrated with lysis buffer and passed through a glass column. The column was then washed with Binding Buffer (2 x 50 mL) and Wash Buffer (2 x 50 mL). The protein was eluted with Elution Buffer in 5 x 5 mL fractions. The eluted fractions from column 1 were pooled and concentrated to 10 mL with a 50 kDa MWCO spin concentrator and injected into an S200 16/60 column (pre-equilibrated in GF Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 0.5 mM TCEP, 5% Glycerol)) at 1.0 mL/min. 1.5 mL-fractions were collected. The eluted protein was pooled and concentrated to 17 mg/mL using a 50 kDa MWCO concentrator.
Regulatory Domain (hsMTHFR348-656)
SGC ID: MTHFRA-c113
Vector: pNIC28-Bsa4
Entry clone accession: BC053509
Cell line: E. coli Rosetta
Tags and additions: N-terminal, TEV protease cleavable hexa-histidine tag
Construct protein sequence:
MHHHHHHSSGVDLGTENLYFQ*SMPWALSAHPKRREEDVRPIFWASRPKSYIYRTQEWDEFPNGRWGNSSSPAFGELKDYYLFYLKSKSPKEELLKMWGEELTSEASVFEVFVLYLSGEPNRNGHKVTCLPWNDEPLAAETSLLKEELLRVNRQGILTINSQPNINGKPSSDPIVGWGPSGGYVFQKAYLEFFTSRETAEALLQVLKKYELRVNYHLVNVKGENITNAPELQPNAVTWGIFPGREIIQPTVVDPVSFMFWKDEAFALWIEQWGKLYEEESPSRTIIQYIHDNYFLVNLVDNDFPLDNCLWQVVEDTLELLNRPTQNARETEAP
(underlined sequence contains vector encoded His-tag and TEV protease cleavage site*)
DNA was transformed into BL21(DE3) cells, plated and then used to inoculate a small-scale LB culture. This was then used to inoculate 6L of TB media. Cultures were induced overnight at 18°C with 0.1mM IPTG. Harvested cells were resuspended in lysis buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 20 mM Imidazole pH 7.5, 0.5 mM TCEP, 1 µL per 1 mL protease inhibitor cocktail EDTA-free).
The cell pellet was dissolved in approximately 200 mL lysis buffer and broken by sonication, performed at 35% amplitude for 5 minutes with cycles of 5 seconds on and 10 seconds off. The cell debris was pelleted at 35000 x g, 1h and the supernatant used for purification with Nickel resin.
Buffers used:
Binding Buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 20 mM Imidazole pH 7.5, 0.5 mM TCEP
Wash Buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 40 mM Imidazole pH 7.5, 0.5 mM TCEP
Elution Buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 250 mM Imidazole pH 7.5, 0.5 mM TCEP
The clarified cell extract was added to 5 ml of Ni-NTA resin pre-equilibrated with lysis buffer and passed through a glass column. The column was then washed with Binding Buffer (2 x 50 mL) and Wash Buffer (2 x 50 mL). The protein was eluted with Elution Buffer in 5 x 5 mL fractions. The eluted fractions from column 1 were pooled and concentrated to 10 mL with a 30 kDa MWCO spin concentrator and injected into an S200 16/60 column pre-equilibrated in GF Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 0.5 mM TCEP, 5% Glycerol) at 1.0 mL/min. 1.5 mL-fractions were collected. The eluted protein was pooled and concentrated to 17 mg/mL using a 30 kDa MWCO concentrator.
Assays
All enzymatic assays were performed using the physiological forward assay described by Suormala et al (21) with modifications as described by Rummel et al (24) and Burda et al (11,25). Only minor adaptations were made for use with pure recombinant protein, including reducing the assay time to 7 minutes and the addition of BSA to keep purified proteins stable. For SAM inhibition, purified SAM (26) was used. The Ki was estimated following a plot of log(inhibitor) vs. response and a four-parameter curve fit as performed by GraphPad Prism (v6.07).
Analytical gel filtration was performed on a Superdex 200 HiLoad 10/30 column (GE Healthcare) pre-equilibrated with 10 mM HEPES pH 7.5, 150 mM NaCl and 5% glycerol in the presence or absence of 250 µM SAH or SAM (both Sigma-Aldrich).
Differential scanning fluorimetry was used to assay shifts in melting temperature caused by ligand binding in a 96-well PCR plate using an LC480 light cyler (Roche). Each well (20 µl) consisted of protein (0.1 mg ml-1), SYPRO-Orange (Invitrogen) diluted 1000X, and buffer (10 mM HEPES pH 7.5, 500 mM NaCl) in the presence of 0 - 250 µM SAM, SAH or analogue compounds.
Surface Plasmon Resonance (SPR)
SPR was used to determine the affinity between hsMTHFR38-644 and (S)-SKI-72. 30 μg of hsMTHFR38-644 was immobilised onto Sensor Chip CM5 sensor (series S) via the protein -NH2 groups employing 1-Ethyl-3-(3-dimethylaminoproppyl)carbodiimide crosslinker (EDC) and N-hydroxysuccinimide (NHS) in acetate buffer pH 4.0. For the binding experiment, (S)-SKI-72 was serially diluted from 6.25 μM to 0.05 μM in 20 mM Hepes pH 7.5, 150 mM NaCl, 0.5 mM TCEP, 0.05% Tween, 5% DMSO. Association and dissociation times were set for 60 seconds and 90 seconds, respectively. Data were analysed using the Biacore S200 Evaluation Software.
Mass spectrometry
Mass spectrometry of hsMTHFR proteins under native conditions was performed on an Agilent 1290 uHPLC system, in accordance with previously published protocol (27).
To prepare samples for phosphorylation mapping, 20-100 µg hsMTHFR1-656 was reduced in 100 µl of 100 mM ammonium bicarbonate buffer, pH 7.5 by addition of 1 µl of 1 M DTT and incubation at 56°C for 40 minutes. Alkylation was performed by addition of 4 µl of saturated iodoacetamide solution and incubated at room temperature in the dark for 20 minutes. Endoprotease digestion was performed using either trypsin, Smart Digest trypsin (Thermo) or pepsin. LC-MSMS analyses were performed using both whole endoprotease digests and metal oxide affinity enriched samples, on a Dionex U3000 nanoHPLC coupled to a Bruker Esquire HCT ion trap mass spectrometer.
Crystallisation & Structure Determination
Proteins were concentrated to 15-20 mg/ml prior to crystallisation. hsMTHFR38-644 crystals were grown by sitting drop vapour diffusion at 20°C, in mother liquor containing 0.1 M Na citrate tribasic, 22.5% PEG4K, 5% 2-propanol. scMET121-302 crystals were grown by sitting drop vapour diffusion at 20°C, in mother liquor containing 0.2M Na/K tartrate, 20% PEG3350. All crystals were cryo-protected in mother liquor containing ethylene glycol (25% v/v) and flash-cooled in liquid nitrogen.
X-ray diffraction data were collected at the Diamond Light Source and processed using XIA2 (28). The hsMTHFR38-644 structure was solved by selenium multi-wavelength anomalous diffraction phasing using autoSHARP (29), and subjected to automated building with BUCCANEER (30). Phases for scMET121-302 were calculated by molecular replacement using the coordinates of Thermus thermophiles MTHFR (PDB code: 3APY) as model.
Ligand docking
To perform a virtual docking experiment employing the compounds identified in the DSF screen and hsMTHFR38-644 (PDB code: 6FCX) coordinates, we used ICM-Pro’s standard docking algorithm and removed crystallographic SAH from MTHFR coordinates prior to docking. As a control, we docked SAH back into hsMTHFR model, which recapitulated the conformation observed in the crystal structure, suggesting that the modelled binding conformation of the compounds represent a good starting point for inferring their interactions with MTHFR.
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