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PDB ID |
Structure Details |
ELOVL7 crystal structure with covalently bound 3-keto-eicosanoyl-CoA |
Protein Expression and Purification
Vector: pFB-CT10HF-LIC (available from The Addgene Nonprofit Plasmid Repository)
Cell line: DH10Bac, Sf9 cells, Expi293F
Tags and additions: C-terminal TEV protease site, followed by 10x His and FLAG tags
Construct sequence: Residues 1 – 281
MAFSDLTSRTVHLYDNWIKDADPRVEDWLLMSSPLPQTILLGFYVYFVTSLGPKLMENRKPFELKKAMITYNFFIVLFSVYMCYEFVMSGWGIGYSFRCDIVDYSRSPTALRMARTCWLYYFSKFIELLDTIFFVLRKKNSQVTFLHVFHHTIMPWTWWFGVKFAAGGLGTFHALLNTAVHVVMYSYYGLSALGPAYQKYLWWKKYLTSLQLVQFVIVAIHISQFFFMEDCKYQFPVFACIIMSYSFMFLLLFLHFWYRAYTKGQRLPKTVKNGTCKNKDNAENLYFQSHHHHHHHHHHDYKDDDDK
(underlined sequence contains vector encoded TEV protease cleavage site, His and FLAG tag)
Expression
The human ELOVL7 gene (HGNC:26292; Gene ID 6505), which encodes the ELOVL7 protein (ELOVL fatty acid elongase 7; residues Met1 to Asn281), was subcloned into the pFB-CT10HF-LIC vector and baculovirus was generated using the Bac-to-Bac system. Briefly, this was performed by transforming into the Escherichia coli strain DH10Bac, to generate bacmid DNA, which was then used to transfect Spodoptera frugiperda (Sf9) insect cells, from which recombinant baculovirus were obtained. Large scale grow-ups of Sf9 cells were infected with baculovirus and incubated for 72 h at 27 °C in shaker flasks. Cells were harvested by centrifugation at 900 g for 10 min. The cell pellets were resuspended in PBS and pelleted again by centrifugation at 900 g for 20 min, then flash-frozen in liquid nitrogen and stored at -80 °C.
For mammalian expression, the same construct was also cloned into the pHTBV1.1-LIC baculovirus transfer vector (The BacMam vector backbone (pHTBV1.1), which was kindly provided by Professor Frederick Boyce, Massachusetts General Hospital, Cambridge, MA and adapted for ligation independent cloning in house) for expression in Expi293F cells (Thermo-Fisher Scientific, Cat. No. A14527). This vector also adds a TEV cleavable His10-FLAG tag to the C-terminus of the protein. For expression, 1 L of Expi293F cell cultures (2 x 106 cells/ml) in Freestyle 293TM Expression Medium (Thermo-Fisher) were transduced with 30 ml of P3 baculovirus (third passage) in the presence of 5 mM sodium butyrate in a 2 L roller bottle (Biofil). Cells were grown in a humidity controlled orbital shaker for 48 hours at 37 °C with 8% CO2 before being harvested using the same process as for Sf9 cells.
Cell Lysis and detergent extraction of membrane proteins
Extraction Buffer (EXB): 50 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 5% v/v glycerol, 1 mM TCEP-NaOH, Roche protease inhibitor cocktail EDTA-free (1 tablet was used for 40 ml resuspended cells, dissolved in 1 ml Extraction buffer per tablet by vortexing prior to addition to the cell pellets).
Cell pellets were resuspended in EXB buffer (50 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 5% v/v glycerol, 1 mM TCEP-NaOH, Roche protease inhibitor cocktail EDTA-free) at the ratio of 50 ml per litre of equivalent original cell culture. The resuspension was then passed twice through an EmulsiFlex-C5 homogenizer (Avestin Inc.) at 10000 psi. Membrane proteins were extracted from the cell lysate with 1% w/v octyl glucose neopentyl glycol (OGNG; Generon, Cat. No. NG311) / 0.1% cholesteryl hemisuccinate tris salt (CHS; Sigma-Aldrich, Cat. No. C6512) and rotated for 2 h. Cell debris was removed by centrifugation at 35,000 x g for 1 h at 4 oC.
Purification
Wash Buffer: 50 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 1 mM TCEP-NaOH, 0.12% w/v OGNG / 0.012% w/v CHS and 20 mM imidazole pH 8.0
Elution Buffer: 50 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 1 mM TCEP-NaOH, 0.12% w/v OGNG / 0.012% w/v CHS and 250 mM imidazole pH 8.0
PD10 Buffer: 50 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 1 mM TCEP-NaOH, 0.15% w/v OGNG / 0.015% w/v CHS
Size exclusion buffer (SEC) Buffer: 20 mM HEPES-NaOH, pH 7.5, 200 mM NaCl, 1 mM TCEP-NaOH, 0.08% w/v OGNG/ 0.008% w/v CHS
Column 1: Co2+ TALON resin
The detergent-extracted supernatant was supplemented with 5 mM imidazole pH 8.0 before incubation with Co2+ charged TALON resin (Clontech) for 1 h on a rotator at 4 oC (1 ml resin slurry per L original culture volume). The Talon resin was collected by centrifugation at 700 x g for 5 mins and washed with 30 column volumes of wash buffer before the target protein was eluted with elution buffer. Peak fractions were combined and passed through PD10 columns, pre-equilibrated with four column volumes of PD10 buffer.
TEV protease cleavage and reverse purification
TEV protease was added at a ratio of 10:1 (ELOVL7:enzyme, wt:wt) and incubated at 4 oC overnight. For each litre of initial cell culture volume, 0.25 ml of a 50% slurry of TALON resin (pre-equilibrated as above) was added and the sample was rotated at 4 oC for 1 hour. The sample was transferred to a gravity column and the flow-through was collected.
Column 2: Superdex 200 Increase 10/300 GL column (GE Healthcare)
The protein sample was concentrated in a 100 kDa MWCO Vivaspin 20 centrifugal concentrator (pre-equilibrated in SEC buffer without detergent) at 3,000 g, with mixing every 5 min, to a final volume of < 1 ml. After centrifugation at 21,000 g for 20 min at 4o C, the sample was subjected to size exclusion chromatography
Iodoacetamide modification
For structural studies, the flow through from the reverse Talon step was incubated with 50 mM iodoacetamide (IAM) (Merck Millipore) for 20 mins at room temperature. IAM was removed by passing the reaction mixture down a PD-10 desalting column prior to concentration. After SEC, fractions containing the highest concentration of ELOVL7 were pooled and concentrated to 12-25 mg/ml using a 100 kDa MWCO Vivaspin 20 centrifugal concentrator.
Crystallisation
Initial protein crystals were grown at 4 °C in condition E10 of the MemGold2-ECO Screen (Molecular Dimensions; 0.05 M Na-acetate pH 4.5, 0.23 M NaCl, 33 % v/v polyethylene glycol (PEG) 400) in 3-well sitting-drop crystallisation plates (SwissCi) with 150 nl drops and 2:1 and 1:1 protein to reservoir ratios. Crystals appeared after 4-7 days and grew to full size within 3-4 weeks. Two microlitre hanging drops were set up in 24-well XRL plates (Molecular Dimensions) at protein to reservoir ratios of 2.5:1, 2:1 and 1.5:1. The best crystals grew in 0.1 M Na-acetate pH 4.5, 0.23 M NaCl, 34-38% v/v PEG400 using the IAM-modified protein at a concentration of 5-8 mg/ml and were harvested after 12-14 days. Prior to vitrification, crystals were sequentially transferred to mother liquor solutions with an increasing amount of PEG400 to a final concentration of PEG400 of 46% v/v over 10-15 mins. For heavy atom derivatisation, crystals were looped into drops containing reservoir solution supplemented with 10 mM mercury chloride and soaked for 10 mins. Hg-soaked crystals were then treated to the same PEG400 escalation strategy using Hg-free solutions before being vitrified in liquid nitrogen. Diffraction data were collected at the I24 microfocus beamline at Diamond Light Source.
Structure Determination
ELOVL7 crystallises in monoclinic space group P21 with two copies of the enzyme in each asymmetric unit. All diffraction data were highly anisotropic and limited to between 3.4 - 4.5 Å resolution in the worse direction and 2.05 – 3 Å in the best direction. Phasing was carried out in PHENIX (22) using SIRAS with the Hg-peak data and a 3Å isomorphous lower resolution native dataset. Two Hg2+ sites were located with phenix.hyss using data to 4.5 Å (23). The resulting 3 Å phased electron density map had clear protein density allowing the identification of the NCS relationship between the two ELOVL7 molecules in the asymmetric unit. After two-fold averaging using RESOLVE, the resultant map was of sufficient quality to manually model all the TM helices. Initial phases were further improved by cross-crystal averaging with a non-isomorphous, less anisotropic and slightly higher resolution dataset using DMMULTI (24). The resultant map was of excellent quality and the majority of the structure could be built automatically with BUCCANEER (25). Model completion was carried out manually in COOT (26) and the structure was refined with BUSTER (27) using all data to 2.05 Å. The final model comprises residues 16-269 (chain B, 14-269), a covalently bound 3-keto-CoA acyl lipid, four OGNG detergent molecules and 112 solvent molecules.
Mass spectrometry
The denaturing intact mass spectrometry measurements were performed using an Agilent 1290 Infinity LC System in-line with an Agilent 6530 Accurate-Mass Q-TOF LC/MS (Agilent Technologies Inc.). The solvent system was consisted of 0.1% OptimaTM LC/MS grade formic acid (Fisher Chemical) in HPLC electrochemical grade water (Fisher Chemical) (solvent A) and 0.1% formic acid in OptimaTM LC/MS grade methanol (Fisher Chemical) (solvent B). Typically, 1-2 µg of protein sample was diluted to 60 µl with 30% methanol in 0.1% formic acid. 60 µl of sample was injected onto a ZORBAX StableBond 300 C3 column (Agilent Technologies Inc.) by an auto sampler. The flowrate of the LC system was set to 0.5 ml/min. 30% of solvent B was applied in the beginning and the sample elution was initiated by a linear gradient from 30% to 95% of solvent B over 7 min. 95% B was then applied for 2 min, followed by 2 min equilibration with 30% B. The mass spectrometer was in positive ion, 2 GHz detector mode and spectra were recorded with capillary, fragmentor and collision cell voltages of 4000 V, 250 V and 0 V, respectively. The drying gas was supplied at 350°C with flow rate of 12 l/min and nebulizer at 60 psi. The data was acquired from 100-3200 m/z. Data analysis was performed using MassHunter Qualitative Analysis Version B.07.00 (Agilent) software.
In order to trap the covalent acyl-enzyme intermediate, the purified, tagged, wild-type ELOVL7 protein at 1.5 mg/mL (obtained after the desalting step that followed IMAC elution) was incubated with 100 μM C18:0-CoA (Avanti Polar Lipids, Cat. No. 870718) for 2 hours at 37 oC, in the presence and absence of 1 mM EDTA, 1 mM EGTA, or 100 μM Malonyl CoA (Sigma-Aldrich, Cat. No. M4263). The reaction was terminated by dilution into 30% methanol in 0.1% formic acid, as described above. Covalent acyl-enzyme intermediate formation was identified by monitoring the presence of a mass shift upon incubation with the substrate, corresponding to the addition of the substrate acyl chain through attachment of the histidine imidazole to the thioester carbonyl, resulting in thioester cleavage and loss of CoA (predicted +266.47 Da upon reaction with C18:0-CoA). The site of covalent modification was probed by carrying out this experiment with the His150Ala and His181Ala mutants, which allowed identification of His150 as the nucleophile involved in covalent intermediate formation.
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