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Horizontal Tabs
PDB ID |
Structure details |
Resolution (Å) |
Structure of DHTKD1 bound with TPP |
1.87 |
|
Structure of DHTKD1 ground-state model |
1.52 |
|
Structure of DHTKD1 bound with TPP and fragment b (x0048): 2-[(1H-benzimidazol-2-ylamino)methyl]phenol |
1.61 |
|
|
Structure of DHTKD1 bound with TPP and fragment c (x0164): N-[(6-methylpyridin-3-yl)methyl]cyclobutanecarboxamide |
1.60 |
|
Structure of DHTKD1 bound with TPP and fragment d (x0173) ((2S,4S)-4-fluoro-1-((2-methylthiazol-4-yl)methyl)pyrrolidin-2-yl)methanamine |
1.67 |
|
Structure of DHTKD1 bound with TPP and fragment e (x0166): 1-(3-fluoropyridin-2-yl)-4,4-dimethylpyrrolidin-3-ol |
1.98 |
Structure of DHTKD1 bound with TPP and fragment f (x0095): 5-ethyl-N-[(1-methylpyrazol-4-yl)methyl]thiophene-2-carboxamide |
1.66 |
|
EMDB ID |
|
|
Cryo-EM map of human dihydrolipoamide succinyltransferase catalytic domain (DLST) |
4.70 |
Protein Expression and Purification
hDHTKD145-919 for crystallisation
SGC ID: DHTKD1A-c002
Vector: pNIC28-Bsa4
Accession: Q96HY7, NM_018706.6
Cell line: E. coli Rosetta
Tags and additions: N-terminal, TEV protease cleavable hexa-histidine tag.
Construct Protein Sequence:
MHHHHHHSSGVDLGTENLYFQ*SMGALERPPVDHGLARLVTVYCEHGHKAAKINPLFTGQALLENVPEIQALVQTLQGPFHTAGLLNMGKEEASLEEVLVYLNQIYCGQISIETSQLQSQDEKDWFAKRFEELQKETFTTEERKHLSKLMLESQEFDHFLATKFSTVKRYGGEGAESMMGFFHELLKMSAYSGITDVIIGMPHRGRLNLLTGLLQFPPELMFRKMRGLSEFPENFSATGDVLSHLTSSVDLYFGAHHPLHVTMLPNPSHLEAVNPVAVGKTRGRQQSRQDGDYSPDNSAQPGDRVICLQVHGDASFCGQGIVPETFTLSNLPHFRIGGSVHLIVNNQLGYTTPAERGRSSLYCSDIGKLVGCAIIHVNGDSPEEVVRATRLAFEYQRQFRKDVIIDLLCYRQWGHNELDEPFYTNPIMYKIIRARKSIPDTYAEHLIAGGLMTQEEVSEIKSSYYAKLNDHLNNMAHYRPPALNLQAHWQGLAQPEAQITTWSTGVPLDLLRFVGMKSVEVPRELQMHSHLLKTHVQSRMEKMMDGIKLDWATAEALALGSLLAQGFNVRLSGQDVGRGTFSQRHAIVVCQETDDTYIPLNHMDPNQKGFLEVSNSPLSEEAVLGFEYGMSIESPKLLPLWEAQFGDFFNGAQIIFDTFISGGEAKWLLQSGIVILLPHGYDGAGPDHSSCRIERFLQMCDSAEEGVDGDTVNMFVVHPTTPAQYFHLLRRQMVRNFRKPLIVASPKMLLRLPAAVSTLQEMAPGTTFNPVIGDSSVDPKKVKTLVFCSGKHFYSLVKQRESLGAKKHDFAIIRVEELCPFPLDSLQQEMSKYKHVKDHIWSQEEPQNMGPWSFVSPRFEKQLACKLRLVGRPPLPVPAVGIGTVHLHQHEDILAKTFA
(underlined sequence contains vector encoded His-tag and TEV protease cleavage site*)
hDHTKD11-919
SGC ID: DHTKD1A-c014
Vector: pCDF-LIC
Accession: Q96HY7, NM_018706.6
Cell line: E. coli Rosetta
Tags and additions: N-terminal, TEV protease cleavable hexa-histidine tag.
Construct Protein Sequence:
MHHHHHHSSGVDLGTENLYFQ*SMASATAAAARRGLGRALPLLWRGYQTERGVYGYRPRKPESREPQGALERPPVDHGLARLVTVYCEHGHKAAKINPLFTGQALLENVPEIQALVQTLQGPFHTAGLLNMGKEEASLEEVLVYLNQIYCGQISIETSQLQSQDEKDWFAKRFEELQKETFTTEERKHLSKLMLESQEFDHFLATKFSTVKRYGGEGAESMMGFFHELLKMSAYSGITDVIIGMPHRGRLNLLTGLLQFPPELMFRKMRGLSEFPENFSATGDVLSHLTSSVDLYFGAHHPLHVTMLPNPSHLEAVNPVAVGKTRGRQQSRQDGDYSPDNSAQPGDRVICLQVHGDASFCGQGIVPETFTLSNLPHFRIGGSVHLIVNNQLGYTTPAERGRSSLYCSDIGKLVGCAIIHVNGDSPEEVVRATRLAFEYQRQFRKDVIIDLLCYRQWGHNELDEPFYTNPIMYKIIRARKSIPDTYAEHLIAGGLMTQEEVSEIKSSYYAKLNDHLNNMAHYRPPALNLQAHWQGLAQPEAQITTWSTGVPLDLLRFVGMKSVEVPRELQMHSHLLKTHVQSRMEKMMDGIKLDWATAEALALGSLLAQGFNVRLSGQDVGRGTFSQRHAIVVCQETDDTYIPLNHMDPNQKGFLEVSNSPLSEEAVLGFEYGMSIESPKLLPLWEAQFGDFFNGAQIIFDTFISGGEAKWLLQSGIVILLPHGYDGAGPDHSSCRIERFLQMCDSAEEGVDGDTVNMFVVHPTTPAQYFHLLRRQMVRNFRKPLIVASPKMLLRLPAAVSTLQEMAPGTTFNPVIGDSSVDPKKVKTLVFCSGKHFYSLVKQRESLGAKKHDFAIIRVEELCPFPLDSLQQEMSKYKHVKDHIWSQEEPQNMGPWSFVSPRFEKQLACKLRLVGRPPLPVPAVGIGTVHLHQHEDILAKTFA
(underlined sequence contains vector encoded His-tag and TEV protease cleavage site*)
hDHTKD125-919
SGC ID: DHTKD1A-c015
Vector: pCDF-LIC
Accession: Q96HY7, NM_018706.6
Cell line: E. coli Rosetta
Tags and additions: N-terminal, TEV protease cleavable hexa-histidine tag.
Construct Protein Sequence:
MHHHHHHSSGVDLGTENLYFQ*SMGYQTERGVYGYRPRKPESREPQGALERPPVDHGLARLVTVYCEHGHKAAKINPLFTGQALLENVPEIQALVQTLQGPFHTAGLLNMGKEEASLEEVLVYLNQIYCGQISIETSQLQSQDEKDWFAKRFEELQKETFTTEERKHLSKLMLESQEFDHFLATKFSTVKRYGGEGAESMMGFFHELLKMSAYSGITDVIIGMPHRGRLNLLTGLLQFPPELMFRKMRGLSEFPENFSATGDVLSHLTSSVDLYFGAHHPLHVTMLPNPSHLEAVNPVAVGKTRGRQQSRQDGDYSPDNSAQPGDRVICLQVHGDASFCGQGIVPETFTLSNLPHFRIGGSVHLIVNNQLGYTTPAERGRSSLYCSDIGKLVGCAIIHVNGDSPEEVVRATRLAFEYQRQFRKDVIIDLLCYRQWGHNELDEPFYTNPIMYKIIRARKSIPDTYAEHLIAGGLMTQEEVSEIKSSYYAKLNDHLNNMAHYRPPALNLQAHWQGLAQPEAQITTWSTGVPLDLLRFVGMKSVEVPRELQMHSHLLKTHVQSRMEKMMDGIKLDWATAEALALGSLLAQGFNVRLSGQDVGRGTFSQRHAIVVCQETDDTYIPLNHMDPNQKGFLEVSNSPLSEEAVLGFEYGMSIESPKLLPLWEAQFGDFFNGAQIIFDTFISGGEAKWLLQSGIVILLPHGYDGAGPDHSSCRIERFLQMCDSAEEGVDGDTVNMFVVHPTTPAQYFHLLRRQMVRNFRKPLIVASPKMLLRLPAAVSTLQEMAPGTTFNPVIGDSSVDPKKVKTLVFCSGKHFYSLVKQRESLGAKKHDFAIIRVEELCPFPLDSLQQEMSKYKHVKDHIWSQEEPQNMGPWSFVSPRFEKQLACKLRLVGRPPLPVPAVGIGTVHLHQHEDILAKTFA
(underlined sequence contains vector encoded His-tag and TEV protease cleavage site*)
hDHTKD145-919 for co-expression in E. coli
SGC ID: DHTKD1A-c016
Vector: pCDF-LIC
Accession: Q96HY7, NM_018706.6
Cell line: E. coli Rosetta
Tags and additions: N-terminal, TEV protease cleavable hexa-histidine tag.
Construct Protein Sequence:
MHHHHHHSSGVDLGTENLYFQ*SMGALERPPVDHGLARLVTVYCEHGHKAAKINPLFTGQALLENVPEIQALVQTLQGPFHTAGLLNMGKEEASLEEVLVYLNQIYCGQISIETSQLQSQDEKDWFAKRFEELQKETFTTEERKHLSKLMLESQEFDHFLATKFSTVKRYGGEGAESMMGFFHELLKMSAYSGITDVIIGMPHRGRLNLLTGLLQFPPELMFRKMRGLSEFPENFSATGDVLSHLTSSVDLYFGAHHPLHVTMLPNPSHLEAVNPVAVGKTRGRQQSRQDGDYSPDNSAQPGDRVICLQVHGDASFCGQGIVPETFTLSNLPHFRIGGSVHLIVNNQLGYTTPAERGRSSLYCSDIGKLVGCAIIHVNGDSPEEVVRATRLAFEYQRQFRKDVIIDLLCYRQWGHNELDEPFYTNPIMYKIIRARKSIPDTYAEHLIAGGLMTQEEVSEIKSSYYAKLNDHLNNMAHYRPPALNLQAHWQGLAQPEAQITTWSTGVPLDLLRFVGMKSVEVPRELQMHSHLLKTHVQSRMEKMMDGIKLDWATAEALALGSLLAQGFNVRLSGQDVGRGTFSQRHAIVVCQETDDTYIPLNHMDPNQKGFLEVSNSPLSEEAVLGFEYGMSIESPKLLPLWEAQFGDFFNGAQIIFDTFISGGEAKWLLQSGIVILLPHGYDGAGPDHSSCRIERFLQMCDSAEEGVDGDTVNMFVVHPTTPAQYFHLLRRQMVRNFRKPLIVASPKMLLRLPAAVSTLQEMAPGTTFNPVIGDSSVDPKKVKTLVFCSGKHFYSLVKQRESLGAKKHDFAIIRVEELCPFPLDSLQQEMSKYKHVKDHIWSQEEPQNMGPWSFVSPRFEKQLACKLRLVGRPPLPVPAVGIGTVHLHQHEDILAKTFA
(underlined sequence contains vector encoded His-tag and TEV protease cleavage site*)
hDLST68-453 for co-expression in E. coli
SGC ID: DLSTA-c019
Vector: pNIC-CT10HStII
Accession: P36957, NM_001933.4
Cell line: E. coli Rosetta
Tags and additions: C-terminal His10 tag and Tandem Strep TagII tag, preceded by a TEV protease cleavage site.
Construct Protein Sequence:
MDDLVTVKTPAFAESVTEGDVRWEKAVGDTVAEDEVVCEIETDKTSVQVPSPANGVIEALLVPDGGKVEGGTPLFTLRKTGAAPAKAKPAEAPAAAAPKAEPTAAAVPPPAAPIPTQMPPVPSPSQPPSGKPVSAVKPTVAPPLAEPGAGKGLRSEHREKMNRMRQRIAQRLKEAQNTCAMLTTFNEIDMSNIQEMRARHKEAFLKKHNLKLGFMSAFVKASAFALQEQPVVNAVIDDTTKEVVYRDYIDISVAVATPRGLVVPVIRNVEAMNFADIERTITELGEKARKNELAIEDMDGGTFTISNGGVFGSLFGTPIINPPQSAILGMHGIFDRPVAIGGKVEVRPMMYVALTYDHRLIDGREAVTFLRKIKAAVEDPRVLLLDL
hDHTKD145-919 for co-infection in Sf9
SGC ID: DHTKD1A-c012
Vector: pFB-Bio5
Accession: Q96HY7, NM_018706.6
Cell line: Sf9
Tags and additions: His6 tag in 22-aa N-terminal fusion peptide, with TEV protease cleavage site and C-terminal biotin attachment site.
Construct Protein Sequence:
MGHHHHHHSSGVDLGTENLYFQ*SMGALERPPVDHGLARLVTVYCEHGHKAAKINPLFTGQALLENVPEIQALVQTLQGPFHTAGLLNMGKEEASLEEVLVYLNQIYCGQISIETSQLQSQDEKDWFAKRFEELQKETFTTEERKHLSKLMLESQEFDHFLATKFSTVKRYGGEGAESMMGFFHELLKMSAYSGITDVIIGMPHRGRLNLLTGLLQFPPELMFRKMRGLSEFPENFSATGDVLSHLTSSVDLYFGAHHPLHVTMLPNPSHLEAVNPVAVGKTRGRQQSRQDGDYSPDNSAQPGDRVICLQVHGDASFCGQGIVPETFTLSNLPHFRIGGSVHLIVNNQLGYTTPAERGRSSLYCSDIGKLVGCAIIHVNGDSPEEVVRATRLAFEYQRQFRKDVIIDLLCYRQWGHNELDEPFYTNPIMYKIIRARKSIPDTYAEHLIAGGLMTQEEVSEIKSSYYAKLNDHLNNMAHYRPPALNLQAHWQGLAQPEAQITTWSTGVPLDLLRFVGMKSVEVPRELQMHSHLLKTHVQSRMEKMMDGIKLDWATAEALALGSLLAQGFNVRLSGQDVGRGTFSQRHAIVVCQETDDTYIPLNHMDPNQKGFLEVSNSPLSEEAVLGFEYGMSIESPKLLPLWEAQFGDFFNGAQIIFDTFISGGEAKWLLQSGIVILLPHGYDGAGPDHSSCRIERFLQMCDSAEEGVDGDTVNMFVVHPTTPAQYFHLLRRQMVRNFRKPLIVASPKMLLRLPAAVSTLQEMAPGTTFNPVIGDSSVDPKKVKTLVFCSGKHFYSLVKQRESLGAKKHDFAIIRVEELCPFPLDSLQQEMSKYKHVKDHIWSQEEPQNMGPWSFVSPRFEKQLACKLRLVGRPPLPVPAVGIGTVHLHQHEDILAKTFASSKGGYGLNDIFEAQKIEWHE
(underlined sequence contains vector encoded His-tag and TEV protease cleavage site*)
hDLST68-453 for co-infection in Sf9
SGC ID: DLSTA-c017
Vector: pFB-LIC-Bse
Accession: P36957, NM_001933.4
Cell line: Sf9
Tags and additions: His6 tag in 22-aa N-terminal fusion peptide, with TEV protease cleavage site.
Construct Protein Sequence:
MGHHHHHHSSGVDLGTENLYFQ*SMDDLVTVKTPAFAESVTEGDVRWEKAVGDTVAEDEVVCEIETDKTSVQVPSPANGVIEALLVPDGGKVEGGTPLFTLRKTGAAPAKAKPAEAPAAAAPKAEPTAAAVPPPAAPIPTQMPPVPSPSQPPSGKPVSAVKPTVAPPLAEPGAGKGLRSEHREKMNRMRQRIAQRLKEAQNTCAMLTTFNEIDMSNIQEMRARHKEAFLKKHNLKLGFMSAFVKASAFALQEQPVVNAVIDDTTKEVVYRDYIDISVAVATPRGLVVPVIRNVEAMNFADIERTITELGEKARKNELAIEDMDGGTFTISNGGVFGSLFGTPIINPPQSAILGMHGIFDRPVAIGGKVEVRPMMYVALTYDHRLIDGREAVTFLRKIKAAVEDPRVLLLDL
(underlined sequence contains vector encoded His-tag and TEV protease cleavage site*)
Site-directed mutations were constructed using the QuikChange mutagenesis kit (Stratagene) and confirmed by sequencing. All primers are available upon request. Wild-type and variant hDHTKD1 proteins were expressed in E. coli BL21(DE3)R3-Rosetta cells from 1-6 L of Terrific Broth culture. Cultures were grown at 37°C until an OD of 1.0, when they were cooled to 18°C and induced with 0.1 mM IPTG overnight. Cultures were harvested at 4000 x g for 30 minutes. Cell pellets were lysed by sonication at 35% amplitude, 5 seconds on 10 seconds off, and centrifuged at 35,000 x g. The clarified cell extract was incubated with Ni-NTA resin pre-equilibrated with lysis buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 20 mM imidazole, 5% glycerol, 0.5 mM TCEP). The column was washed with 80 ml Binding Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 20 mM imidazole, 0.5 mM TCEP), 80 ml Wash Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 40 mM imidazole, 0.5 mM TCEP) and eluted with 15 ml of Elution Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 250 mM Imidazole, 0.5 mM TCEP). The eluant fractions were concentrated to 5 ml and applied to a Superdex 200 16/60 column pre-equilibrated in GF Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 0.5 mM TCEP, 5% glycerol). Eluted protein fractions were concentrated to 10-15 mg/ml.
Co-expression of human DHTKD1 and DLST
The hDHTKD1-hDLST complex used in this study was co-expressed in E. coli and insect Sf9 cells. For E. coli co-expression, hDHTKD145-919 was subcloned into the pCDF-LIC vector (incorporating His-tag) and co-transformed with the plasmid encoding untagged hDLST68-453 in the pNIC-CT10HStII vector. Co-transformed cultures were grown and protein purification was performed as described above for hDHTKD1 alone. For co-expression in insect cells, two baculoviruses were produced by transformation of DH10Bac cells, one expressing His-tagged hDHTKD145-919 in pFB-Bio5 vector and one expressing His-tagged hDLST68-453 in pFB-LIC-Bse vector. Viruses were amplified by infecting Sf9 insect cells in 250 ml of sf900II serum free, protein-free insect cell medium (Thermo Fisher Scientific) and grown for 65 h at 27°C. Large-scale Sf9 culture was then co-infected in a 1:1 ratio with the two viruses each at 1.5 ml/L. One baculovirus pFB-Bio5 vector expresses His-tagged hDHTKD145-919 and the other baculovirus pFB-LIC-Bse vector expresses His-tagged hDLST68-453. The cultures were grown at 27°C for 72 hr in 3L flasks before harvesting at 900 x g for 30 minutes. The purification of Sf9 expressed proteins was performed mostly as above, differing only in the addition of 1:1000 benzonase to the lysis buffer and a gentler sonication cycle of 4 seconds on, 12 seconds off.
Structural biology and biophysical assays
Crystallisation and structure determination of DHTKD1
Crystals were grown by vapour diffusion method. To crystalise hDHTKD145-919, concentrated protein was incubated for 30 minutes on ice with 3 mM MgCl2 and 3 mM ThDP before being centrifuged for 10 minutes at 13500xg to remove any precipitation. Sitting drops containing 75 nL protein (10 mg/mL) and 75 nL well solution containing 20% (w/v) PEG 3350, 0.1 M bis-tris-propane pH 8.5, 0.2 M sodium formate and 10% (v/v) ethylene glycol were equilibrated at 4°C. Crystals were mounted and frozen without additional cryo-protectant, as the crystallisation condition contains 10% (v/v) ethylene glycol. Diffraction data were collected at the Diamond Light Source beamline i04, and processed using the CCP4 program suite (14). hDHTKD145-919 crystallised in the primitive space group P1 with two molecules in the asymmetric unit. The structure was solved by molecular replacement using the program PHASER (15) and the E. coli OGDH structure (PDB code 2JGD) as search model. The structure was refined using PHENIX (16), followed by iterative cycles of model building in COOT (17).
Small angle X-ray scattering
SAXS experiments were performed at 0.99 Å wavelength Diamond Light Source at beamline B21 coupled to the appropriate size exclusion column (Harwell, UK) and equipped with Pilatus 2M two-dimensional detector at 4.014 m distance from the sample, 0.005 < q < 0.4 Å-1 (q = 4π sin θ/λ, 2θ is the scattering angle). hDHTKD145-919 at 20 mg/ml in 10 mM Hepes-NaOH pH 7.5, 200 mM NaCl, 0.5 mM TCEP and 2% glycerol was applied onto the Shodex KW404-4F column.
SAXS measurements were performed at 20 ̊C, using an exposure time of 3 seconds per frame. SAXS data were processed and analyzed using the ATSAS program package (20) and Scatter (http://www.bioisis.net/scatter). The radius of gyration Rg and forward scattering I(0) were calculated by Guinier approximation. The maximum particle dimension Dmax and P(r) function were evaluated using the program GNOM (21).
Solution analysis
Analytical gel filtration was performed on a Superdex 200 Increase 10/300 GL column or Superose 6 Increase 10/300 GL (GE Healthcare) pre-equilibrated with 20 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM TCEP.
Differential scanning fluorimetry (DSF)
DSF was performed in a 96-well plate using an Mx3005p RT-PCR machine (Stratagene) with excitation and emission filters of 492 and 610 nm, respectively. Each well (20 µl) consisted of protein (2 mg/ml in 100 mM Hepes pH 7.5, 150 mM NaCl, 5% glycerol), SYPRO-Orange (Invitrogen, diluted 1000-fold of the manufacturer’s stock). Fluorescence intensities were measured from 25 to 96 °C with a ramp rate of 1 °C/min. Tm was determined by plotting the intensity as a function of temperature and fitting the curve to a Boltzmann equation. Temperature shifts, Δ Tm , were determined as described (22) and final graphs were generated using GraphPad Prism (v.7; Graph-Pad Software). Assays were carried out in technical triplicate
MIDAS protein-metabolite screening
Protein-metabolite interaction screening using an updated MIDAS platform was performed similar to (11). Briefly, a flow injection analysis-mass spectrometry (FIA-MS) validated library of 412 metabolite standards were combined into four defined screening pools in 150 mM ammonium acetate pH 7.4. For each metabolite pool, 5 µl of target protein was arrayed in triplicate across a SWISSCI 10 MWC 96-well microdialysis plate (protein chambers). To the trans side of each dialysis well, 300 µl of a 50 µM metabolite pool supplemented with 1 mM ThDP and 1 mM MgCl2 were arrayed in triplicate per hDHTKD145-919 protein (metabolite chambers). Dialysis plates were placed in the dark at 4°C on a rotating shaker (120 rpm) and incubated for 40 hours. Post-dialysis, protein and metabolite chamber dialysates were retrieved, normalised and diluted 1:10 in 80% methanol, incubated 30 min on ice, and centrifuged at 3200 rcf for 15 min to remove precipitated protein. Analytes were aliquoted across a 384-well microvolume plate and placed at 4°C in a Shimadzu SIL-20ACXR autosampler for FIA-MS analysis. 2 µl of each sample was analysed in technical triplicate by FIA-MS on a SCIEX X500R QTOF MS with interspersed injections of blanks.
MIDAS data analysis
FIA-MS spectra collected from MIDAS protein-metabolite screening was qualitatively and quantitatively processed in SCIEX OS 1.5 software to determine relative metabolite abundance by integrating the mean area under the curve (AUC) across technical triplicates. Log2(fold change) for each metabolite was calculated from the relative metabolite abundance in the protein chamber (numerator) and metabolite chamber (denominator) from dialysis triplicates. For each technical triplicate, up to one outlier was removed using a z-score cut-off of five (< 0.1% of observations). The corrected technical replicates were collapsed to one mean fold-change summary per protein-metabolite pair. To remove fold-change variation that was not specific to a given metabolite-protein pair, the first three principal components of the cumulative screening dataset were removed (~75% of observed variance) creating Log2(corrected fold change). Protein-metabolite z-scores were determined by comparing the target protein-metabolite Log2(corrected fold change) to a no-signal model for that metabolite using measures of the central tendency (median) and standard deviation (sd extrapolated from the 25%-75% quantiles) which are not biased by the signals in the tails of a metabolite’s fold-change distribution. Z-scores were false-discovery rate controlled using Storey’s q-value (http://github.com/jdstorey/qvalue). Protein-metabolite interactions with p-values < 0.05 and q-values < 0.1 were considered significant.
Grid preparation and EM data collection
3 µl of 0.4 mg/ml purified hDHTKD145-919-hDLST68-453 sample from Sf9 cells expression were applied to the glow-discharged Quantifoil Au R1.2/1.3 grid (Structure Probe). Blotting and vitrification in liquid ethane was carried out using a Vitrobot Mark IV (FEI Company) at 4 °C, 95% humidity with a nine second wait and a three second blot at zero blotting force from both sides. Cryo grids were loaded into a Glacios transmission electron microscope (ThermoFisher Scientific) operating at 200 keV with a Falcon3 camera. Movies were recorded in linear mode with a pixel size of 0.96 Å, a defocus range of -1 to -3.1 μm (steps of 0.3 μm), a total dose of 32.52 e/A^2 with 1s exposure over 19 frames.
EM data processing
A total of 619 dose-fractioned movies were corrected for drift using Relion’s MotionCor2 (23) with the dose-weighting option. Contrast transfer function (CTF) parameters were determined by ctffind4.1 (24). A subset of 1,244 particles were manually picked and extracted with a box size of 344 pixels rescaled to 172 pixels. 8 classes were selected from one round of 2D classification for reference based auto picking using Relion 3.0 (25). 165,739 particles were extracted with a box size of 344 pixels rescaled to 172 pixels. All downstream particle classification, refinement and post-processing steps were performed in Relion 3.0. The resulting 3,356 particles were refined to a global resolution of 4.7 Å based on the FSC 0.143 threshold criterion.
EM Model building and refinement
Model building and refinement was carried out using the suite of programmes in CCP-EM (26). To fit a template to the final map the E. coli DLST orthologue structure (PDB 1SCZ) was used. The sequence was humanised and residues truncated to the alpha carbon using Chainsaw (27). The oligomeric structure was docked into the density map, sharpened with a B-factor of -281 Å2, using Molrep. One round of refinement using refmac5 was carried out with ProSMART restraints generated from the E. coli DLST orthologue to avoid overfitting. Overfitting was monitored through simultaneous refinement against the two half maps from the final 3D refinement. FSC between map and model was calculated using model validation in CCP-EM.
Crystallography-based fragment screening
To grow crystals for fragment screening campaign, hDHTKD145-919 at 10mg/mL was incubated for 30 minutes on ice with 3mM MgCl2 and 3mM ThDP before being centrifuged for 10 minutes at 13500xg to remove any precipitation. Crystals were grown by vapour diffusion in 400 nL sitting drops in the presence of seeds, at 20°C equilibrated against well solutions of 0.1M MgCl2, 0.1M Hepes pH 7.0, 20% PEG 6K, 10% ethylene glycol.
For soaking, 50 nL of each fragment compound from the XChem fragment library (final concentration of 50 mM) was added to a crystallisation 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 XChem Explorer pipeline (28) was used for structure solution with parallel molecular replacement using DIMPLE (29), followed by map averaging and statistical modelling to identify weak electron densities generated from low occupancy fragments using PANDDA software (30). Coordinates and structure factors for exemplary data sets with bound fragments are deposited in the RCSB Protein Data Bank.
Assays
DHTKD1 enzyme assay
The enzymatic activity assay was performed in triplicates, in a buffer containing 35 mM potassium phosphate (KH2PO4), 0.5 mM EDTA, 0.5 mM MgSO4, 2 mM 2OA or 2OG, 1 mM ThDP, 5 mM sodium azide (NaN3) and 60 µM 2,6-dichlorphenol indophenol (DCPIP), pH 7.4. The activity was determined as a reduction of DCPIP at λ= 610-750 nm, 30°C (18) with and without 2OA or 2OG. The dye DCPIP changes colour from blue to colourless, when being reduced (19). To obtain Km and Vmax different concentrations of 2OA (0.1 mM, 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1 mM, 2 mM) and no substrate were measured in a 96-well micro titre plate (total well volume 300 µl). The ensuing OD values were plotted on a graph (slope= 1/Vmax; Y-intercept= Km/Vmax) to calculate Km and Vmax using the Hanes Woolf plot:
V0= initial velocity; [S]= substrate concentration; Vmax= maximum velocity.
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