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Horizontal Tabs
PDB ID |
Structure Details |
Supplier |
hHAO1-FMN, bound with glyoxylate, disordered loop 4 |
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hHAO1-FMN, bound with glycolate, ordered loop 4 |
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hHAO1-FMN, bound with CCPST inhibitor |
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hHAO1-FMN, bound with fragment 2 (crystal x208) |
Crea-Chim UAB |
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hHAO1-FMN, bound with fragment 5 (crystal x368) |
Diamond Light Source XChem facility |
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hHAO1-FMN, bound with fragment 6 (crystal x438) |
Diamond Light Source XChem facility |
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hHAO1-FMN, bound with fragment 7 (crystal x518) |
Diamond Light Source XChem facility |
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hHAO1-FMN, bound with fragment 3 (crystal x566) |
Diamond Light Source XChem facility |
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hHAO1-FMN, bound with fragment 4 (crystal x603) |
Diamond Light Source XChem facility |
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hHAO1-FMN, bound with fragment 1 (crystal x604) |
Diamond Light Source XChem facility |
Non-SGC resources
Commercially available CRISPR/Cas9 knockout plasmids |
SCBT: Cat # sc-407493 |
Genscript: Cat # 54363 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. |
Commercially available antibodies |
Thermofisher: MA5-24346 (monoclonal) |
SCBT: sc-517552 (monoclonal) |
Protein expression and purification of hHAO1
Vector: pNIC28-Bsa4
Entry Clone Accession: NM_017545
Cell line: BL21 (DE3)-R3/Rosetta
Tags and additions: N-terminal, TEV protease cleavable hexahistidine tag
Construct protein sequence:
Mhhhhhhssgvdlgtenlyfq*sMLPRLICINDYEQHAKSVLPKSIYDYYRSGANDEETLADNIAAFSRWKLYPRMLRNVAETDLSTSVLGQRVSMPICVGATAMQRMAHVDGELATVRACQSLGTGMMLSSWATSSIEEVAEAGPEALRWLQLYIYKDREVTKKLVRQAEKMGYKAIFVTVDTPYLGNRLDDVRNRFKLPPQLRMKNFETSTLSFSPEENFGDDSGLAAYVAKAIDPSISWEDIKWLRRLTSLPIVAKGILRGDDAREAVKHGLNGILVSNHGARQLDGVPATIDVLPEIVEAVEGKVEVFLDGGVRKGTDVLKALALGAKAVFVGRPIVWGLAFQGEKGVQDVLEILKEEFRLAMALSGCQNVKVIDKTLVRKNPLAVSKI
(Underlined sequence contains vector encoded His-tag and TEV protease cleavage site*)
An overnight culture (20 mL LB) was used to inoculate 2L auto-induction TB containing 50 µg/ml each of kanamycin and chloramphenicol. Cells were cultured at 37°C for 6 hours followed by 48 h incubation at 18°C.
Extraction Buffer: 500 mM NaCl, 50 mM HEPES pH 7.5, 20 mM imidazole, 0.5 mM TCEP, 5% glycerol, 1:1000 of Merck Protease Cocktail II, 0.5 mg/mL lysozyme and 0.2 µg/mL benzonase.
Binding Buffer: 500 mM NaCl, 50 mM HEPES pH 7.5, 20 mM imidazole, 0.5 mM TCEP, 5% glycerol.
Washing Buffer: 500 mM NaCl, 50 mM HEPES pH 7.5, 40 mM imidazole, 0.5 mM TCEP, 5% glycerol.
Elution Buffer: 500 mM NaCl, 50 mM HEPES pH 7.5, 250 mM imidazole, 0.5 mM TCEP, 5% glycerol.
GF buffer: 500 mM NaCl, 550 mM HEPES pH 7.5, 0.5 mM TCEP, 5% glycerol.
Cell pellet from 2 L of culture was re-suspended in 150 mL Extraction Buffer, lysed by sonication for 15 minutes, and centrifuged at 37000 x g for 1 hour at 4°C. The clarified cell extract was incubated with 5 mL of Ni-NTA resin pre-equilibrated with lysis buffer before applying to 1.5 x 10 cm column by gravity flow. The column was washed with 10 column volumes of Binding Buffer, 10 column volumes of Wash Buffer, and eluted with 5 x 5 mL Elution Buffer. Fractions containing hHAO1 were loaded onto gel filtration column (Superdex 200 Hiload 16/60) pre-equilibrated with GF buffer. Fractions containing hHAO1 were pooled.
Activity assay
The catalytic activity of hHAO1 was measured by the Amplex Red assay. We adopted the assay in 96 well format for detection using the PheraStar FSX fluorescence reader (BMG Labtech)(Excitation/Emission = 560/590 nm). Michaelis-Menten kinetics were determinedy by incubating 100 nM protein with substrate (0.5 µM - 10 mM) at room temperature for 10 minutes, adding Amplex Red reagent (0.01U horseradish peroxidase and 50 µM Amplex Red), incubating for a further 10 minutes, and measuring the resultant fluorescence. The reaction buffer consists of 50 mM sodium phosphate, pH 7.4, 200 mM KCl, 2 mM MgCl2 and 0.01% TritonX. This assay gave a linear response with protein concentration up to 200 nM and H2O2 production up to 50 µM. Standard curve ranging from 0 – 50 µM H2O2 was used to calculate reaction rate (µM H2O2 / min). To test fragments for their effect on hHAO1 activity, protein was pre-incubated with fragment compound (0.1 µM - 10 mM) for 10 minutes before addition of substrate (720 µM glycolate) and following procedures above.
Differential scanning fluorimetry
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 20 µL reaction consisted of 2 µL protein (final concentration of 2 µM) and (if applicable) 2 µL ligand at various concentrations in DSF buffer (150 mM KCl, 10 mM HEPES pH 7.5) to which 2 µL SYPROrange, diluted 500-fold in DSF buffer from the manufacturers stock (Invitrogen), was added. Fluorescence intensities were measured at each 1°C temperature increase from 25 to 96°C with a ramp rate of 3°C/min.
Crystallization and structure determination
For the glyoxylate-bound structure, crystals were grown by vapour diffusion at 20°C in a sitting drop consisting of 50 nL of protein (12 mg/ml) and 100 nl well solution, equilibrated against well solution containing 30% PEG 1000, 0.1 M Malate-MES-Tris pH 7.0. For the glycolate-bound structure, crystals were grown by vapour diffusion at 4°C in sitting drops consisting 100 nL protein (13.7 mg/mL) and 50 nL well solution, equilibrated against well solution containing either 20% PEG3350, 0.1 M bis-tris-propane pH 7.5, 10% ethylene glycol, 0.2 M sodium nitrate. Crystals for the CCPST-bound structure were grown from protein pre-incubated with 2 mM CCPST, equilibrated against well solution containing 30% PEG1000, sodium malonate-imidazole-boric acid pH 8.0 (6GMC). Crystal was cryo-protected with 20-25% ethylene glycol before flash-cooling in liquid nitrogen.
The initial structure of hHAO1 (PDB 2NZL) was solved by molecular replacement with the program PHASER (11), using published structure of spinach glycolate oxidase (PDB 1GOX) as search model (54.6% sequence identity). Bound FMN and glyoxylate in the active site were identified by difference Fourier method and manually placed into the electron density using Coot (12). Iterative cycles of refinement with SHELXL (13) followed by manual model building using Coot were performed. All subsequent hHAO1 structures were solved by molecular replacement with PHASER using 2NZL as the search model, followed by iterative cycles of REFMAC5 (14) including TLS refinement, and manual model building in Coot.
Crystallography-based fragment screening
To launch fragment soaking campaign, crystals were grown by vapour diffusion at 4°C in sitting drops of 13.7 mg/mL protein equilibrated against well solution containing 25-35% PEG1000, sodium malonate-imidazole-boric acid pH 8.0. Approximately 50 crystals of suitable size were identified per 96-well crystallisation plate, and a total of 10 plates generated sufficient crystals for the entire fragment campaign.
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 (15) was used for structure solution with parallel molecular replacement using DIMPLE (template 2NZL) (16), followed by map averaging and statistical modelling to identify weak electron densities generated from low occupancy fragments using Pandda software (17). Coordinates and structure factors for all data sets with bound fragments are deposited in the RCSB Protein Data Bank. The crystal structures displaying conformational changes in loop 4 (fragment 1, 5QIB; fragment 2, 5QIH; and fragment 5, 5QIC) were deposited containing coordinates for both the fragment-bound and ground states for the loop.
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