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Horizontal Tabs
PDB ID |
Structure Details |
Resolution (Å) |
Structure of hsALAS2 bound with PLP |
2.30 | |
Structure of hsALAS2 bound with PLP and fragment 1 (x1131): |
1.49 | |
Structure of hsALAS2 bound with PLP and fragment 2 (x1154): |
1.44 | |
Structure of hsALAS2 bound with PLP and fragment 3 (x1277): |
1.73 | |
Structure of hsALAS2 bound with PLP and fragment 4 (x1311): |
1.83 | |
Structure of hsALAS2 bound with PLP and fragment 5 (x1317): |
1.76 | |
Structure of hsALAS2 bound with PLP and fragment 6 (x1084): |
1.56 | |
Structure of hsALAS2 bound with PLP and fragment 7 (x1097): |
1.51 | |
Structure of hsALAS2 bound with PLP and fragment 8 (x1359): |
1.68 | |
Structure of hsALAS2 bound with PLP and fragment 9 (x1082): |
1.55 | |
Structure of hsALAS2 bound with PLP and fragment 10 (x1036): |
1.93 | |
Structure of hsALAS2 bound with PLP and fragment 11 (x1147): |
1.66 | |
Structure of hsALAS2 bound with PLP and fragment 12 (x1235): |
1.63 | |
Structure of hsALAS2 bound with PLP and fragment 13 (x1208): |
1.75 | |
Structure of hsALAS2 bound with PLP and fragment 14 (x1080): |
1.67 | |
Structure of hsALAS2 bound with PLP and fragment 15 (x1070): |
1.85 | |
Structure of hsALAS2 bound with PLP and fragment 16 (x1141): |
1.55 | |
Structure of hsALAS2 bound with PLP and fragment 17 (x1185): |
1.57 | |
Structure of hsALAS2 bound with PLP and fragment 18 (x1196): |
1.49 | |
Structure of hsALAS2 bound with PLP and fragment 19 (x1281): |
1.72 |
Non-SGC Resources
Commercially available CRISPR/Cas9 knockout plasmids | Commercially available antibodies |
---|---|
SCBT: Cat # sc-403752 | Thermofisher: TA810000 (monoclonal) |
Genscript: Cat # 212 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: 166739 (monoclonal) |
Protein expression and purification of hsALAS2ΔN78
Vector: pFB-LIC-Bse
Entry clone accession: BC030230
Cell line: DH10Bac
Tags and additions: N-terminal, TEV protease cleavable hexahistidine tag
Construct protein sequence (underlined sequence contains vector encoded His-tag and TEV protease cleavage site*):
hsALAS2ΔN78 (SGC ID: hALAS2-c016; aa 79-587)
MGHHHHHHSSGVDLGTENLYFQ*SMFSYDQFFRDKIMEKKQDHTYRVFKTVNRWADAYPFAQHFSEASVASKDVSVWCSNDYLGMSRHPQVLQATQETLQRHGVGAGGTRNISGTSKFHVELEQELAELHQKDSALLFSSCFVANDSTLFTLAKILPGCEIYSDAGNHASMIQGIRNSGAAKFVFRHNDPDHLKKLLEKSNPKIPKIVAFETVHSMDGAICPLEELCDVSHQYGALTFVDEVHAVGLYGSRGAGIGERDGIMHKIDIISGTLGKAFGCVGGYIASTRDLVDMVRSYAAGFIFTTSLPPMVLSGALESVRLLKGEEGQALRRAHQRNVKHMRQLLMDRGLPVIPCPSHIIPIRVGNAALNSKLCDLLLSKHGIYVQAINYPTVPRGEELLRLAPSPHHSPQMMEDFVEKLLLAWTAVGLPLQDVSVAACNFCRRPVHFELMSEWERSYFGNMGPQYVTTYA
hALAS2-c017 (aa 79-545)
MGHHHHHHSSGVDLGTENLYFQ*SMFSYDQFFRDKIMEKKQDHTYRVFKTVNRWADAYPFAQHFSEASVASKDVSVWCSNDYLGMSRHPQVLQATQETLQRHGVGAGGTRNISGTSKFHVELEQELAELHQKDSALLFSSCFVANDSTLFTLAKILPGCEIYSDAGNHASMIQGIRNSGAAKFVFRHNDPDHLKKLLEKSNPKIPKIVAFETVHSMDGAICPLEELCDVSHQYGALTFVDEVHAVGLYGSRGAGIGERDGIMHKIDIISGTLGKAFGCVGGYIASTRDLVDMVRSYAAGFIFTTSLPPMVLSGALESVRLLKGEEGQALRRAHQRNVKHMRQLLMDRGLPVIPCPSHIIPIRVGNAALNSKLCDLLLSKHGIYVQAINYPTVPRGEELLRLAPSPHHSPQMMEDFVEKLLLAWTAVGL
Bacmid DNA was prepared from DH10Bac cells and used to transfect Sf9 insect cells for the preparation of initial baculovirus. hsALAS2 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).
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 are detailed hereafter;
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 10 mg/mL using a 30 kDa mwco concentrator.
Activity assay
Reaction mixtures consisted of 50 mM potassium phosphate buffer, pH 7.4, 50 mM PLP, 1 mM DTT, and 10 mM MgCl2, various concentrations of glycine and succinyl-CoA (Sigma), and 1-4 µg/mL fresh purified ALAS2 enzyme (175 µL total). After incubation at 37°C for 15 minutes (previously checked for linear ALA formation with each enzyme concentration), reactions were terminated with 100 µL trichloroacetic acid and centrifuged at 13,000xg for 5 min to remove protein. Supernatants (240 µL) were added to freshly prepared mixtures of 240 µL of 1 M sodium acetate, pH 4.7, and 20 µL acetylacetone (500 µL total), and boiled for 10 min to derivatize the ALA product. Samples were cooled and three 150 µL aliquots per reaction (three technical replicates) were further derivatized with 150 µL modified Ehrlich’s reagent and monitored at 554 nm every 60 s in a CLARIOstar microplate reader (BMG Labtech). Absorbance values collected after 5 minutes were converted to molar quantities of ALA using an extinction coefficient of 60.4 mM-1 cm-1. For kinetic studies, apparent Vmax, Km, and kcat values were determined by titrating 2.5-50 mM glycine in the presence of 100 µM succinyl-CoA and 5-100 µM succinyl-CoA in the presence of 50 mM glycine. Michaelis-Menten nonlinear regression analysis was subsequently carried out on data for two separate protein preparations (two biological replicates) with Prism software (GraphPad 8.0).
Crystallisation
To crystalise hsALAS2ΔN142, sitting drops containing 75 nL protein (17 mg/mL) and 75 nL well solution containing 25% (w/v) PEG 3350, 0.1 M bis-tris pH 6.7 and 0.3 M MgCl2 were equilibrated at 20 oC by vapour diffusion. Crystals were cryo-protected using 25% (v/v) ethylene glycol and flash-cooled in liquid nitrogen. Diffraction data were collected at the DLS beamlines i04 and i24.
Structure Determination
hsALAS2ΔN142 crystallised in the monoclinic space group C2 with two molecules (chains A & B) in the asymmetric unit. The data was processed using the Xia2 autoprocessing dials pipeline. Structure was solved by molecular replacement using the program PHASER (19) and the rcALAS structure (PDB code 2BWN) (17) as search model. The final model was produced by iterative cycles of restrained refinement and model building using COOT (20), REFMAC5 (21) and Phenix.refine (22). The final model consists of Phe143-Met578 of chains A and B. In both chains the last 9 residues aa 579-587 and a loop between Ser182 and Ser188 are not visible in the electron density map indicating that this region is largely unstructured.
Crystallography-based fragment screening
To grow crystals for the fragment screening campaign, 10 mg/mL of ALAS2 was pre-incubated with 5 mM hydroxylamine for 30 minutes on ice to convert PLP into the homogenous, non-covalent form. Crystals were grown by vapour diffusion in 400 nL sitting drops in the presence of seeds, at 20°C equilibrated against well solutions of 0.1 M bis-tris pH 7.0, 0.3 M magnesium chloride and 23% PEG3350.
For soaking, 50 nL of each fragment compound from the XChem fragment library (final concentration of 125 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 (23) was used for structure solution with parallel molecular replacement using DIMPLE (24), followed by map averaging and statistical modelling to identify weak electron densities generated from low occupancy fragments using PANDDA software (25). Coordinates and structure factors for exemplary data sets with bound fragments are deposited in the RCSB Protein Data Bank.
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