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Horizontal Tabs
PDB ID |
Structure Details | Resolution (Å) |
Structure of KEAP1 BTB-3-box in complex with CUL3NTD |
3.45 | |
Structure of apo-Kelch domain of KEAP1 |
2.16 | |
Structure of apo-Kelch domain of KLHL2 |
1.99 | |
Structure of the KLHL2 Kelch domain with WNK4 peptide |
1.56 | |
Structure of the KLHL3 Kelch domain with WNK4 peptide |
1.84 | |
Structure of the KLHL3 Kelch domain with WNK3 peptide |
2.80 | |
Structure of the apo-Kelch domain of KLHL7 |
1.63 | |
Structure of the BTB-BACK domains of KLHL11 |
2.60 | |
Structure of the KLHL11 BTB-BACK domains with CUL3NTD |
2.80 | |
Structure of the KLHL11 BTB-BACK with CUL3NTDΔN22 |
3.10 | |
Structure of the apo-Kelch domain of KLHL12 |
1.85 | |
Structure of the apo-Kelch domain of KLHL17 |
2.60 | |
Structure of the KLHL20 Kelch domain with DAPK1 peptide | 1.09 | |
Structure of the apo-Kelch domain of KLHL40 (KBTBD5) | 1.78 |
Mass Spectrometry
Protein masses were determined using an Agilent LC/MSD TOF system with reversed-phase high-performance liquid chromatography coupled to electrospray ionization and an orthogonal time-of-flight mass analyser. Proteins were desalted prior to mass spectrometry by rapid elution off a C3 column with a gradient of 5-95% isopropanol in water with 0.1% formic acid. Spectra were analysed using the MassHunter software (Agilent).
Protein Expression and Purification
Human KEAP1 Kelch domain
Boundaries: residues 321-624
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
Expressed protein sequence:
MHHHHHHSSGVDLGTENLYFQSMAPKVGRLIYTAGGYFRQSLSYLEAYNPSDGTWLRLADLQVPRSGLAGCVVGGLLYAVGGRNNSPDGNTDSSALDCYNPMTNQWSPCAPMSVPRNRIGVGVIDGHIYAVGGSHGCIHHNSVERYEPERDEWHLVAPMLTRRIGVGVAVLNRLLYAVGGFDGTNRLNSAECYYPERNEWRMITAMNTIRSGAGVCVLHNCIYAAGGYDGQDQLNSVERYDVETETWTFVAPMKHRRSALGITVHQGRIYVLGGYDGHTFLDSVECYDPDTDTWSEVTRMTSGRSGVGVAVTMEPCRKQIDQQNCTC
The Kelch domain of human KEAP1 was expressed in E. coli (BL21(DE3)-R3-pRARE2) cells with a TEV-cleavable 6xHis tag. Cell cultures were grown in TB medium at 37 °C until the OD600 reached 0.8. Protein expression was induced with 1 mM IPTG for 16 h at 18 °C. Cells were spun at 5000 rpm for 10 min, then resuspended in binding buffer (20mM TRIS pH 7.5, 300 mM NaCl, 5 mM Imidazole) and frozen at -80°C. After thawing, the cells were lysed using an Emulsiflex C5 homogeniser by sonication. After centrifugation at 4 °C, the supernatant was applied to a His GraviTrap column (GE healthcare) equilibrated with binding buffer. After washing with binding buffer, KEAP1 was eluted with binding buffer supplemented to 500 mM imidazole. The eluted protein was applied to a PD-10 desalting column (GE Healthcare) and eluted with binding buffer. The N-terminal affinity tag was removed by TEV cleavage overnight and uncleaved protein was removed by applying it again to a His GraviTrap column. The flow-through was concentrated and purified further by size exclusion chromatography using a HiLoad 26/600 Superdex 75 pg (GE Healthcare Life Sciences) column equilibrated with gel filtration buffer (20mM TRIS pH 7.5, 300 mM NaCl, 1 mM TCEP). Fractions containing protein were pooled, concentrated to 12 mg/mL and stored at -80°C.
Human KEAP1 BTB-3-box and CUL3NTD complex
Boundaries: KEAP1 residues 48-213 and CUL3 residues 1-388
Vector: pNIC28-Bsa4 (KEAP1) and pNIC-CTHF (CUL3NTD)
Tag and additions: TEV-cleavable N-terminal hexahistidine tag (KEAP1); TEV-cleavable C-terminal hexahistidine and Flag tags (CUL3NTD)
Expression cell: E. coli BL21(DE3)R3-pRARE2
Expressed protein sequence (KEAP1):
MHHHHHHSSGVDLGTENLYFQSMGNRTFSYTLEDHTKQAFGIMNELRLSQQLCDVTLQVKYQDAPAAQFMAHKVVLASSSPVFKAMFTNGLREQGMEVVSIEGIHPKVMERLIEFAYTASISMGEKCVLHVMNGAVMYQIDSVVRACSDFLVQQLDPSNAIGIANFAEQIGCVELHQRAREYIYMHFGE
Expressed protein sequence (CUL3NTD):
MSNLSKGTGSRKDTKMRIRAFPMTMDEKYVNSIWDLLKNAIQEIQRKNNSGLSFEELYRNAYTMVLHKHGEKLYTGLREVVTEHLINKVREDVLNSLNNNFLQTLNQAWNDHQTAMVMIRDILMYMDRVYVQQNNVENVYNLGLIIFRDQVVRYGCIRDHLRQTLLDMIARERKGEVVDRGAIRNACQMLMILGLEGRSVYEEDFEAPFLEMSAEFFQMESQKFLAENSASVYIKKVEARINEEIERVMHCLDKSTEEPIVKVVERELISKHMKTIVEMENSGLVHMLKNGKTEDLGCMYKLFSRVPNGLKTMCECMSSYLREQGKALVSEEGEGKNPVDYRQGLDDLKSRFDRFLLESFNNDRLFKQTIAGDFEYFLNLNSRSPEYLAENLYFQSHHHHHHDYKDDDDK
Human KEAP1 (Uniprot Q14145, residues 48-213 (BTB-3-box)) was subcloned into the vector pNIC28-Bsa4, and human CUL3NTD (Uniprot Q13618, residues 1-388, or where desired residues 23-388 (CUL3NTDΔN22), was subcloned into the vector pNIC-CTHF using ligation-independent cloning. Two point mutations (I342R and L346D) were introduced into the CUL3 sequence to stabilize the isolated N-terminal domain (NTD). Proteins were expressed separately overnight at 18°C in BL21(DE3)R3-pRARE2 cells in the presence of kanamycin and chloramphenicol, with 0.25 mM IPTG for induction. The harvested cells (2-3 L) were resuspended in 40 mL Binding Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 % glycerol, 5 mM imidazole and 0.5 mM TCEP) per 2 L cell culture. Lysozyme (1 mg/mL), PEI (1 mL of 5% stock) and protease inhibitors were added and the cells sonicated. After centrifugation of the lysate at 50 000 g the supernatant was filtered (1.2 µm) and incubated with 3 mL Ni2+ sepharose resin equilibrated in Binding Buffer, for 30 min. The column was washed with 50-80 mL Wash Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 % glycerol, 30 mM imidazole and 0.5 mM TCEP) and the proteins eluted in 10 mL fractions with each of Elution buffers 1-4 (Binding Buffer with increasing concentrations of imidazole: 50, 100, 150 and 250 mM). The fractions were pooled, diluted one third with binding buffer and tobacco etch virus (TEV) protease added for overnight incubation to remove the hexahistidine tags. The samples were concentrated, KEAP1 and CUL3 mixed together in a 1:1 molar ratio and incubated for 2 hours on ice, before size exclusion purification on a 16/60 Superdex S200 column at 4°C in Gel Filtration (GF) Buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP). Peak fractions were pooled, concentrated with 10 mM DTT and used in crystal trials.
Human KEAP1 BTB-BACK Kelch domains
Boundaries: residues 48-624
Vector: pFB-LIC-Bse
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: Sf9
Expressed protein sequence:
MGHHHHHHSSGVDLGTENLYFQSMGNRTFSYTLEDHTKQAFGIMNELRLSQQLCDVTLQVKYQDAPAAQFMAHKVVLASSSPVFKAMFTNGLREQGMEVVSIEGIHPKVMERLIEFAYTASISMGEKCVLHVMNGAVMYQIDSVVRACSDFLVQQLDPSNAIGIANFAEQIGCVELHQRAREYIYMHFGEVAKQEEFFNLSHCQLVTLISRDDLNVRCESEVFHACINWVKYDCEQRRFYVQALLRAVRCHSLTPNFLQMQLQKCEILQSDSRCKDYLVKIFEELTLHKPTQVMPCRAPKVGRLIYTAGGYFRQSLSYLEAYNPSDGTWLRLADLQVPRSGLAGCVVGGLLYAVGGRNNSPDGNTDSSALDCYNPMTNQWSPCAPMSVPRNRIGVGVIDGHIYAVGGSHGCIHHNSVERYEPERDEWHLVAPMLTRRIGVGVAVLNRLLYAVGGFDGTNRLNSAECYYPERNEWRMITAMNTIRSGAGVCVLHNCIYAAGGYDGQDQLNSVERYDVETETWTFVAPMKHRRSALGITVHQGRIYVLGGYDGHTFLDSVECYDPDTDTWSEVTRMTSGRSGVGVAVTMEPCRKQIDQQNCTC
KEAP1 BTB-BACK Kelch domains were cloned into the baculoviral transfer vector pFB-LIC-Bse. Bacmid was prepared from this vector in DH10Bac cells. Baculovirus was then prepared from this using Sf9 cells. Large scale baculoviral expression was performed for 72 hours at 27°C. The harvested cells were resuspended in 40 mL binding buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 % glycerol, 5 mM imidazole and 0.5 mM TCEP) per 2L cell culture. PEI (1 mL) and protease inhibitors were added and the cells sonicated. After centrifugation of the lysate at 50 000 g the supernatant was filtered and incubated with 3 ml Ni2+ resin for 30 min. The column was washed with 50-80 ml wash buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 % glycerol, 30 mM imidazole and 0.5 mM TCEP) and the protein eluted in 10 mL fractions of each of Elution buffers 1-4 (Binding buffer with increasing concentrations of imidazole – 50, 100, 150 and 250 mM). The fractions were pooled, diluted one third with binding buffer and TEV added for overnight incubation. The samples were concentrated, KEAP1 and Cul3 added together in a 1:1 molar ratio and incubated for 2 hours, before size exclusion purification on a 16/60 Superdex S200 column. Peak fractions were pooled, concentrated and stored at 4°C or -80°C.
Biotinylated KEAP1 BTB-3-box domains (used for assays)
Boundaries: residues 48-213
Vector: pNIC-Bio3
Tag and additions: TEV-cleavable N-terminal hexahistidine tag plus C-terminal avi tag for biotinylation
Expression cell: E. coli BL21(DE3)R3-pRARE2
Expressed protein sequence:
MHHHHHHSSGVDLGTENLYFQSMGNRTFSYTLEDHTKQAFGIMNELRLSQQLCDVTLQVKYQDAPAAQFMAHKVVLASSSPVFKAMFTNGLREQGMEVVSIEGIHPKVMERLIEFAYTASISMGEKCVLHVMNGAVMYQIDSVVRACSDFLVQQLDPSNAIGIANFAEQIGCVELHQRAREYIYMHFGESSKGGYGLNDIFEAQKIEWHE
Human KEAP1 BTB-3-box domains were cloned into the pNIC-Bio3 vector containing a His6 tag in the 22-aa N-terminal fusion peptide, with TEV protease cleavage site, and an avi tag sequence for biotinylation in the C-terminal fusion. The protein was co-expressed in E.coli containing a BirA vector for in cell biotinylation. Cells were grown at 37°C, 180 rpm for 2-3 hours in 2xYT medium containing 100 μM D-biotin until OD600 = 0.6 and were then induced with 0.4 mM IPTG and incubated with shaking at 18°C overnight. Biotin was prepared at 2.4 mg/mL (10 mM) in 10 mL of 10 mM Bicine, pH 8.3 and filter sterilised. A further 10 mL of this solution was added to the cell cultures the next morning for one hour before harvesting at 7000 g for 15 minutes at 4 °C. Protein was purified sequentially using Ni-affinity and size exclusion chromatography as for the KEAP1 BTB-3-box above.
Kelch selectivity panel proteins (used for crystallography and assays)
All Kelch domains were expressed from either a pNIC28-Bsa4 vector overnight at 18 °C in E. coli BL21(DE3)R3-pRARE2 cells (KEAP1, KLHL2, KLHL3, KLHL7, KLHL11, KLHL12, KLHL17, KLHL20, KBTBD5, KBTBD10, KLHDC4), with 0.4 mM IPTG induction, or utilising a pFB-LIC-Bse baculoviral transfer vector system in Sf9 baculoviral cultures (KLHL6, KLHL21, KBTBD7, KBTBD8, KLHDC5, KLHDC9) at 27°C for 72 hours in glass shaker flasks. Construct residue ranges are listed in Fig. 9B. All constructs were His-tagged and therefore purified using Nickel sepharose chromatography followed by TEV cleavage of the tag and gel filtration. Briefly, the harvested cells (2-3 L) were resuspended in 40 mL Binding Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 % glycerol, 5 mM imidazole and 0.5 mM TCEP) per 2 L cell culture. PEI (0.125%) and protease inhibitors were added and the cells sonicated. After centrifugation of the lysate at 50 000 g the supernatant was filtered (1.2 µm) and incubated rotating at 4 °C with 3 mL Ni2+ resin equilibrated in Binding Buffer, for 1 hour. The column was washed with 50-80 mL Wash Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 % glycerol, 30 mM imidazole and 0.5 mM TCEP) and the protein eluted in 5-10 mL fractions with each of Elution buffers 1-4 (Binding Buffer with increasing concentrations of imidazole: 50, 100, 150 and 250 mM). The fractions were pooled, diluted one third with binding buffer and tobacco etch virus (TEV) protease added for overnight incubation to remove the hexahistidine tag. The samples were concentrated in centrifugal concentrators (Sartorius Vivaspin 10 000 MWCO) before size exclusion purification on a 16/60 Superdex S200 column at 4°C in Gel Filtration (GF) Buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP). Peak fractions were pooled and concentrated, subjected to a nickel rebind step if necessary, then snap frozen and stored at -80 °C.
Structure Determination
Human KEAP1 Kelch domain (PDB: 6ROG, 2.16 Å)
KEAP1 Kelch domain was crystallized using the vapour-diffusion technique in 150 nL sitting drops containing 50 nL protein (12 mg/mL) and 100 nL of a reservoir solution containing 4M sodium formate at 4 °C. Crystals were directly flash frozen in liquid nitrogen without addition of a cryo-protectant. Diffraction data were collected at 100K on Diamond Light Source beamline I04-1. Data were indexed and integrated using XDS (18) and scaled using AIMLESS (19) as part of the XIA2 auto-processing pipeline (20). The structure was solved by molecular replacement with PHASER (21) and PDB ID 1U6D as a search model. Refinement was carried out with REFMAC5 (22) and manual rebuilding was performed with COOT (23). The refined structure was validated with MolProbity (24).
Human KEAP1 BTB-3-box and CUL3NTD complex (PDB: 5NLB, 3.45 Å)
Crystallisation was achieved at 20°C using the sitting drop vapour diffusion method. The protein complex crystallised in 150 nL drops at 9.4 mg/mL at a 1:2 ratio of protein to precipitant (20% PEG 3350, 10 % ethylene glycol, 0.2 M potassium citrate tribasic), using 20 nL of seeds previously prepared in a similar condition. Crystals were cryoprotected in 20 % ethylene glycol in well precipitant and then vitrified in liquid nitrogen. Diffraction data were collected at the Diamond Light Source, station I03 using monochromatic radiation at wavelength 0.97626 Å. Automated diffraction data reduction was performed using xia2 3d (20), and the indexed, integrated, scaled and merged data was phased using Phaser-MR in Phenix (25) with a structure of KLHL11 BTB-BACK complexed to CUL3 as the search model (PDB 4AP2). The molecular replacement (MR) structure solution was refined using Phenix (25) and Buster (26) with manual rebuilding with Coot (23). Molprobity (24) was used to verify the geometrical correctness of the structure.
Assays
Biolayer interferometry
Biolayer interferometry (BLI) was used to determine the affinity of binding between KEAP1 BTB-3-box and CUL3NTD. Biotinylated KEAP1 (residues G48-E213) buffered in 50 mM HEPES, 300 mM NaCl, 0.5 mM TCEP, 10 mM DTT, was used at 0.16 mg/mL to immobilise 7-8 nm response units of protein to streptavidin-coated Octet sensors (ForteBio). Serial dilutions of CUL3 NTD (from 0.1563 to 5 μM) in the same buffer plus 0.01 % Tween were placed in the relevant wells, with buffer in the reference wells. Association and dissociation were set for 120 s each. Data were analysed using ForteBio Data Analysis 9.0 with local, partial fitting and double referencing.
Differential scanning fluorimetry
Differential scanning fluorimetry (DSF) was performed in triplicate using an Mx3005p real time PCR machine with 2 μM protein buffered in 10 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM TCEP, and 1:1000 dilution of Sypro orange. Compounds to be tested were added to a final concentration of 12.5 μM. 20 μL of each sample were placed in a 96-well plate and heated from 25 to 96°C. Fluorescence was monitored with excitation and emission filters set to 465 and 590 nm, respectively. Data were analysed with the MxPro software and curves fit in Microsoft Excel using the Boltzmann equation to determine the midpoint of thermal denaturation (Tm). Thermal shift values (ΔTm) induced by inhibitor binding were calculated relative to control wells containing protein and 2.5% DMSO.
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