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Horizontal Tabs
PDBID | Structure Details | Resolution (Å) |
6GY5 | structure of KLHL20 Kelch domain with DAPK1 peptide | 1.09 |
Mass Spectrometry
Protein masses were determined using an Agilent LC/MSD TOF system with reversed-phase high-performance liquid chromatography coupled to electrospray ionisation 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 KLHL20 Kelch domain
Boundaries: residues 303-605
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
Expressed protein sequence:
MHHHHHHSSGVDLGTENLYFQSMQGPRTRPRKPIRCGEVLFAVGGWCSGDAISSVERYDPQTNEWRMVASMSKRRCGVGVSVLDDLLYAVGGHDGSSYLNSVERYDPKTNQWSSDVAPTSTCRTSVGVAVLGGFLYAVGGQDGVSCLNIVERYDPKENKWTRVASMSTRRLGVAVAVLGGFLYAVGGSDGTSPLNTVERYNPQENRWHTIAPMGTRRKHLGCAVYQDMIYAVGGRDDTTELSSAERYNPRTNQWSPVVAMTSRRSGVGLAVVNGQLMAVGGFDGTTYLKTIEVFDPDANTWRLYGGMNYRRLGGGVGVIKMTHCE
The Kelch domain of human KLHL20 (Uniprot Q9Y2M5 isoform 1, M303-E605) was cloned using ligation-independent cloning into the bacterial expression vector pNIC28-Bsa4 (GenBank accession number EF198106) which provides for an N-terminal hexahistidine tag and TEV cleavage site. Plasmid DNA was transformed into E. coli strain BL21(DE3)R3-pRARE2. Cells were cultured in LB broth at 37°C until OD600 reached 0.6. Recombinant protein expression was then induced by addition of 0.4 mM isopropyl β-D-1-thiogalactopyranoside followed by 18 hours continuous shaking at 18°C. Cells were harvested by centrifugation and lysed by sonication in binding buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 5 mM imidazole) supplemented with 0.5 mM TCEP. Recombinant proteins were captured on nickel sepharose resin, washed with binding buffer and eluted by a stepwise gradient of 30-250 mM imidazole. Further clean-up was performed by size exclusion chromatography using a HiLoad 16/600 S200 Superdex column buffered in 50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP. Finally, the eluted protein was purified by anion exchange chromatography using a 5 mL HiTrap Q column. Protein masses were confirmed by intact LC-MS mass spectrometry. Where required, the hexahistidine tag was cleaved overnight at 4°C using TEV protease.
Human KLHL3 Kelch domain
Boundaries: residues 298-587
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
Expressed protein sequence:
MHHHHHHSSGVDLGTENLYFQSMSLPKVMIVVGGQAPKAIRSVECYDFEEDRWDQIAELPSRRCRAGVVFMAGHVYAVGGFNGSLRVRTVDVYDGVKDQWTSIASMQERRSTLGAAVLNDLLYAVGGFDGSTGLASVEAYSYKTNEWFFVAPMNTRRSSVGVGVVEGKLYAVGGYDGASRQCLSTVEQYNPATNEWIYVADMSTRRSGAGVGVLSGQLYATGGHDGPLVRKSVEVYDPGTNTWKQVADMNMCRRNAGVCAVNGLLYVVGGDDGSCNLASVEYYNPVTDKWTLLPTNMSTGRSYAGVAVIHKSL
The Kelch domain of human KLHL3 (Uniprot Q9UH77 isoform 1, residues S298–L587) was cloned into the bacterial expression vector pNIC28-Bsa4. Plasmid DNA was transformed into E. coli strain BL21(DE3)R3-pRARE2. Cells were cultured in LB broth at 37°C until OD600 reached 0.6. Recombinant protein expression was then induced by addition of 0.4 mM isopropyl β-D-1-thiogalactopyranoside, followed by 18 hours continuous shaking at 18°C. Cells were harvested by centrifugation and lysed by sonication in binding buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 5 mM imidazole) supplemented with 0.5 mM TCEP. Recombinant proteins were captured on nickel sepharose resin, washed with binding buffer and eluted by a stepwise gradient of 30-250 mM imidazole. Further clean-up was performed by size exclusion chromatography using a HiLoad 16/600 S200 Superdex column buffered in 50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP. Finally, the eluted protein was purified by anion exchange chromatography using a 5 mL HiTrap Q column. Protein masses were confirmed by intact LC-MS mass spectrometry. Where required, the hexahistidine tag was cleaved overnight at 4°C using TEV protease.
Structures
Structure of the KLHL20-DAPK1 complex
The purified KLHL20 Kelch domain was concentrated to 12 mg/mL using a 10 kDa molecular-mass cut-off centrifugal concentrator in 50 mM HEPES pH 7.5, 300 mM NaCl and 5 mM TCEP buffer. The 11-residue DAPK1 peptide (LGLPDLVAKYN) was purchased from LifeTein and added in the same buffer to a final concentration of 3 mM. The protein-peptide mixture was incubated on ice for 1 hour prior to setting up sitting-drop vapour-diffusion crystallisation plates. Micro-seed stocks were prepared from small KLHL20 crystals grown during previous rounds of crystal optimisation. Those early crystals were transferred into an Eppendorf tube containing 50 µL reservoir solution and a seed bead (Hampton Research), then vortexed for 2 min. Seed stocks were diluted 500-fold before use. The best-diffracting crystals of the KLHL20 complex were obtained at 20°C by mixing 75 nL protein, 20 nL diluted seed stock and 75 nL of a reservoir solution containing 2 M sodium chloride and 0.1 M acetate buffer pH 4.5. Prior to vitrification in liquid nitrogen, crystals were cryoprotected by direct addition of reservoir solution supplemented with 25 % ethylene glycol. Diffraction data were collected on beamline I03 at Diamond Light Source, Didcot, UK. Data were processed in PHENIX version 1.9 (18). Molecular replacement was performed with PHENIX.Phaser-MR using KLHL12 (PDB 2VPJ chain A) as the search model. PHENIX.Autobuild was used to build the initial structural model. COOT(19) was used for manual model building and refinement whereas PHENIX.REFINE was used for automated refinement. TLS parameters were included at later stages of refinement. Tools in COOT, PHENIX and MolProbity (20) were used to validate the structure.
Homology model
A homology model for the death domain of human DAPK1 was built in Molsoft ICM-Pro software using MyD88 (PDB 3MOP chain A, 25% sequence identity) as the structural template. The initial model was refined by energy minimisation and side chain optimisation in ICM-Pro (Molsoft) (21).
Assays
Peptide arrays (SPOT assay)
Cellulose-bound peptide arrays were prepared employing standard Fmoc solid phase peptide synthesis using a MultiPep-RSi-Spotter (INTAVIS, Köln, Germany) as previously described (22). After array synthesis, membranes were incubated with 5% BSA to block free sites. The arrays were then incubated with 1 µM recombinant hexahistidine-tagged KLHL20 Kelch domain in PBS at 4°C overnight. Unbound protein was washed off in PBS buffer with 0.1% Tween 20 and bound protein was detected using HRP-conjugate anti-His antibody Merck Millipore (Cat #71840).
Surface Plasmon Resonance
Assays were performed at 25°C using a BIACORE S200 (GE Healthcare) surface plasmon resonance (SPR) instrument. The Kelch domains of KLHL20 and KLHL3 were immobilised on sensor chip CM5 (GE Healthcare) using amine coupling. Reference flow cells had no immobilised protein. Binding was monitored using a flow rate of 30 µL/min. The peptide analytes were prepared in HBS-P buffer (GE Healthcare). Data reported were after reference flow cell signal subtraction. Data were analysed by one-site steady-state affinity analysis using the Biacore S200 Evaluation software and the fitting equation Req = CRmax/(Kn+C) + RI (RI, bulk refractive index contribution; KD, dissociation constant; C, analyte concentration; Rmax, maximum response). Peptides were purchased from Severn Biotech.
Co-immunoprecipitation of KLHL20 and DAPK1
HEK293T cells were cultured in high glucose Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich) with 5% Penicillin Streptomycin (ThermoFisher) and 10% Fetal Bovine Serum (Sigma-Aldrich) inside a 5% CO2 incubator at 37°C. KLHL20 Kelch domain (residues M303-T602) and full length DAPK1 constructs were transfected into HEK293T cells at 60% confluency with polyethylenimine. 40 hours after transfection, cells were harvested and lysed in the presence of protease and phosphatase inhibitors. Flag immunoprecipitation was performed using ANTI-FLAG® M2 Affinity Gel (Sigma-Aldrich). Results were analysed using Western blotting (Flag antibody – Sigma-Aldrich, F1804; HA antibody – Biolegend, 901501; Myc antibody – Cell signalling, 2040S). Full length DAPK1 was cloned into pcDNA3.1(+). KLHL20 Kelch domain was cloned into pcDNA3-N-Flag-LIC.
Cycloheximide chase assay
HEK293T cells were cultured as described above until 60% confluency. Full length KLHL20 and DAPK1 constructs were transfected with polyethylenimine. 24 hours after transfection, 100 µg/mL cycloheximide was added to inhibit protein synthesis. Cells were harvested at different time points – 0, 0.5h, 1h, 2h, 4h and 6h. Results were analysed using Western blotting with corresponding antibodies. GAPDH level in each sample was also detected for control (anti-GAPDH antibody; Thermo Fisher, MA5-15738). Western blot band intensities were quantified using Image Studio Lite Ver 5.2 and normalised for the GADPH control. Full length KLHL20 was cloned into pRK5 (myc tag).
Peptide Displacement Assays
Assays were set up with biotinylated peptide (Biotin-LLAMNLGLPDLVAKYNTSNGA, btn-peptide) and N-terminally hexahistidine tagged protein (6His-protein). For detection, two orthogonal technologies were used; i. AlphaScreen technology from Perkin Elmer and ii. HTRF from Cisbio. To determine optimal assay conditions for each protein prep, 6His-protein and btn-peptide were titrated against each other in a 16 by 16 matrix in 1:1 dilutions, starting from 800 nM. For the final ratio of 6His-protein and btn-peptide to use in the assay, the point representing the EC90 in the two-dimensional titration was chosen. Typically, final assay concentrations for 6His-protein and btn-peptide fell between 20 and 200 nM. For the AlphaScreen assay, AlphaScreen Histidine (Nickel Chelate) Detection Kit donor and acceptor beads were used at a 1:2500 dilution from purchased stock; for HTRF, SA-XL665 and anti-6His antibody were used at 50 nM and 0.125 nM, respectively. Assays were performed on 384-well ProxiPlates (Perkin Elmer) at a final volume of 20 µL and plates were read using a Pherastar FSX plate reader (BMG Labtech). For inhibitor screening, compounds were dispensed in duplicate at single concentration (typically 30-50 µM) for the initial screen and as 11-point dose response curves starting from 200 µM for IC50 value determination.
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