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Preparation of the Human LIMK1 LIM-LIM domains
Boundaries: residues 18-172
Vector: pNIC-Zb
Tag and additions: TEV-cleavable N-terminal hexahistidine and Z-basic tags
Expression cell: E. coli BL21(DE3)R3-pRARE2
Expression was performed in LB media containing 50 µg/mL kanamycin and 34 µg/mL chloramphenicol. Cultures were grown at 37°C with shaking until OD600 = 0.5 and then induced overnight at 18°C with 0.5 mM IPTG. Cells harvested and lysed by ultrasonication. Recombinant LIMK1 protein was purified on
Nickel-sepharose buffered in 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP) and eluted with an imidazole step gradient. Further purification was achieved using size-exclusion and anion exchange chromatography. Affinity tags were cleaved from LIMK1 using TEV protease.
Preparation of Human BMPR2 kinase domain
Boundaries: residues 189-517
Vector: pNIC-CH
Tag and additions: non-cleavable C-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
The kinase domain of human BMPR2 (residues 189-517) was subcloned into the pNIC-CH vector and transformed into E. coli strain BL21(DE3)R3-pRARE2 for expression. This vector provides a non-cleavable
C-terminal hexahistidine tag. Cultures in LB media, 50 µg/mL kanamycin and 34 µg/mL chloramphenicol, were induced with 1 mM IPTG overnight at 18°C and the cells harvested and lysed by ultrasonication. Recombinant proteins were purified by Ni-affinity, size-exclusion and anion exchange chromatography. Proteins were stored at 4°C buffered in 50 mM HEPES pH 7.4, 300 mM NaCl, 10% glycerol, 10 mM DTT, 50 mM L-arginine, 50 mM L-glutamate.
Preparation of the Human LIMK1 kinase domain
Boundaries: residues 330-637
Vector: pFB-LIC-Bse
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: Sf9 insect cells
Bacmid DNA was prepared from DH10Bac cells and used to transfect Sf9 insect cells for the preparation of initial baculovirus. LIMK1 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 7.4, 500 mM NaCl, 20 mM imidazole, 0.5 mM TCEP, 5% glycerol) and lysed by sonication. The lysate was cleared by centrifugation and purified by Ni-affinity chromatography. LIMK1 was eluted in buffer supplemented to 300 mM imidazole. The eluted protein was cleaved overnight by TEV protease and purified further by size exclusion chromatography using an S200 16/600 column buffered in 20 mM HEPES pH 7.4, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP. Additional clean-up was performed by reverse Ni-affinity chromatography and if necessary a cation exchange chromatography step using a HiTrap SP column. The final yield was 2 mg LIMK1 protein from 1L culture. It was observed that the inclusion of protease inhibitors during purification was helpful to maintain the intact LIMK1 protein.
Preparation of the Human LIMK1-CFL1 complex
For co-crystallisation the purified LIMK1 and CFL1 (Ser3Cys) proteins were mixed at a 1:1 molar ratio and incubated for 2 hours on ice. The complex was then purified by size exclusion chromatography using an AKTAxpress system with an S200 16/600 column buffered in 20 mM HEPES pH 7.4, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP.
Preparation of the Human LIMK2 kinase domain
Boundaries: residues 330-632
Vector: pFB-LIC-Bse
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: Sf9
Bacmid DNA was prepared from DH10Bac cells and using to transfect Sf9 insect cells for the preparation of initial baculovirus. LIMK2 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 7.4, 500 mM NaCl, 20 mM imidazole, 0.5 mM TCEP, 5% glycerol) and lysed by sonication. The lysate was clarified by centrifugation and purified by Ni-affinity chromatography. LIMK2 was eluted in buffer supplemented to 300 mM imidazole. The eluted protein was cleaved overnight by TEV protease whilst being dialysed to remove imidazole. The cleaved protein was purified further using a reverse Ni-affinity purification step and then by size exclusion chromatography using an S200 16/600 column buffered in 20 mM HEPES pH 7.4, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP. The final yield was 0.4 mg LIMK2 protein from 1L culture.
Preparation of full length Human CFL1 cofilin protein (wild-type and Ser3Cys mutant)
Boundaries: Full-length
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
A similar construct containing a Ser3Cys mutation was produced by site directed mutagenesis. Plasmids were transformed into E. coli strain BL21(DE3)R3-pRARE2. Expression was performed in LB media containing 50 µg/mL kanamycin and 34 µg/mL chloramphenicol. Cultures were grown at 37°C with shaking until OD600=0.5 and then induced overnight at 18°C with 0.5 mM IPTG. Cells were spun at 5000 rpm for 10 mins and the pellets frozen at -80°C. On thawing, cells were lysed by sonication and purified on Ni-sepharose resin in binding buffer (50 mM HEPES pH 7.4, 500 mM NaCl, 20 mM Imidazole, 5% glycerol, 0.5 mM TCEP). After washing with buffer supplemented to 50 mM imidazole, the CFL1 protein was eluted with buffer supplemented to 300 mM imidazole. The N-terminal affinity tag was removed by TEV cleavage overnight and the CFL1 protein purified further by size exclusion chromatography using an S200 16/600 column buffered in 20 mM HEPES pH 7.4, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP. Fractions containing protein were pooled, concentrated and stored at
-80°C.
Structure Determination of the BMPR2 kinase domain (PDB: 3G2F)
Crystallization of the BMPR2-ADP complex was achieved at 4°C using the sitting-drop vapour diffusion method. BMPR2 was pre-incubated with 10 mM ADP at a protein concentration of 5.8 mg/mL, and crystallized using a precipitant containing 25% PEG 8K, 0.3 M ammonium sulphate, 0.05 M MgCl2, and 0.1 M sodium cacodylate pH 6.0. The diffraction quality co-crystals grew in a 150 nL crystallization drop containing equal volume of the protein and reservoir solution. Crystals were cryoprotected with mother liquor plus 20% ethylene glycol for the BMPR2-ADP complex and vitrified in liquid nitrogen. Diffraction data were collected at Swiss Light Source, station PX10 using monochromatic radiation at wavelength 1.000 Å. Data were processed with MOSFLM and subsequently scaled using the program SCALA from the CCP4 suite. Initial phases were obtained by molecular replacement using the program PHASER and the structure of ACVR2B (PDB 2QLU) as a search model. Density modification and NCS averaging were performed using the program DM, and the improved phases were used in automated model building with the programs ARP/wARP and Buccaneer. The resulting structure solution was refined using REFMAC5 from the CCP4 suite and manually rebuilt with COOT. Appropriate TLS restrained refinement using the tls tensor files calculated from the program TLSMD was applied at the final round of refinement. The complete structure was verified for geometric correctness with MolProbity.
Structure Determination of the LIMK1 complex with staurosporine (PDB: 3S95)
Crystallization of LIMK1 was achieved by vapour diffusion using the sitting drop method at 20°C. The protein was pre-incubated with 1.5 mM staurosporine and concentrated to 8 mg/mL. Crystals were obtained mixing protein and reservoir at 3:1 volume ratio in 24% MPD, 100 mM Tris pH 7.2 and 10 mM phenol and were cryoprotected in 30% MPD, 10% glycerol. A diffraction dataset was collected at Diamond Light Source synchrotron, beamline I03, and indexed and integrated with MOSFLM. Scaling and merging were performed using the program SCALA. The structure was solved by molecular replacement using PHASER with the initial search models SRC (PDB 1YI6) and EPHA3 (PDB ID: 2QO9). Further manual model building used COOT, alternated with refinement using REFMAC5 in the CCP4 suite. Optimal TLS refinement was performed in the last refinement stages using parameters generated by the TLSMD server. The final model was validated with MOLPROBITY.
Structure Determination of the LIMK1 complex with PF-477736 (PDB: 5NXC)
10 mg/mL LIMK1 protein was mixed with 0.5 mM PF-477736. Crystals were grown at 4°C in sitting drops mixing 100 nL of the protein-ligand complex with 50 nL of a precipitant solution containing 0.1 M HEPES pH 7.0,
0.2 M MgCl2, 10% PEG8K. Crystals appeared overnight and did not change appearance after 7 days. They were mounted in precipitant solution cryoprotected with 25% ethylene glycol. Data were collected at Diamond Light Source, analysed, scaled and merged with Xia2. The structure was solved by molecular replacement with PHASER using a SRC model as a template (PDB ID 1YI6) and refined with REFMAC. The model was validated using MOLPROBITY.
Structure Determination of the LIMK1-CFL1 complex with ATPγS (PDB: 5L6W)
10 mg/mL LIMK1-CFL1 protein complex was mixed with 1.2 mM ATPγS and 2.5 mg/mL MgCl2. Crystals were grown at 4°C in sitting drops mixing 50 nL protein with 100 nL of a precipitant solution containing 0.1 M HEPES pH 7.5, 0.2 M KCl, 35% pentaerythritol propoxylate 5/4. Crystals were cryo-protected by equilibration into precipitant solution containing 20% ethylene glycol and vitrified in liquid nitrogen. Diffraction data were collected at Diamond, beamline I02, analysed, scaled and merged with Xia2. The structure was solved by molecular replacement with PHASER using a SRC model as a template (PDB ID: 1YI6) and refined with REFMAC5. The model was validated using MOLPROBITY.
Structure Determination of the LIMK2 complex with TH300 (PDB: 5NXD)
7.5 mg/mL LIMK2 protein was mixed with 0.5 mM TH300. Crystals were grown at 4°C in sitting drops mixing 75 nL of the protein-ligand complex with 75 nL of a precipitant solution containing 0.1 M BisTrisPropane
pH 7.5, 0.2 M Na2SO4, 10% ethylene glycol, 17% PEG3.35K. Crystals appeared after 4 days and did not change appearance after 6 days. They were mounted in precipitant solution cryoprotected with an additional 20% ethylene glycol. Diffraction data were collected at Diamond Light Source, analysed, scaled and merged with Xia2. The structure was solved by molecular replacement with PHASER using a LIMK2 model as a template (PDB ID: 4TPT) and refined with REFMAC5. The model was validated using MOLPROBITY.
Thermal Shift Assay
A fluorescence-based thermal shift assay (differential scanning fluorimetry (DSF)) was performed as a screen to identify potential LIMK1 inhibitors. Ligands in this assay increase a protein’s melting temperature (Tm shift) by an amount proportional to their binding affinity. A solution of 2 mM LIMK1 protein in assay buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 5% glycerol) was mixed 1:1000 with SYPRO Orange (Sigma). Compounds to be tested were added to a final concentration of 10 µM. 20 µL of each sample were placed in a 96-well plate and heated from 25 to 95°C. Fluorescence was monitored using a Mx3005P real-time PCR instrument (Stratagene) 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 (DTm) induced by inhibitor binding were calculated relative to control wells containing protein and 2.5% DMSO.
RapidFire Mass Spectrometry Kinase Assay
An assay for inhibitor IC50 determination was developed using an Agilent 6530 Rapidfire QTOF Mass Spectrometer to follow LIMK1/2 kinase activity against the CFL1 substrate. RF-MS assays were performed in a 384-well plate format using polypropylene plates (Greiner, code 781280) and an assay buffer containing 50 mM TRIS pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA. All bulk liquid handling steps were performed using a multidrop combi reagent dispenser (Thermo Scientific, Code 5840300) equipped with a small tube plastic tip dispensing cassette (Thermo Scientific, Code 24073290). After each reagent transfer, assay plates were sealed with an aluminium foil plate seal (Costar, Code 6570) and centrifuged briefly for 5 sec at 1000 rpm. For inhibitor IC50 determinations an 11-point and 3-fold serial dilution in DMSO was prepared from a 50 mM stock solution in DMSO and 250 nL of each concentration was transferred in duplicate using an ECHO 550 acoustic dispenser (Labcyte). A DMSO control (250 nL) was transferred into column 12 and the potent LIMK inhibitor, LIMKi3, was dispensed into column 24 as a no enzyme control. LIMK1 (80 nM, 2 X final concentration in assay buffer) and LIMK2 (400 nM, 2 X final concentration in assay buffer) was dispensed into each well of the assay plate (25 ml per well) and allowed to pre-incubate with inhibitor for 10 minutes at room temperature. After 10 minutes
25 ml of 2 X final concentration of substrate (1.6 mM ATP, 10 mM MgCl2, 4 mM CFL1 in assay buffer) was dispensed into each well to initiate the reaction and the enzyme reaction was allowed to proceed for 1 hour (LIMK1) and 4 hour (LIMK2) at room temperature. The enzyme reaction was stopped by addition of 5 ml of 10% formic acid and the plate transferred to a RapidFire RF360 high throughput sampling robot. Samples were aspirated under vacuum and loaded onto a C4 solid phase extraction (SPE) cartridge and the SPE washed for 5.5 sec with 0.1% (v/v) formic acid in LCMS grade water to remove non-volatile buffer components. After the aqueous wash, analytes of interest were eluted from the C4 SPE onto an Agilent 6530 accurate mass Q-TOF in an organic elution step (85% acetonitrile in LCMS grade water containing 0.1% formic acid). Ion data for the CFL1 substrate and phosphorylated CFL1 product were extracted and peak area data integrated using RapidFire integrator software (Agilent). % conversion of CFL1 to phosphorylated CFL1 was calculated in excel and IC50 curves generated using graphpad prism version 7.0. The assay had a Z score of 0.69.
Isothermal Titration Calorimetry (ITC) using BMPR2 peptide
Experiments were performed at 15°C using a Microcal VP-ITC microcalorimeter. Proteins were buffered in
50 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM TCEP. 1 mM BMPR2 peptide (SNLKQVETGVAKMNTINAA) was titrated into a 100 µM solution of the LIMK1 LIM-LIM domains (a.a. 18-172). Data were analyzed using a single binding site model implemented in the Origin software package provided with the instrument.
Isothermal Titration Calorimetry (ITC) using TH251 inhibitor
Measurements were performed at 15°C on a MicroCal iTC200 (GE Healthcare). LIMK1 kinase domain was dialysed overnight into assay buffer (20 mM HEPES pH 7.4, 500 mM NaCl, 0.5 mM TCEP, 5% glycerol). The syringe was loaded with 150 µM LIMK1, the cell was filled with 15 µM TH-251. Every 2.5 minutes, 2 μL of the protein solution were injected into the cell for a total of 19 injections. The heat flow data were analysed with the MicroCal ORIGIN software package employing a single binding site model.
Intact mass spectrometry
Protein masses of all purified proteins 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).
Drosophila models of Fragile X syndrome
A Drosophila model for fragile X syndrome was developed by the group of Akiko Hata. Hyperactive locomotion was demonstrated as a reliable behavioral marker for an in vivo drug screen. Full experimental details have been published (Sci Signal. 2017; 10, 477: eaai8133).
- Kashima, R., Roy, S., Ascano, M., Martinez-Cerdeno, V., Ariza-Torres, J., Kim, S., Louie, J., Lu, Y., Leyton, P., Bloch, K. D., Kornberg, T. B., Hagerman, P. J., Hagerman, R., Lagna, G., and Hata, A. (2016) Augmented noncanonical BMP type II receptor signaling mediates the synaptic abnormality of fragile X syndrome. Science signaling 9, ra58
- Sivadasan, R., Hornburg, D., Drepper, C., Frank, N., Jablonka, S., Hansel, A., Lojewski, X., Sterneckert, J., Hermann, A., Shaw, P. J., Ince, P. G., Mann, M., Meissner, F., and Sendtner, M. (2016) C9ORF72 interaction with cofilin modulates actin dynamics in motor neurons. Nature Neuroscience 19, 1610
- Kashima, R., Redmond, P. L., Ghatpande, P., Roy, S., Kornberg, T. B., Hanke, T., Knapp, S., Lagna, G., and Hata, A. (2017) Hyperactive locomotion in a Drosophila model is a functional readout for the synaptic abnormalities underlying fragile X syndrome. Science signaling 10
- Goodwin, N. C., Cianchetta, G., Burgoon, H. A., Healy, J., Mabon, R., Strobel, E. D., Allen, J., Wang, S., Hamman, B. D., and Rawlins, D. B. (2015) Discovery of a Type III Inhibitor of LIM Kinase 2 That Binds in a DFG-Out Conformation. ACS medicinal chemistry letters 6, 53-57
- Hamill, S., Lou, H. J., Turk, B. E., and Boggon, T. J. (2016) Structural Basis for Noncanonical Substrate Recognition of Cofilin/ADF Proteins by LIM Kinases. Molecular cell 62, 397-408
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