Protein Expression and Purification
Recombinant baculoviruses encoding an N-terminal His6-tagged C-terminal fragment of human Bub1 (Bub1C) containing residues 724–1085 were constructed using the Bac-to-Bac system (Invitrogen) according to manufacturer’s protocols. Sf9 cells were infected with the Bub1C baculovirus and harvested about 50 hrs post-infection. Cells were resuspended in lysis buffer (25 mM phosphate pH 7.4, 150 mM KCl, 20 mM imidazole, 0.1% (v/v) Triton X-100, 1 mM PMSF, 5 mM DTT, and a protease inhibitor cocktail). After sonication and centrifugation, the supernatant was filtered through 0.45 µm filters and passed through a 5-ml HisTrap Chelating HP column (GE Healthcare). After washing with the wash buffer (25 mM phosphate pH 7.4, 150 mM KCl, 20 mM imidazole, 1 mM PMSF, and 5 mM DTT), the bound proteins were eluted with a linear gradient (0–70%) of the elution buffer (25 mM phosphate pH 7.4, 300 mM KCl, 300 mM imidazole, 1 mM PMSF, 5 mM DTT). The His6-Bub1C-containing fractions were pooled, desalted and purified using ion exchange chromatography (resource S 5/5 and resource Q 5/5, GE Healthcare). His6-Bub1C was incubated with tobacco etch virus (TEV) protease and passed through a HisTrap Chelating HP column (GE Healthcare). Bub1C was further purified by a gel filtration column (Superdex 200, GE Healthcare), concentrated to 15 mg/ml in the purification buffer (50 mM Tris pH 7.7, 100 mM KCl, 5 mM MgCl2, 5 mM DTT, and 1 mM PMSF), flash frozen in liquid nitrogen, and stored at −80°C for crystallization or kinase assays.
The seleno-methionine variant of Bub1C protein was expressed in Sf9 cells as described (Zhou et al., 2004
). Briefly, Sf9 cells cultured in the EX-420 medium (JRH Bioscience) were infected with the Bub1C baculovirus. The cells were harvested at 20 hrs post-infection, washed twice with the EX-420 medium without L-methionine, and resuspended into this medium. The cells were incubated for 6 hrs to deplete the remaining L-methionine, harvested again, and resuspended into the EX-420 medium supplied with 100 mg/l seleno-methionine. After an additional 24–30 hrs, the cells were harvested and the seleno-methionine Bub1C protein was purified as described above.
Crystallization, Data Collection, and Structure Determination
The Bub1C protein was thawed in ice-cold water, and ATP was added to a final concentration of 10 mM. Crystals were grown at 20° C by the vapor diffusion method in sitting drop mode by mixing 1.5 µl protein with 1.5 µl crystallization solution (20% (w/v) polyethylene glycol 3350, 0.1 M sodium formate (pH 6.29), and 25 mM DTT) and equilibrating against 200 µl of reservoir solution. Large single crystals were obtained by repeated seeding. The crystals were cryo-protected in reservoir solution supplemented with 18% (v/v) glycerol and then flash-cooled in liquid propane. The crystals exhibit the symmetry of space group C2221 with cell dimensions of a = 109 Å, b = 148 Å, c = 47 Å, and contained one molecule of Bub1C in the asymmetric unit with a solvent content of 46%. The seleno-methionine-derivatized Bub1C crystals were obtained in the same way and had similar cell parameters.
Diffraction data were collected at beamline 19-ID (SBC-CAT) of the Advanced Photon Source (Argonne National Laboratory, Argonne, Illinois, USA) and processed with HKL2000 (Otwinowski and Minor, 1997
). Native and seleno-methionine-derivatized crystals diffracted to a minimum Bragg spacing of about 2.3 Å and about 3.5 Å, respectively (Table S1
). All crystals showed significant anisotropic diffraction. Single-wavelength anomalous diffraction data on the seleno-methionine Bub1C crystals were collected at 3.2 Å with weak anomalous signal present to about 3.5 Å.
Phases for the seleno-methionine variant were obtained from a single anomalous dispersion (SAD) experiment. Using data to 3.5 Å, twelve of the 15 expected seleno-methionine sites were identified using SHELX (Schneider and Sheldrick, 2002
; Sheldrick, 2002
) and refined using SHARP (Bricogne et al., 2003
). The resulting electron density map clearly showed several α-helices, which allowed placing the similar structures of the kinase domains of c-Jun, CK2, and twitchin (PDB codes: 1jnk, 1daw, and 1kob, respectively). Using these structures, the phases from the selenium-SAD experiment, and the known positions of twelve selenium atoms as guides, a model was constructed manually in an iterative fashion using the program XtalView and automatically rebuilt and refined with the programs ARP/wARP and Refmac5 of the CCP4 package (Consortium, 1994
; Murshudov et al., 1997
; Perrakis et al., 1999
). Completion of the model and final refinement were then carried out using the native data to 2.32 Å. The final model contains residues 735–806, 814–931, 939–1080 of Bub1C, one molecule Mg2+
-ATP, two chloride ions and 20 water molecules. After final refinement, the model had an Rwork
of 23.2% and an Rfree
of 30.2%. The model has good geometry, except for three residues that are outliers in a Ramachandran plot as defined by MolProbity (Davis et al., 2007
). One of them, D946, is a direct ligand of the bound ATP. Its electron density is well defined. The other two residues, P776 and T778, are located in a surface loop with weak electron density.
Cell Culture and Transfection
HeLa tet-on cells were cultured in DMEM (Invitrogen) supplemented with fetal bovine serum. The Bub1 ΔKEN1 (with K535, E536, and N537 mutated to alanines), Bub1 ΔKEN2 (with K625, E626, and N627 mutated to alanines), and Bub1 ΔKEN (with both KEN motifs mutated to alanines) mutants were constructed using the QuikChange mutagenesis kit (Qiagen). Plasmid transfections were performed using the Effectene reagent (Qiagen). To establish stable cell lines, HeLa tet-on cells were transfected with the pIRES-mCherry-Bub1 WT or ΔKEN plasmids and incubated with 0.5 µg/ml puromycin. Surviving clones that expressed mCherry-Bub1 WT or ΔKEN at similar levels were transfected with Bub1 siRNA for 24 hr and treated with 100 nM Taxol for 18 hr. After a 30-min incubation with Hoechst 33342, the cells were analyzed using an inverted fluorescence microscope. Round cells with condensed chromosomes were counted as mitotic cells.
To determine the IC50 values of 2OH-BNPP1 for Bub1, Bub1C, Aurora B and p38, about 0.1 µg of various kinases were incubated with 2 µg of substrates for 30 min at RT in 25 µl of kinase buffer I (50 mM Tris, pH 7.5, 0.2 M NaCl, 1 mM DTT, 0.1 mM ATP, 0.1 µCi/µl γ-32
P-ATP) containing various concentrations of 2OH-BNPP1. Reaction mixtures were quenched with SDS sample buffer, separated on SDS-PAGE, and analyzed using a phosphoimager. The Immunoprecipitation (IP) kinase assays were performed as described previously (Kang et al., 2007
For Bub1 localization, HeLa tet-on cells stably expressing mCherry-Bub1 WT or ΔKEN were grown in 6-well plates, treated with Taxol for 18 hr, and sedimented on slides using Shandon cytospin 4 (Thermo electron). The cells were fixed in 4% (w/v) paraformaldehyde for 10 min and incubated with the appropriate primary antibodies (1 µg/ml) in IF buffer I (PBS with 0.1% (v/v) Triton X-100) containing 3% (w/v) BSA for 2 hr. The cells were further washed with IF buffer I, incubated with the appropriate FITC-, Cy3-, or Cy5-conjugated secondary antibodies (4 µg/ml, Molecular Probes) in IF buffer I containing 3% (w/v) BSA for 1 hr, washed again with IF buffer I containing 1 µg/ml DAPI, and analyzed using a 63X objective on a Zeiss Axiovert 200M inverted fluorescence microscope. The images were acquired with the Intelligent Imaging software and processed with Photoshop.
For Mad1 and BubR1 localization, HeLa tet-on cells were grown in 4-well chamber slides, permeabilized with IF buffer II (60 mM PIPES, pH 7.0, 20 mM HEPES, pH 7.5, 5 mM EGTA, pH 8.0, 2 mM MgCl2, and 4 M glycerol) containing 0.2% (v/v) Triton, fixed in IF buffer II containing 4% (w/v) paraformaldehyde and 0.05% (v/v) glutaraldehyde for 10 min, and incubated with the appropriate primary antibodies (1 µg/ml) in IF buffer I containing 3% (w/v) BSA for 2 hr. The cells were then processed and analyzed as described above.