Cloning
All cloning was performed as described previously (
Klenchin et al., 2011). In brief, a modified QuikChange mutagenesis protocol was used to introduce all point mutants, and QuikChange cloning was used to amplify and insert the Kar3 and Vik1 genes. All constructs generated were sequence verified over the entire open reading frame insert.
Constructs
All Kar3 constructs were amplified from
S. cerevisiae genomic DNA and introduced into a modified pET24d (EMD) plasmid by QuikChange cloning. Vik1 constructs were also amplified from
S. cerevisiae genomic DNA and introduced into a Strep II tag version of pKLD37, a modified pET31b plasmid (EMD), by QuikChange cloning (
Rocco et al., 2008). Construct designs are listed in .
Preparation of the streptavidin column
A mutant streptavidin optimized to bind the Strep II tag was purified as described previously (
Schmidt and Skerra, 1994;
Voss and Skerra, 1997). In brief, native streptavidin was expressed as inclusion bodies in an
Escherichia coli BL21-CodonPlus (DE3)-RIL cell line (Agilent Technologies). Inclusion bodies were solubilized in 6 M guanidine × HCl, pH 1.5, and protein was refolded by rapid dilution into 75 mM Hepes, pH 7.5, with 100 mM NaCl. Folded streptavidin was then concentrated by precipitation in an 80% saturated ammonium sulfate solution at 4°C, resuspended in Milli-Q water, and dialyzed against Milli-Q water to remove remaining ammonium sulfate. Protein was lyophilized and stored at −20°C.
Streptavidin was coupled to Sepharose CL-4B using periodate activation (
Sanderson and Wilson, 1971;
Wilson and Nakane, 1976). Sepharose CL-4B (Pharmacia) was activated by adding two column volumes of 20 mg/ml NaIO
4 and allowed to react with gentle mixing for 2 h at room temperature. The activated Sepharose was washed with 10 column volumes of Milli-Q water and equilibrated in carbonate buffer (100 mM Na
2CO
3, pH 10.5). Purified streptavidin was added to a final concentration of 5 mg streptavidin per milliliter of activated Sepharose and allowed to couple for 16 h at room temperature with gentle mixing. The protein-coupled Sepharose was then washed with two column volumes of carbonate buffer. One column volume of freshly prepared 3 mg/ml NaBH
4 in carbonate buffer was added and allowed to react with gentle mixing at room temperature for 5 min. The column was then washed with 10 column volumes of carbonate buffer and equilibrated in a suitable buffer for protein purification. Protocol courtesy of V.V. Sinitsyn.
Protein expression and purification
Kar3 and Vik1 plasmids were coexpressed in an E. coli BL21-CodonPlus (DE3)-RIL cell line (Stratagene). Cells were grown in lysogeny broth (LB) medium to an A600 of ~0.9, then cooled on ice for 15 min; at this point, 0.5 mM IPTG was added and the cells were grown for 16 h at 16°C with shaking before harvesting by centrifugation. The Kar3Vik1 heterodimers were purified as follows; all procedures were performed at 4°C. 20 g of cell paste in 200 ml of lysis buffer (20 mM Tris, pH 8.0, 300 mM NaCl, 30 mM imidazole, 2 mM MgCl2, 0.1 mM EGTA, 0.5 mM TCEP, and 0.2 mM ATP) with 1 mM PMSF, 50 nM Leupeptin (Peptide International), 70 nM E-65 (Peptide International), 2 nM Aprotinin (ProSpec), and 2 µM AEBSF (Gold BioTechnology) were lysed by sonication and centrifuged at 125,000 g for 30 min at 4°C. The supernatant was loaded onto a 7 ml Ni-NTA column (QIAGEN) at a rate of 1 ml/min and washed with 15 column volumes of lysis buffer. Protein was eluted in four column volumes of elution buffer (lysis buffer with 200 mM imidazole). The elution from the Ni-NTA column was directly loaded onto a 50-ml streptavidin agarose column at a rate of 2 ml/min, washed with three column volumes of wash buffer (20 mM Tris buffer, 300 mM NaCl, 2 mM MgCl2, 0.1 mM EGTA, 0.5 mM TCEP, and 0.2 mM ATP), and eluted in three column volumes wash buffer with 2.5 mM Desthiobiotin.
The octa-Histidine and Strep II tag were removed by incubation with a 1:40 molar ratio of rTEV protease to Kar3Vik1 for 16 h at 4°C for proteins used in crystallization and EM studies (
Blommel and Fox, 2007). The cleaved protein was then loaded onto a 2-ml Ni-NTA column equilibrated in rTEV buffer (20 mM Tris, pH 8.0, 300 mM NaCl, 2 mM MgCl
2, 0.1 mM EGTA, 0.5 mM TCEP, and 0.2 mM ATP); Kar3Vik1 was eluted in three column volumes of rTEV buffer with 30 mM imidazole, and rTEV was eluted with rTEV buffer with 200 mM imidazole. All protein constructs were concentrated in a Centriprep YM-50 (Millipore) to 10–15 mg/ml. Proteins for crystallization and EM were dialyzed into 20 mM Tris, pH 8.0, 100 mM NaCl, 2 mM MgCl
2, 1 mM EGTA, 0.2 mM ATP, and 0.5 mM TCEP, then frozen as 30-µl drops into liquid nitrogen and stored at −80°C.
Protein cross-linking
All procedures were performed at 4°C. Concentrated protein was exchanged into cross-linking buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and 0.2 mM ATP degassed and then sparged with Argon) using a PD-10 column (Pharmacia). A 10-fold molar excess of M4M, EBI (Toronto Research Company), or DPDPB (Thermo Fisher Scientific) was immediately added and allowed to react with stirring for 30 min (or 16 h in the dark for EBI). After cross-linking, protein was concentrated to 10–15 mg/ml and dialyzed into 20 mM Tris, pH 8.0, 100 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and 0.2 mM ATP.
Crystallization of cross-linked SHD-Kar3Vik1
Crystals were grown by small-scale batch crystallization, where 4 µl of concentrated protein was mixed with 5 µl PEG solution (16% monomethyl polyethylene glycol 2000, 100 mM Hepes, pH 7.5, and 50 mM sodium citrate;
Rayment, 2002). Drops were immediately streak-seeded, then allowed to grow at 4°C for 3 wk. Crystals grew to maximal dimensions of 1 mm × 100 µm × 100 µm. For freezing, crystals were transferred to a synthetic mother liquor (12% monomethyl polyethylene glycol 5000, 75 mM Hepes, pH 7.5, 37.5 mM sodium citrate, 5 mM Tris, pH 8.0, 25 mM NaCl, 0.5 mM MgCl
2, 0.25 mM EGTA, and 80 µM ATP) and then transferred stepwise to a solution of 18% polyethylene glycol 8000, 16% glycerol, 100 mM Hepes, pH 7.5, 0.2 mM ATP, 2 mM MgCl
2, and 250 mM sodium citrate. Crystals were flash-frozen in liquid nitrogen.
X-ray data collection and structural refinement
X-ray diffraction data for the cross-linked SHD-Kar3Vik1 crystals were collected at the SBC 19-ID beam line (Advanced Photon Source). The datasets were integrated and scaled with the program HKL2000 (
Otwinowski and Minor, 1997). X-ray data collection statistics are given in . The structure of Kar3Vik1 was solved by molecular replacement using the program Phaser (
Collaborative Computational Project 4, 1994;
McCoy et al., 2007) and the structure with PDB accession nos.
3KAR and
2O0A as the search models (
Gulick et al., 1998;
Allingham et al., 2007). Parrot was used for density modification, and these phases were used to build an initial model in Buccaneer (
Cowtan, 2008,
2010). This was followed by iterative cycles of manual model building in Coot and restrained and TLS refinement in Refmac 5.6 (
Emsley and Cowtan, 2004;
Skubák et al., 2004). Data processing and refinement statistics are presented in . All structural alignments were done with the program Superpose using secondary structure matching (
Krissinel and Henrick, 2004).
| Table 2.Crystal structure data collection and refinement statistics |
High-resolution metal shadowing
Decoration of microtubules with dimeric Kar3Vik1 was performed after adsorbing microtubules to plain, glow-discharged carbon grids for 1 min in the presence of 10 mM ADP. The initial solution of microtubules during absorption to EM grids was at 0.4 mg/ml, but excess liquid was briefly blotted away from the grids before adding 4 µl of a solution of Kar3Vik1 at 0.8 mg/ml. The microtubules that remained on the grids were incubated with the motor solution for 10 min. Then excess liquid was again blotted away from the grids, which were immediately plunged into liquid nitrogen for rapid freezing. The frozen grids were freeze-dried for 90 min in the so-called Midilab system (a unique freeze-drying/metal shadowing unit located at the Eidgenössische Technische Hochschule Zürich, Hoenggerberg, Switzerland), and subsequently shadowed unidirectionally with a 3–4-Å-thick layer of tantalum-tungsten at an elevation angle of 45° (
Hoenger and Gross, 2008).
Microtubule polymerization for cryo-EM studies
Microtubules were polymerized in vitro from purified bovine brain tubulin (Cytoskeleton) with 80 mM Pipes, pH 6.8, 1 mM MgCl
2, 1 mM EGTA, 1 mM GTP, 10 µM paclitaxel, and 8% DMSO for 30 min at 37°C, then allowed to stabilize overnight at room temperature (
Cope et al., 2010).
Vitrification of MT•Kar3Vik1 for cryo-EM
MT•Kar3Vik1 complexes were assembled directly on holey carbon C-flat grids (Protochips) to prevent microtubules from bundling. Polymerized microtubules at 3.75 µM were adsorbed to a holey carbon grid for 35–60 s. Excess liquid was blotted away, and 5 µl of 5–8 µM Kar3Vik1 in the appropriate nucleotide state was immediately added to the microtubules for 90–120 s before blotting away excess liquid and plunging the grid into liquid ethane using a homemade plunge-freezing device. To achieve the nucleotide-free state, Kar3Vik1 was incubated on ice with 1 U of apyrase grade VII (Sigma-Aldrich) for 30–45 min. To mimic the ATP state, Kar3Vik1 was incubated on ice with the ATP analogue AMPPNP (Sigma-Aldrich) for 10–30 min.
Cryo-EM data collection and helical reconstruction
Plunge-frozen samples were transferred under liquid nitrogen to a Gatan-626 cryo-holder (Gatan). Two-dimensional projections of vitreously frozen MT•Kar3Vik1 complexes were acquired on an FEI Tecnai F20 FEG transmission electron microscope (FEI Company) operating at 200 kV. Single-frame images were taken at a nominal magnification of 29,000× and a defocus of −2.5 µm with a total electron dose of 15 electrons/Å2. Images were recorded without binning on a 4,000 × 4,000 charge-coupled device camera (Ultrascan 895; Gatan) with the resulting pixel size corresponding to 3.8 Å on the specimen.
Helical processing was performed using PHOELIX essentially as described previously (
Whittaker et al., 1995). In brief, each microtubule was computationally straightened, and layer-line information was extracted. Layer-line data from a large number of microtubules were shifted to a common phase origin using a reference and subsequently averaged. IMOD (
Kremer et al., 1996) and UCSF Chimera (
Pettersen et al., 2004) were used for visualization and surface rendering of the electron density maps of the averages.
The number of individual datasets and approximate number of asymmetric units included in each of the helical reconstructions is shown in Fig. S3.
Docking of crystal structures into cryo-EM maps
The crystal structure of the αβ-tubulin dimer (PDB accession no.
1JFF;
Löwe et al., 2001) was docked manually into the electron density maps using UCSF Chimera (
Pettersen et al., 2004). The Kar3Vik1 heterodimer crystal structure (PDB accession no.
4ETP) was docked initially into the maps manually using Chimera by positioning Kar3 onto the microtubule based on the results of
Hirose et al. (2006). The Kar3Vik1 crystal structure was then docked quantitatively into the map of the nucleotide-free state using the FFT-Accelerated 6D Exhaustive Search tool in Situs (
Chacón and Wriggers, 2002). This program was used to perform a rigid-body, exhaustive search around translational and rotational space to obtain the best global fit of Kar3Vik1 into the electron density map. The fit that gave the highest correlation score from this quantitative docking was in very close agreement with our initial manual docking result. To dock Kar3Vik1 into the AMPPNP state maps, the structure was divided into two rigid-body components that could be manipulated separately: (1) the Kar3MD core (residues G385-K729) and (2) the complete Vik1MHD domain with the coiled coil stalk. Each component was docked interactively by eye into the maps to obtain the best fit.
MT•Kar3Vik1 cosedimentation assays
Reactions of 200 µl were formed with 2 µM Kar3Vik1 plus microtubules (0–10 µM tubulin polymer and 40 µM paclitaxel in ATPase buffer: 20 mM Hepes, pH 7.2, with KOH, 5 mM magnesium acetate, 0.1 mM EDTA, 0.1 mM EGTA, 50 mM potassium acetate, 1 mM dithiothreitol, and 5% sucrose) in the presence of either 1 mM MgADP, 1 mM MgAMPPNP, or 100 µM MgADP followed by apyrase treatment (0.02 U/ml, grade VII; Sigma-Aldrich) to generate the nucleotide-free state as described previously (
Chen et al., 2011). The samples were incubated at room temperature for 30 min followed by centrifugation. The supernatant and pellet for each reaction were analyzed by SDS-PAGE, followed by Coomassie Blue staining. To determine the fraction of Kar3Vik1 that partitions to the pellet, the gels were scanned, and ImageJ (National Institutes of Health) was used to convert the scanned image to a TIFF file. Image Gauge version 4 software (FUJIFILM Science Laboratory) was used to box the Kar3Vik1 gel bands as a unit for conversion to pixel number. A box of comparable size was used to obtain a background sample. The background total was subtracted from each Kar3Vik1 sample. For each microtubule concentration, the Image Gauge sum for Kar3Vik1 partitioning to the pellet was divided by the sum of the Kar3Vik1 in the supernatant + pellet, thereby providing the fraction. Note that each sample is independent of the others, with supernatant + pellet = 100% of 2 µM Kar3Vik1. This approach avoids errors associated with small differences in loading volumes. The fraction of Kar3Vik1 that partitioned with the microtubule pellet as a function of microtubule concentration was plotted, and the data were fit to the following quadratic equation because of the stoichiometric binding conditions of the experiment: [(MT•E)/(E)] = 0.5 × {(E
0 +
Kd + MT
0) − [(E
0 +
Kd + MT
0)
2 − (4E
0MT
0)]
1/2}. MT•E/E is the fraction of Kar3Vik1 that partitioned with the microtubules, E
0 is total Kar3Vik1,
Kd is the apparent dissociation constant, and MT
0 is the microtubule concentration. shows representative experiments at each condition. Each experiment was repeated multiple times with the
Kd constants reported as the mean ± SEM.
Polarity marked microtubules and microtubule gliding assay
Polarity marked paclitaxel-stabilized rhodamine-labeled microtubules were assembled, and the motility assays were performed as described previously (
Allingham et al., 2007;
Sardar et al., 2010). Before each experiment, Kar3Vik1 was clarified and the protein concentration was determined by the Bio-Rad Protein assay (IgG as standard). Protein concentrations are expressed as the Kar3Vik1 heterodimer concentration with one nucleotide binding site per Kar3Vik1.
The MT•Kar3Vik1 complex (1 µM Kar3Vik1, 0.5 µM polarity marked rhodamine-microtubules, and 20 µM paclitaxel) was preformed in the presence of 0.5 mM MgAMPPNP in ATPase buffer: 20 mM Hepes, pH 7.2, with KOH, 5 mM magnesium acetate, 0.1 mM EDTA, 0.1 mM EGTA, 50 mM potassium acetate, 1 mM dithiothreitol, and 5% sucrose. The microscope perfusion chamber was rinsed with PME-80 buffer (80 mM Pipes, pH 6.9, with KOH, 5 mM MgCl2, and 1 mM EGTA) and coated with Penta-His antibodies (QIAGEN) at 100 µg/ml, followed by a blocking buffer containing 1 mM MgAMPPNP and an oxygen scavenger mix (OSM; 1× PME-80 buffer, 1 mg/ml casein, 0.2 mg/ml glucose oxidase, 0.175 mg/ml catalase, 25 mM glucose, and 20 µM paclitaxel). The MT•Kar3Vik1•AMPPNP complex was then perfused into the chamber and became immobilized to the slide by the N-terminal His tag of Kar3Vik1 bound to the Penta-His antibodies. After a 5-min incubation, PME-80 + 0.5 mM MgAMPPNP buffer wash was flowed into the chamber to remove unbound microtubules and motors. microtubule gliding was initiated by adding 1.5 mM MgATP with an ATP regeneration system (0.3 µg/µl creatine phosphokinase and 2 mM phosphocreatine in OSM buffer). The final mix was: 1× PME-80 buffer, 1.5 mM ATP, 1.5 mM magnesium acetate, 0.2 mg/ml glucose oxidase, 0.175 mg/ml catalase, 25 mM glucose, 0.5% β-mercaptoethanol, and 20 µM paclitaxel. Note that reducing agents (DTT, β-mercaptoethanol) were excluded from the buffers and reaction mix for experiments with cross-linked Kar3Vik1 motors.
Total internal reflection fluorescence (TIRF) microscopy was used to visualize movement of polarity-marked rhodamine microtubules at 25°C. Imaging was performed on an AxioObserver Z1 Inverted Laser TIRF3 microscope system (Carl Zeiss) using the TIRF DsRed filter set. Images were recorded with the electron multiplier (EM-CCD) digital camera (Hamamatsu Photonics) using the 100×/1.46 α-Plan-Apochromat oil objective lens. The Definite Focus module (Carl Zeiss) was used to stabilize image capture. Images were collected every 20 s for 45 min with an exposure time of 180 ms. All movies and images were analyzed and processed with the AxioVision Release 4.8.2 software and Photoshop (Adobe). microtubules that were scored met the following criteria: microtubules were entirely within the field of view, were ≥ 3 µm but ≤ 8 µm in length, and travelled in a clearly defined path. Because the Kar3Vik1 motors were immobilized on the coverslip, microtubule minus-end–directed motility is observed with the bright microtubule minus end trailing with the dimmer microtubule plus end leading.
Steady-state ATPase kinetics
The turnover of α-[
32P]ATP to α-[
32P]ADP•P
i during steady-state was determined in ATPase buffer as described previously (
Gilbert and Mackey, 2000). These experiments were performed by preparing two tubes, each with 50 µl for a total reaction volume of 100 µl. The Kar3Vik1 tube A contained the specific Kar3Vik1 motor, microtubules, ATPase buffer, and 40 µM paclitaxel. The ATP tube B contained 1 µl of α-[
32P]ATP, unlabeled MgATP, and ATPase buffer. The reactions were initiated by the addition of 5 µl of tube A to 5 µl of tube B and incubated at 25°C for varying times (eight time points per 100-µl reaction). At each time point, the 10-µl reaction was terminated by the addition of 20 µl of 10 N formic acid. The zero time points were determined by denaturing the Kar3Vik1 (5 µl of tube A) with 20 µl formic acid before mixing with 5 µl of tube B. An aliquot (1 µl) of the terminated reaction mixture for each time point was spotted onto a polyethyleneimine cellulose F TLC plate (20 × 20 cm, plastic-backed; EM Science) and developed in 0.6 M potassium phosphate buffer, pH 3.4, with phosphoric acid to separate the products α-[
32P]ADP + P
i from α-[
32P]ATP. Each TLC plate was scanned, and the radiolabeled nucleotide was quantified using Image Gauge V4.0 software (Fujifilm) to box the α-[
32P]ADP and α-[
32P]ATP spots for conversion to pixel number.
For each time point, the sum of ADP divided by the sum of ADP + ATP provided the fraction of ATP that was hydrolyzed during the time of the reaction. This fraction is converted to micromolar ADP formed at each time point based on the ATP concentration in the reaction and corrected for the contaminating α-[32P]ADP using the zero time point data. The ADP product formed was plotted as a function of time, and the linear fit of each time course provided the observed rate of the reaction (micromolar ADP produced per Kar3Vik1 per second) for each ATP concentration. The observed rates (s−1) plotted as a function of increasing MgATP concentration were fit to the Michaelis-Menten equation, and the microtubule concentration dependence data were fit to the following quadratic equation because the enzyme concentration was not 10-fold less than the K1/2,MT. Rate = 0.5 × kcat × {(E0 + K1/2,MT + MT0) − [(E0 + K1/2,MT + MT0)2 − (4E0S0)]1/2}. The rate is the product of ADP•Pi formed per second per Kar3Vik1 active site, kcat is the maximum rate constant of steady-state turnover at saturating microtubules, E0 is the Kar3Vik1 concentration in (μM), K1/2,MT is the concentration of tubulin polymer that yields half the kcat, and MT0 is the tubulin polymer concentration (μM).
Online supplemental material
Fig. S1 elaborates on the Kar3Vik1 construct design. Fig. S2 displays motor properties of GCN4-Kar3Vik1 and SHD-Kar3Vik1. Fig. S3 confirms the registration of Kar3Vik1’s coiled coil within the x-ray crystal structure. Fig. S4 presents the cryo-EM and helical reconstructions of cross-linked Kar3Vik1. Fig. S5 demonstrates the cross-linking efficiency of cross-linked Kar3Vik1 motors. Videos 1–7 show the motility of WT SHD-Kar3Vik1, non-cross-linked SHD-Kar3Vik1, cross-linked SHD-Kar3Vik1, WT GCN4-Kar3Vik1, non-cross-linked GCN4-Kar3Vik1, 10-Å cross-linked Kar3Vik1, and 20-Å cross-linked Kar3Vik1, respectively. Online supplemental material is available at
http://www.jcb.org/cgi/content/full/jcb.201201132/DC1.
2 and Ivan Rayment1
This article has been 
