The kinesin-5 tail domain directly modulates the mechanochemical cycle of the motor domain for anti-parallel microtubule sliding
- University of California, Davis, United States
- Scripps Research Institute, United States
- University of Vermont, United States
- Rensselaer Polytechnic Institute, United States
- Lerner Research Institute, Cleveland Clinic, United States
- Mayo Clinic, United States
- Yale University, United States
- Ben-Gurion University of the Negev, Israel
Figures
Figure 1 with 1 supplement
The kinesin-5 tail domain inhibits the motor domain MT-stimulated ATPase activity through stabilization of the MT bound nucleotide-free state.
(A) Top, domain organization for the kinesin-5 motors, Dm KLP61F and Hs FL-Eg5 consisting of conserved N-terminal motor domain, central BASS domain and C-terminal tail domain. Bottom, homotetrameric …
Figure 1—figure supplement 1
The kinesin-5 tail domain inhibits motor domain MT-stimulated ATPase activity through stabilization of ADP-bound and nucleotide-free state.
(A) Affinity co-purification of the KLP61F motor using the tail-StrepII construct in solution in the presence of three nucleotide states at 15 μmol mixture of motor+ tail-StrepII, which is …
Figure 2 with 1 supplement
Cryo-EM reveals the kinesin-5 tail engages the motor domain in the nucleotide-free state and stabilizes an open ATP active site.
(A-C) (A) Side view of 4.0 Å cryo-EM structure of Klp61F motor domain decorated MT unit in the AMPPNP state. A single kinesin motor-bound αβ-tubulin unit is shown. The segmented motor domain …
Figure 2—figure supplement 1
Cryo-EM reveals the kinesin-5 tail domain engages the motor domain directly through the α0-helix and stabilizes an open ATP active site.
(A) Top panel (I): Colores view of the Klp61F motor MT AMPPNP map, colored based on resolution scale. Bottom Panel (II): A gold standard Fourier Shell correlation (FSC) curve for the MT-decorated …
Figure 3 with 1 supplement
The kinesin-5 tail domain decreases velocity for homotetrameric motors along MTs.
(A) Human Eg5 constructs used in reconstitution studies. Top panel, domain organization of FL-Eg5-GFP. Second panel, domain organization of Eg5-Δtail-GFP with its tail domain deleted (residues …
Figure 3—figure supplement 1
The tail domain decreases the velocity of homotetrameric kinesin-5 motors along MTs.
(A) Right panel, Size exclusion chromatography (SEC) for recombinant FL-Eg5-GFP (red) and SDS-PAGE lane for peak fraction. Left panel, SEC for recombinant Eg5-Δtail-GFP and SDS-PAGE lane for peak …
Figure 4 with 1 supplement
The kinesin-5 tail is critical for the zippering of two sliding MTs via slow directional motility within the overlap zones.
(A) Left panel, scheme for TIRF microscopy reconstitution of MT sliding assays where FL-Eg5-GFP motors (green) recruit free MTs (orange) along single surface anchored AlexaF-633 and biotin labeled …
Figure 4—figure supplement 1
The kinesin-5 tail domain regulates the zippering of two sliding MTs via slow directional motility within the overlapping zones.
(A) Left side panels, wide field view of reconstituting 10–20 nM FL-Eg5-GFP mediated MT sliding events. Free MTs (yellow) can be seen recruited along anchored MTs (red) mediated by the FL-Eg5-GFP …
Figure 5
Kinesin-5 tail is critical in generating pushing forces during MT sliding.
(A) Scheme for MT sliding and optical trapping to measure the MT sliding pushing forces. Hilyte 649 labeled and biotins labeled MTs (purple) were attached to glass surfaces via neutravidin-biotin. …
Figure 6
Deletion of the kinesin-5 tail domain disrupts localization of the motor to the mitotic spindle in metaphase and anaphase.
(A) Western blot for mCherry (mCh, green) and GAPDH (red) indicating the expression of FL-Eg5-mCherry (FL Eg5-mCh, green) and Eg5-Δtail-mCherry (Eg5-Δtail-mCh, green) in HeLa cells. Results are …
Figure 7
A revised model for kinesin-5 tail-motor interaction during stepping motility and critical role for force generation during MT sliding.
(A) Model for kinesin-5 homotetramers with their motor and tail domains at each end of the bipolar minifilament (60 nm). The motors and tail domains at each end may form assemblies where the tail …
Videos
Video 1
Structural transition of the kinesin-5 motor domain from AMPPNP to nucleotide state and its effect on binding of the tail domain.
View of the kinesin-5 motor domain map with AMPPNP showing the motor domain model, transition to the motor nucleotide state map showing the site of binding for the tail domain density, and model for …
Video 2
Wide view of FL-Eg5-GFP and Eg5-Δtail-GFP (green) along single MTs (Red).
Video 3
close up views of FL-Eg5-GFP (left) and Eg5-Δtail-GFP (right) along single MTs at 25, 50 and 100 mM KCl conditions.
Video 4
left, close up views of FL-Eg5-GFP motors (green) mediating zippering of free MT (yellow) along anchored MT (red).
Right close up view of Eg5-Δtail-GFP motors crosslinking but unable to zipper MTs leading to scissoring defect.
Video 5
Top left, close up view of 1 nM FL-Eg5-GFP motor (green) spiking during free MT (yellow) sliding along anchored MT (red) mediated by 20 nM unlabeled FL-Eg5.
Top right, same event without the free MT revealing FL-Eg5-GFP motors along the anchored MT. Bottom left, close up view of 1 nM Eg5-Δtail-GFP motor (green) spiking during free MT (yellow) sliding …
Video 6
left, optical trapping of MT sliding experiments revealing the bead attached to sliding free MT (red) along anchored MT (purple) mediated by FL-Eg5-GFP motors.
Right, optical trapping of MT sliding experiments revealing the bead attached to sliding free MT (red) along anchored MT (purple) mediated by Eg5-Δtail-GFP motors.
Tables
Table 1
Steady kinetic parameters for MT-activated ATP hydrolysis.
| Construct | Source | Ionic Strength | kcat (sec−1) | K0.5,MT (nM) |
|---|---|---|---|---|
| Motor | Dm KLP61F | 50 mM K Acetate | 7.1 ± 0.1 | 680 ± 48 |
| Motor + Tail | Dm KLP61F | 50 mM K Acetate | 3.5 ± 0.5 | 757 ± 327 |
| Motor-Tail fusion | Dm KLP61F | 50 mM K Acetate | 3.3 ± 0.1 | 39 ± 11 |
| Motor | Hs Eg5 | 20 mM KCl | 7.3 ± 0.2 | 334 ± 14 |
| Motor-Tail fusion | Hs Eg5 | 20 mM KCl | 3.4 ± 0.2 | 158 ± 41 |
| Motor + Tail | Hs Eg5 | 20 mM KCl | 5.4 ± 0.3 | 209 ± 56 |
| Motor | Hs Eg5 | 50 mM K Acetate | 6.71 ± 0.7 | 3849 ± 800 |
| Motor-Tail fusion | Hs Eg5 | 50 mM K Acetate | 5.87 ± 0.23 | 391 ± 59 |
Table 2
Cryo-EM KLp61F motor and tail MT structures: collection and reconstruction.
| Dm Klp61F motor-AMPPNP (15 protofilaments) | Dm Klp61F- motor AMPPNP (14 protofilaments) | Dm KLP61F motor-tail- nucleotide free (15 protofilaments) | Dm Klp61F5 motor-tail-nucleotide free (14-protofilaments) | |
|---|---|---|---|---|
| Data collection | ||||
| Microscope | Titan Krios (FEI) | Titan Krios (FEI) | Titan Krios (FEI) | Titan Krios (FEI) |
| Voltage (kV) | 300 | 300 | 300 | 300 |
| Ls | 22,500X | 22,500X | 22,500X | 22,500X |
| Cumulative exposure dose (e- Å−2) | 38 | 38 | 40 | 40 |
| Exposure rate (e-/pixel/sec) | 7.9 | 7.9 | 8.3 | 8.3 |
| Detector | K2 Summit | K2 Summit | K2 Summit | K2 Summit |
| Pixel size (Å)* | 1.31 | 1.31 | 1.31 | 1.31 |
| Defocus range (µm) | 0.3–3.78 | 0.7–3.78 | 0.19–5.12 | 0.19–5.12 |
| Average defocus (m) | 1.75 | 1.75 | 1.86 | 1.86 |
| Micrographs Used | 1260 | 1260 | 955 | 955 |
| Total extracted helical segment (no.) | 73,451 | 73,451 | 44,081 | 44,081 |
| Refined helical segment (no.) | 21,004 | 39,001 | 9490 | 27,433 |
| Reconstruction | ||||
| Final helical segments (no.) | 21,004 | 39,220 | 9490 | 14,570 |
| Symmetry imposed | HP | HP | HP | HP |
| Resolution (global) FSC 0.143 | 4.2 | 4.4 | 4.2 | 4.3 |
Table 3
Cryo-EM refinement and Structure model statistics.
| Dm-Klp61F motor AMPPNP-MT | Dm-Klp61F motor -Nucleotide free-MT | |
|---|---|---|
| Data collection | ||
| Microscope/detector | Titan Krios/Gatan K2 | Titan Krios/Gatan K2 |
| Magnification | 22,500x | 22,500x |
| Voltage (keV) | 300 | 300 |
| Dose rate (electrons/pixel/second) | 7.96 | 8.3 |
| Pixel size (Å/pixel) | 1.31 | 1.31 |
| Map resolution (Å) | 4.4 | 4.4 |
| FSC threshold | 0.143 | 0.143 |
| Refinement | ||
| Model resolution cutoff (Å) | 4.4 | 4.4 |
| FSC threshold | 0.143 | 0.143 |
| Protein residues | 1173 | 1174 |
| Ligands | 3 (GTP/GDP/AMPPNP) | 2 (GTP/GDP) |
| Map CC | 0.8 | 0.78 |
| B factor (Å) | 216 | 208 |
| R.M.S deviations | ||
| Bond lengths (Å) | 0.003 | 0.006 |
| Bond angles (°) | 0.53 | 1.14 |
| Validation | ||
| All-atom clash score | 14.05 | 11.71 |
| MolProbity score | 2.52 | 1.9 |
| Ramachandran plot | ||
| Favored (%) | 96.19 | 94.65 |
| Allowed (%) | 3.81 | 5.18 |
| Outliers (%) | 0.00 | 0.17 |
Table 4
Motility parameters for FL-Eg5-GFP and Eg5-Δtail-GFP along single MTs.
| FL-Eg5-GFP | Motility (nm/s) | Motor Fluorescence (Au) | Run length (μm) |
|---|---|---|---|
| 25 mM KCl | 7 ± 0.5 n = 149 | 1080 ± 30 n = 100 | N/A |
| 50 mM KCl | 26 ± 4 n = 200 | N/A | 13.7 ± 0.6 |
| 100 mM KCl | 26 ± 5 (60%) 41 ± 4 (40%) n = 149 | 2277 ± 100 4467 ± 630 n = 92 | 13.08 ± 0.6 |
| Eg5-Δtail-GFP | |||
| 25 mM KCl | 32 ± 5 n = 421 | 960 ± 20 n = 95 | N/A |
| 50 mM KCl | 33 ± 4 n = 420 | N/A | 14.9 ± 0.6 |
| 100 mM KCl | 36 ± 5 (85%) 55 ± 10 (15%) n = 149 | 1450 ± 3 n = 95 | 8.0 ± 0.6 |
Table 5
Motor motility and MT sliding parameters in vitro MT sliding assays.
| Single motor velocities in relation to free MT sliding motility | ||
|---|---|---|
| FL-Eg5-GFP | Free MT sliding motility (nm/s) | Motility in sliding zones (nm/s) |
| 25 mM KCl | 13.8 ± 1.0 n = 26 | 13.9 ± 1.0 n = 32 |
| 50 mM KCl | 31.2 ± 1.2 n = 33 | 22.7 ± 1.2 n = 71 |
| Single motor motility within MT sliding zones | ||
| Eg5-Δtail-GFP motors (nm/s) | FL-Eg5-GFP motors (nm/s) | |
| Overlap Zone | 8.6 ± 0.9 n = 32 | 3.4 ± 0.3 n = 67 |
| Single MT | 9.6 ± 0.8 n = 52 | 5.6 ± 0.3 n = 45 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifier | Additional Information |
|---|---|---|---|---|
| Chemical compound, drug | ATP | Sigma | A-2383 | Figures 1, 3, 4, 5 and 6 |
| Chemical compound, drug | ADP | Sigma | A-2754 | Figure 1 |
| Chemical compound, drug | GTP | Sigma | G-8877 | Figures 1, 3, 4 and 5 |
| Chemical compound, drug | GMPCPP | Jenna Biosciences | NU-405L | Figures 3, 4 and 5 |
| Chemical compound, drug | AMPPNP | Sigma | A-2647 | Figure 1 |
| Chemical compound, drug | Paclitaxel | Sigma | T7402 | Figure 1, 2 |
| Other | Streptactin XT | IBA-life sciences | 2-1003-100 | Figure 1, 3 |
| Chemical compound, drug | d-Biotin | Sigma | B-4501 | Figure 1, 3 |
| Commercial assay or kit | EnzCheck ATPase assay kit | Thermofisher | E6646 | Figure 1 |
| Chemical compound, drug | NeutrAvidin | Thermofisher | 31000 | Figure 3, 4 |
| Chemical compound, drug | biotin-PEG-3400-silane | Laysan Bio | Biotin-PEG-SIL-3400–500 mg | Figure 3, 4 |
| Chemical compound, drug | PEG-2000-silane | Laysan Bio | MPEG-SIL-2000–1 g | Figure 3, 4 |
| Chemical compound, drug | Pluronic-F127 | Sigma | P2443 | Figure 3, 4 |
| Antibody | anti-GAPDH (mouse monoclonal) | Thermo-Fisher | 437000 | Western blot: 1:10,000 |
| Antibody | anti-mCherry | Abcam | ab167453 | Western blot: 1:1000 |
| Antibody | anti-mouse IRDye680 (goat polyclonal) | LI-COR | 92568070 | Western blot: 1:10,000 |
| Antibody | anti-rabbit IRDye800 (goat polyclonal) | LI-COR | 92632211 | Western blot: 1:10,000 |
| Antibody | anti-tubulin DM1α (mouse monoclonal) | Sigma | T9026 | Immunofluorescence: 1:750 |
| Antibody | anti-mouse AlexaFluor 488 (goat polyclonal) | Invitrogen | A-11029 | Immunofluorescence: 1:500 |
| Antibody | anti-mouse AlexaFluor 647 (goat polyclonal) | Invitrogen | A-21236 | Immunofluorescence: 1:500 |
| Commercial assay or kit | Nucleofector Cell Line SE Kit | Lonza | V4XC-1024 | |
| Commercial assay or kit | Phusion Site-Directed Mutagenesis | Thermo Scientific | F541 | Figure 6 |
| Chemical compound, drug | BRD-9876 | Tocris Bioscience | 5454/50 | Figure 6 |
| Chemical compound, drug | MG-132 | Selleckchem | S2619 | Figure 6 |
| Peptide, recombinant protein | Drosophila KLP61F | UniprotKB/Swiss-Prot | P46863 | |
| Peptide, recombinant protein | Human Eg5 (KIF11) | UNiportKB/Swiss-Prot | P52732 | |
| Peptide, recombinant protein | Porcine alpha tubulin | UniprotKB/Swiss-Prot | Q2XVP4 | |
| Peptide, recombinant protein | Porcine beta-tubulin | UniprotKB/Swiss-Prot | P02550 | |
| Cell line (E. coli) | SoluBL21 bacterial expression | AmsBio | C700200 | Figure 1, 2 |
| Cell line (S. frugiperda) | Spodoptera frugiperda-9 (Sf-9 cells) | Thermofisher | 11496–015 | Figures 3, 4 and 5 |
| Cell line (Homo sapiens) | HeLa cell line | ATCC | CCL-2 | Figure 6 |
| Recombinant DNA reagent | pLIC_V2-Dm-KLp61F motor- H6(1–369) | This paper | Figure 1, 2 | |
| Recombinant DNA reagent | pLIC_V2-Dm-KLP61F tail H6 (913–1016) | This paper | Figure 1, 2 | |
| Recombinant DNA reagent | pLIC_V2-Dm KLP61F motor-tail fusion (residues 1–360, GSGSGS-linker, residues 913–1016) | This paper | Figure 1 | |
| Recombinant DNA reagent | pET21a human Eg5 motor (residues 1–360) | This paper | Synthetic | Figure 1 |
| Recombinant DNA reagent | pET21a human Eg5 tail (residues 920–1056) | This paper | Synthetic | Figure 1 |
| Recombinant DNA reagent | pET21a human Eg5 motor-tail fusion (residues 1–360 GSGSGS-linker residues 920–1056) | This paper | Synthetic | Figure 1 |
| Recombinant DNA reagent | pFastbac-human FL-Eg5-GFP (residues 1–1056-msfGFP-StrepII) | This paper | Figure 3 | |
| Recombinant DNA reagent | pFastbac-human FL-Eg5 (residues 1–1056-StrepII) | This paper | Figure 3 | |
| Recombinant DNA reagent | pFastbac-human Eg5-Δ-tail-GFP (residues 1–920-msfGFP-StrepII) | This paper | Figure 3 | |
| Recombinant DNA reagent | pcDNA3.1 FL-Eg5-mCh (residues 1–1056, mCherry) siRNA resistant (T2124C, C2130T, G2133T, and G2136A) | This paper | Figure 6 | |
| Recombinant DNA reagent | pcDNA3.1 Eg5- Δtail-mCh (residues 1–920, mCherry) siRNA resistant (T2124C, C2130T, G2133T, and G2136A) | This paper | Figure 6 | |
| Recombinant DNA reagent | GFP-Tubulin | Clonetech | Stock #61171 | Figure 6 |
| Recombinant DNA reagent | pCMV-mCh | Peris et al., 2009 | ||
| Peptide, recombinant protein | αβ-tubulin purified from porcine brains | This paper Castoldi and Popov, 2003 | Figures 1, 3 and 4 | |
| Software, algorithm | ImageLab | Biorad | https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10000076953.pdf | |
| Software, algorithm | FIJI (ImageJ) | Schindelin et al., 2012 | https://fiji.sc | |
| Software, algorithm | Prism | GraphPad | https://www.graphpad.com/scientific-software/prism/ | |
| Software, algorithm | Motioncor2 | Zheng et al., 2017 | https://emcore.ucsf.edu/ucsf-motioncor2 | |
| Software, algorithm | CTFFIND4 | Rohou and Grigorieff, 2015 | https://grigoriefflab.umassmed.edu/ctf_estimation_ctffind_ctftilt | |
| Software, algorithm | EMAN2 | Tang et al., 2007 | http://blake.bcm.edu/emanwiki/EMAN2 | |
| Software, algorithm | FREALIGN | Grigorieff, 2007 | https://grigoriefflab.umassmed.edu/frealign | |
| Software, algorithm | B-factor | Grigorieff, 2007 | https://grigoriefflab.umassmed.edu/bfactor | |
| Software, algorithm | UCSF-Chimera | Pettersen et al., 2004 | https://www.cgl.ucsf.edu/chimera/ | |
| Software, algorithm | CCP4 suite | Collaborative Computational Project, Number 4, 1994 | http://www.ccp4.ac.uk/html/dmmulti.html | |
| Software, algorithm | GCTF | Zhang, 2016 | https://www.mrc-lmb.cam.ac.uk/kzhang/ | |
| Software, algorithm | Phyre protein homology model | Kelley et al., 2015 | www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index | |
| Software, algorithm | Relion 2.2 | Emsley et al., 2010 | https://www2.mrc-lmb.cam.ac.uk/relion/index.php | |
| Software, algorithm | MolProbity | Chen et al., 2010 | http://molprobity.biochem.duke.edu | |
| Software, algorithm | Coot | Emsley et al., 2010 | http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ |
