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. 2019 Feb 15;431(4):864-872.
doi: 10.1016/j.jmb.2019.01.011. Epub 2019 Jan 16.

Cryo-EM Structure (4.5-Å) of Yeast Kinesin-5-Microtubule Complex Reveals a Distinct Binding Footprint and Mechanism of Drug Resistance

Ottilie von Loeffelholz  1 , Alejandro Peña  1 , Douglas Robert Drummond  2 , Robert Cross  2 , Carolyn Ann Moores  3
Affiliations

Cryo-EM Structure (4.5-Å) of Yeast Kinesin-5-Microtubule Complex Reveals a Distinct Binding Footprint and Mechanism of Drug Resistance

Ottilie von Loeffelholz et al. J Mol Biol. .

Erratum in

Abstract

Kinesin-5s are microtubule-dependent motors that drive spindle pole separation during mitosis. We used cryo-electron microscopy to determine the 4.5-Å resolution structure of the motor domain of the fission yeast kinesin-5 Cut7 bound to fission yeast microtubules and explored the topology of the motor-microtubule interface and the susceptibility of the complex to drug binding. Despite their non-canonical architecture and mechanochemistry, Schizosaccharomyces pombe microtubules were stabilized by epothilone at the taxane binding pocket. The overall Cut7 footprint on the S. pombe microtubule surface is altered compared to mammalian tubulin microtubules because of their different polymer architectures. However, the core motor-microtubule interaction is tightly conserved, reflected in similar Cut7 ATPase activities on each microtubule type. AMPPNP-bound Cut7 adopts a kinesin-conserved ATP-like conformation including cover neck bundle formation. However, the Cut7 ATPase is not blocked by a mammalian-specific kinesin-5 inhibitor, consistent with the non-conserved sequence and structure of its loop5 insertion.

Keywords: 3D reconstruction; Cut7; cytoskeleton; mitosis; motor.

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Figures

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Graphical abstract
Fig. 1
Fig. 1
The 4.5-Å resolution reconstruction of Schizosaccharomyces pombe Cut7MD-AMPPNP bound to Sp_tub MT shows epothilone bound at the β-tubulin taxane site of Sp_tub MTs. (a) Inter-PF lateral contacts viewed from the MT lumen, highlighting key secondary structure features and bound epothilone (Epo, orange). (b) The taxane binding pocket in β-tubulin where density corresponding to epothilone is visible (top), compared with our previous structure of Sp_tub MTs without epothilone (middle) and the difference density of these two reconstructions ± epothilone (bottom) . An epothilone molecule is docked for comparison in the epothilone density (middle) but lies outside this cryo-EM density. *The top and bottom panels indicate unassigned density that may reflect mobility or alternate conformations of the drug in the pocket. (c) Top, Sp_tub β-tubulin E-site with β-tubulin in light green ribbon and α-tubulin in dark green ribbon, showing density consistent with bound GDP (in sticks); bottom, ribbon depiction of the atomic model of the Sp_tub MT E-site with density corresponding to the bound nucleotide shown in surface representation. This density is the calculated difference between our cryo-EM reconstruction and simulated 4.4-Å resolution density from the atomic models, calculated using Chimera. This supports the conclusion that the E-site nucleotide in Sp_tub MTs is GDP and thus that GTP hydrolysis has occurred in these MTs. S. pombe MTs were assembled from tag-free, dual isoform purified endogenous tubulin in PEM buffer [100 mM Pipes–KOH, 1 mM MgSO4, 2 mM EGTA, adjusted to pH 6.9 with KOH) mixed 1:1 with Mes polymerization buffer [100 mM Mes (pH 6.5), 1 mM MgCl2, 1 mM EGTA, 1 mM DTT). Tubulin (30 μM) was polymerized in the presence of 5 mM GTP together with 25 μM monomeric Mal3 (residues 1–143), expressed in Escherichia coli and purified as previously described , except that the N-terminal His6 purification tag was removed by TEV protease cleavage. Monomeric Mal3 was added to bias the MT population to 13 PF architecture during polymerization in order to facilitate subsequent structure determination, but Mal3 itself is not visible in the final reconstruction, presumably due to dissociation during sample preparation. MTs were polymerized at 32 °C for 1 h. Epothilone B [in DMSO (Stratech UK)] at a final concentration of 50 μM was added in the final 15 min of polymerization. Sp_tub MT (6 μM) was mixed with 100 μM Cut7MD-AMPPNP at room temperature and 4 μl of the mixture was applied immediately onto glow-discharged Quantifoil R 2/2 holey carbon grids, which were blotted and plunge frozen into liquid ethane using a Vitrobot IV (FEI) operating at room temperature and 100% humidity. Movies were collected manually on a 300 kV Tecnai G2 Polara (FEI) microscope equipped with a Quantum energy filter and K2 Summit direct electron detector (Gatan) in counting mode, recording a total of 606 movies with a total dose in each of 30e−/Å2 fractioned into 50 frames at a pixel size of 1.39 Å/px. Initial frame alignment was performed using IMOD . A second local alignment step was performed with Scipion using the optical flow method . In the final reconstruction only frames 2–21 were included resulting in a total dose of 12e−/Å2. A total of 12,543 MT segments were selected in 908-Å2 boxes in Boxer using the helix option and choosing an overlap with three tubulin dimers (240 Å) unique in each box. Of the 748 MTs that were initially boxed, 669 MTs with 13_3 architecture were selected. The final 3D reconstruction contained 33,007 segments, reboxed with one unique tubulin dimer per box, and was calculated using a semi-automated single particle approach for pseudo-helical assemblies in SPIDER and FREALIGN . The MT Fourier transform layer lines were used to calculate the average helical repeat distance for the Sp_tub heterodimer. The final reconstruction was automatically B-factor sharpened in RELION with an automated calculated B-factor of − 234 . As expected from the substoichiometric concentrations present during sample preparation, no density for the Mal3 added during MT polymerization was present in the final reconstruction. The overall resolution of the masked reconstruction was 4.5 Å (0.143 Fourier Shell Correlation). Atomic models were calculated refined (see Table 1) and structures were visualized using Chimera .
Fig. 2
Fig. 2
The 4.5 A resolution reconstruction of S. pombe Cut7MD-AMPPNP bound to Sp_tub MT reveals the distinct footprint of Cut7 on Sp_tub MTs. (a) The asymmetric unit (αβ-tubulin + Cut7MD) of the reconstruction viewed toward the nucleotide binding pocket with the Cut7MD homology model and Sp_tub atomic model docked into the density. The position of the disordered loop 10 is indicated by the dotted line. (b) View of the Cut7MD nucleotide binding pocket showing helix-α4 at the MT surface, the conserved nucleotide coordinating loops, P-loop (brown), loop9 (yellow) and loop11 (pink), as well as loop5 (purple) emerging away from the nucleotide binding site. The pink arrow indicates the separation of density corresponding to loop11 and the MT surface. (c) Ribbon depiction of longitudinal slices through the Cut7MD model (left) and Sp_tub αβ-tubulin (right) showing the structural elements involved in binding between motor and MT track. The αβ-tubulin ribbon is colored according to sequence conservation with mammalian tubulin; note, while loop2 (L2) lies close to the MT surface, there is no evidence in the EM density of a direct connection between this loop and the MT. (d) View of the MT binding surface of Cut7MD (left) and view of the Cut7MD binding surface of Sp_tub αβ-tubulin (right) with residues < 4 Å distant from each binding partner labeled and colored green and purple, respectively. This interface was evaluated using the model coordinates. (e) View of the MT binding surface of Cut7MD (left) and view of the Cut7MD binding surface of Mam_tub αβ-tubulin (right) with residues < 4 Å distant from each binding partner labeled and colored yellow, respectively. As above, this interface was evaluated using the model coordinates. (f) Cut7MD steady-state ATPase rate as a function of [MT] for Sp_tub (green) and Mam_tub (gray). Data were fit to a Michaelis–Menten kinetic yielding values for Cut7MD Vmax and K0.5MT, respectively, of 1.14 ± 0.02 ATP/s and 13.9 ± 1.1 nM on Sp_tub MTs (R2 = 0.990) and 1.25 ± 0.1 ATP/s and 19.9 ± 5.8 nM on Mam_tub MTs (R2 = 0.918), differences that are not statistically significant (p > 0.99). Inset, the same data presented as Hanes–Woolf plot, yielding values for Cut7MD Vmax = 1.17 ± 0.06 ATP/s and K0.5MT = 13.4 ± 1.1 nM on Sp_tub MTs (R2 of fit = 0.999) and 1.19 ± 0.20 ATP/s and K0.5MT = 14.3 ± 3.4 nM on Mam_tub MTs (R2 = 0.993), which are not statistically significantly different (p > 0.99). (g) Cut7MD MT binding interface colored by surface charge. The approximate position of the adjacent negatively charged β-tubulin C-terminal tail is marked by a circle. A recombinant His6-tagged Cut7 monomeric construct, residues 67–432 lacking its N-terminal extension (Cut7MD) in a pET151D-TOPO vector (Invitrogen), was expressed in BL21*(DE3) E. coli cells as previously described . In brief, cells were grown in LB medium, supplemented with 2% (w/vol) glucose with induction of protein expression by 0.5 IPTG at 18 °C for 5 h. Cells were resuspended in lysis buffer [50 mM Tris–HCl (pH 8.0), 400 mM NaCl, 1 mM MgCl2, 1 mM ATP, 5 mM 2-mercaptoethanol, 10% (vol/vol) glycerol and EDTA-free Protease Inhibitor Cocktail (Roche), 50 μM PMSF] and lysed using a French press. His6-tagged Cut7MD was purified from the clarified cell supernatant using nickel affinity chromatography, and the His6 tag was removed using TEV protease during overnight dialysis into 50 mM Tris–HCl (pH 8.0), 400 mM NaCl, 1 mM MgCl2, 1 mM ATP, 5 mM 2-mercaptoethanol and 10% (vol/vol) glycerol). Immediately prior to use, Cut7MD was buffer exchanged into BrB25 + [25 mM Pipes–KOH (pH 6.8), 30 mM NaCl, 0.5 mM EGTA, 5 mM MgCl2, 1 mM 2-mercaptoethanol, 5 mM AMPPNP] using a Vivaspin® column (Sartorius). To measure the Cut7MD ATPase activity, S. pombe MTs were assembled and stabilized by the addition of epothilone as for cryo-EM sample preparation, except that monomeric Mal3 was excluded from the polymerization mix. Mam_tub MTs were polymerized for 1 h at 37 °C, as previously described using bovine tubulin (Cytoskeleton Inc., Denver, CO) and stabilized by the addition of 1 mM paclitaxel (Calbiochem). Cut7MD ATPase activity was measured using an enzyme-coupled assay in a buffer consisting of 50 mM Tris (pH 8.0), 50 mM NaCl, 1 mM MgCl2, 0.5 mM phosphoenolpyruvate, 0.25 mM NADH, ~ 10 U/ml pyruvate kinase and ~ 14 U/ml lactate dehydrogenase and 5 mM ATP (all reagents from Sigma). The reactions, with each condition performed with 4–6 replicates, were initiated by the addition of Cut7MD at a final concentration of 1.5 μM. Activity was measured by the decrease in NADH absorbance at 340 nm for 10 min at 32 °C in a SpectraMax Plus 384 Microplate Reader (Molecular Devices).
Fig. 3
Fig. 3
Distinctive conformation of Cut7MD and its resistance to small molecule inhibition. (a) Cut7MD CNB formation from the docked neck linker (blue)—directed toward the MT plus end—and the N-terminus (red). (b) Close-up view of the distinct conformation of Cut7MD loop5 (pink) compared to human kinesin-5 [HsK5-AMPPNP (PDB:3HQD), in gray]. (c) Steady-state MT-stimulated ATPase rates of Cut7MD (3.8 μM) and the motor domain of human kinesin-5 (1.3 μM) measured in the absence and presence of 2 μM and 20 μM of the inhibitor STLC plotted as a % of their uninhibited rates. Cut7MD is not detectably inhibited by STLC at concentrations that almost completely inhibit human kinesin-5. Mam_tub MTs (100 nM) were used in each case and 6 and 4 replicates respectively, for control and + STLC were performed. STLC was purchased from Sigma with stocks dissolved in DMSO.
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References

    1. Goulet A., Moores C. New insights into the mechanism of force generation by kinesin-5 molecular motors. Int. Rev. Cell Mol. Biol. 2013;304:419–466. - PubMed
    1. Hagan I., Yanagida M. Novel potential mitotic motor protein encoded by the fission yeast cut7 + gene. Nature. 1990;347:563–566. - PubMed
    1. Kapitein L.C., Peterman E.J., Kwok B.H., Kim J.H., Kapoor T.M., Schmidt C.F. The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks. Nature. 2005;435:114–118. - PubMed
    1. Roostalu J., Hentrich C., Bieling P., Telley I.A., Schiebel E., Surrey T. Directional switching of the kinesin Cin8 through motor coupling. Science. 2011;332:94–99. - PubMed
    1. Gerson-Gurwitz A., Thiede C., Movshovich N., Fridman V., Podolskaya M., Danieli T. Directionality of individual kinesin-5 Cin8 motors is modulated by loop 8, ionic strength and microtubule geometry. EMBO J. 2011;30:4942–4954. - PMC - PubMed

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