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. 2006 May 17;25(10):2263-73.
doi: 10.1038/sj.emboj.7601108. Epub 2006 Apr 27.

A structural model for monastrol inhibition of dimeric kinesin Eg5

Troy C Krzysiak  1 , Thomas Wendt, Lisa R Sproul, Peter Tittmann, Heinz Gross, Susan P Gilbert, Andreas Hoenger
Affiliations

A structural model for monastrol inhibition of dimeric kinesin Eg5

Troy C Krzysiak et al. EMBO J. .

Abstract

Eg5 or KSP is a homotetrameric Kinesin-5 involved in centrosome separation and assembly of the bipolar mitotic spindle. Analytical gel filtration of purified protein and cryo-electron microscopy (cryo-EM) of unidirectional shadowed microtubule-Eg5 complexes have been used to identify the stable dimer Eg5-513. The motility assays show that Eg5-513 promotes robust plus-end-directed microtubule gliding at a rate similar to that of homotetrameric Eg5 in vitro. Eg5-513 exhibits slow ATP turnover, high affinity for ATP, and a weakened affinity for microtubules when compared to monomeric Eg5. We show here that the Eg5-513 dimer binds microtubules with both heads to two adjacent tubulin heterodimers along the same microtubule protofilament. Under all nucleotide conditions tested, there were no visible structural changes in the monomeric Eg5-microtubule complexes with monastrol treatment. In contrast, there was a substantial monastrol effect on dimeric Eg5-513, which reduced microtubule lattice decoration. Comparisons between the X-ray structures of Eg5-ADP and Eg5-ADP-monastrol with rat kinesin-ADP after docking them into cryo-EM 3-D scaffolds revealed structural evidence for the weaker microtubule-Eg5 interaction in the presence of monastrol.

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Figures

Figure 1
Figure 1
Eg5 motor proteins. (A) The domain structure of the human gene as predicted by PAIR-COIL with Eg5-367 and Eg5-513. (B) Analytical gel filtration was carried out to compare Eg5-513, Eg5-513-5His, dimeric Kinesin-1 K401, and monomeric Eg5-367. The void volume eluted at 16 min and the included volume at 51.5 min. (C) Gel filtration of Eg5-513 at the protein concentration range used for the reported experiments indicates that the proposed dimeric state of Eg5-513 remained stable over the range of protein concentrations used in this study (0.5–4 μM). The inset shows an SDS–Coomassie blue stained gel of Eg5-513.
Figure 2
Figure 2
Shadowgraphs of freeze-dried MTs decorated with Eg5 motors. Unidirectional shadowing as shown here reveals specifically the surface features of MTs in the absence of Eg5 (A) and MTs complexed with monomeric Eg5 (Eg5-367) and dimeric Eg5 (Eg5-513), either attempting full saturation (B–E) or undersaturation (F–I). Full saturation was achieved with Eg5-367 in the presence of MgAMPPNP (B) and MgAMPPNP+S-monastrol (C). Dimeric Eg5 allows complete surface decoration in the presence of MgAMPPNP (D) but not in the presence of S-monastrol+MgAMPPNP (E) owing to a strikingly reduced affinity of motors to MTs. The repetitive surface pattern in panel D indicates that Eg5-513 dimers bind with both heads to MTs and do not leave one head unbound and tethered (e.g. as seen with Ncd). At subsaturating conditions (F, G), monomeric Eg5-367 binds randomly along the MT lattice, indicated by the red dots in (G) to mark the motor head position. (H, I) Eg5-513 also shows stochastic binding, but here the motors are clearly visible as double-densities that mostly align along protofilaments. The Eg5-513 head positions are marked in red with potential dimers as double-red dots connected by a yellow marker (I).
Figure 3
Figure 3
Eg5-513-5His promotes plus-end-directed MT gliding in the presence of 1.5 mM MgATP. Polarity-marked MTs exhibit a more highly fluorescent MT minus-end. ▴ indicates a MT that is changing its position over time in comparison to the stationary MT denoted by ★. Conventional Kinesin-1 (KHC) was used to assess the polarity of the MTs. See Supplementary data for a movie.
Figure 4
Figure 4
Steady-state ATPase kinetics and monastrol inhibition. A preformed MT·Eg5 (•) or MT·Eg5-monastrol (⧫) complex was rapidly mixed with [α-32P]ATP. (A) Final concentrations: 0.5 μM Eg5-513 motor domain, 20 μM tubulin, 20 μM Taxol, 150 μM S-monastrol, and 1–200 μM MgATP. The fit of the data to the Michaelis–Menten equation provided the steady-state parameters: kcat=0.44±0.01 s−1, Km,ATP=7.5±0.7 μM and kcat-Mon=0.034±0.001 s−1, Km,ATP-Mon=4.5±0.9 μM. (B) Final concentrations: 1 μM Eg5-513 motor domains, 0.1–40 μM tubulin, 20 μM Taxol, 150 μM S-monastrol, and 400 μM MgATP. The data were fit to equation (1): kcat=0.48±0.01 s−1, K1/2,MT=1.8±0.1 μM and kcat-Mon=0.048±0.002 s−1, K1/2,MT -Mon=1.7±0.3 μM. (C) Final concentrations: 0.5 μM Eg5-513 motor domain, 20 μM tubulin, 20 μM Taxol, 150 μM MgATP, and 0–150 μM S-monastrol. The data were fit to equation (2): Kd-Mon=2.88±0.04 μM.
Figure 5
Figure 5
Eg5 motor binding to MTs. Cryo-EM analysis of monomeric MT·Eg5-367 complexes (A–C) and dimeric MT·Eg5-513 complexes (D–F) are shown in different nucleotide states. Diffraction patterns were taken from the averaged data sets. The graphs on the right display the projected total power of each layer line. The leftmost peak is not the equator, but represents the first layer line marking the protofilament supertwist. The 1/4 nm cluster (marked in panel A) relates mainly to the tubulin monomer repeat along the MT axis, whereas 1/8 nm marks the repeat of the αβ-tubulin dimer or a tubulin dimer complexed with one motor head domain. Eg5-367 binds regularly along the MTs in the absence of nucleotides (A), in the presence of MgAMPPNP (B), and in the presence of MgAMPPNP+S-monastrol (C). Dimeric Eg5-513 in the presence of MgAMPPNP also reveals a strong MT affinity (E) that is, however, significantly reduced in the absence of nucleotides (D). AMPPNP+S-monastrol show an even further reduced affinity (F, also see Figure 2E). MT·Eg5 cosedimentation (G) has been performed to compare Eg5-367 (left) and Eg5-513 (right). The MT binding patterns were not significantly different for monomeric Eg5-367 with MgAMPPNP, apyrase-induced nucleotide-free state, or AMPPNP+S-monastrol. In contrast, dimeric Eg5-513 exhibited a sigmoidal MT binding behavior. The AMPPNP state was more tightly bound (KHill=0.49±0.06 μM3, n=2.8±0.3) than the nucleotide-free state (KHill=0.96±0.07 μM2, n=1.7±0.1). Monastrol treatment with AMPPNP (KHill=1.0±0.05 μM2, n=2.0±0.1) resulted in a binding pattern more similar to the apyrase-induced nucleotide-free state.
Figure 6
Figure 6
3-D analysis of MT·Eg5 complexes. Here, we show surface-rendered 3-D volumes and a selected axial cross-section of helically reconstructed MTs decorated with monomeric (A–D) and dimeric (E, F) Eg5 under various nucleotide conditions. The MTs are oriented with the plus-end at the top of the reconstruction (tubulin: blue; motors: yellow). The maps are normalized to the MT volume. In contrast to Kinesin-1 (Skiniotis et al, 2003), the structural differences for Eg5 according to nucleotide state and/or presence of S-monastrol were not resolved, and all maps obtained with monomeric Eg5-367 appear identical. Reconstructions of MT·Eg5-513 complexes treated with apyrase (nucleotide-free; (E)) show reduced density in the head portion, indicating incomplete surface decoration due to a lower MT affinity of dimeric motors under these conditions. In the presence of MgAMPPNP (F), the density of the head portion is identical to monomers, whereas addition of monastrol abolishes binding almost completely. Hence, no meaningful 3-D map could be calculated. The head shape on reconstructions with dimeric motor constructs (E, F) reveals an average over trailing and leading heads (see Figure 7), but the close similarity to monomer reconstructions confirms the findings with monomers that these heads do not change configuration upon changes in nucleotide conditions. There was no obvious density in the reconstruction with the dimeric Eg5 that could be attributed to the second head being tethered but detached from the MT.
Figure 7
Figure 7
Molecular docking of the atomic resolution X-ray structures of monomeric Eg5 in the presence of ADP and monastrol (left; Yan et al, 2004) and ADP (right; Turner et al, 2001) into our EM-derived 3-D map of the MT·Eg5-513·AMPPNP complex. The plus-end of the MT is on the right. One major structural difference in the two heads that is relevant to MT binding is the position of helix α4 that is rotated by about 20° from the monastrol to ADP structure. Monastrol (green in (A)) binds close to the ATP pocket. The other relevant feature is the locked neck linker (red) in the monastrol structure. Hence, the arrangement as shown here could mimic a dimer with the trailing head in an ATP state and the leading head ready to release ADP and assume a nucleotide-free state. (B) Crystal structures of monomeric Eg5 in the presence of monastrol and ADP, Eg5 with ADP, and rat kinesin with ADP displayed in aligned orientations according to their internal β-sheet.
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References

    1. Berger B, Wilson DB, Wolf E, Tonchev T, Milla M, Kim PS (1995) Predicting coiled coils by use of pairwise residue correlations. Proc Natl Acad Sci USA 92: 8259–8263 - PMC - PubMed
    1. Beuron F, Hoenger A (2001) Structural analysis of the microtubule–kinesin complex by cryo-electron microscopy. Methods Mol Biol 164: 235–254 - PubMed
    1. Blangy A, Lane HA, d'Herin P, Harper M, Kress M, Nigg EA (1995) Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. Cell 83: 1159–1169 - PubMed
    1. Cochran J, Gatial JI, Kapoor T, Gilbert SP (2005) Monastrol inhibition of mitotic kinesin Eg5. J Biol Chem 280: 12658–12667 - PMC - PubMed
    1. Cochran JC, Gilbert SP (2005) ATPase mechanism of Eg5 in the absence of microtubules: insight into microtubule activation and allosteric inhibition by monastrol. Biochemistry 44: 16633–16648 - PMC - PubMed

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