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A structural model for monastrol inhibition of dimeric kinesin Eg5.

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.

SUBMITTER: Krzysiak TC

PROVIDER: S-EPMC1462975 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Effect of N-1 arylation of monastrol on kinesin Eg5 inhibition in glioma cell lines.

Project description:An original and focused library of two sets of dihydropyrimidin-2-thiones (DHPMs) substituted with N-1 aryl groups derived from monastrol was designed and synthesized in order to discover a more effective Eg5 ligand than the template. Based on molecular docking studies, four ligands were selected to perform pharmacological investigations against two glioma cell lines. The results led to the discovery of two original compounds, called 20h and 20e, with an anti-proliferative effects, achieving IC50 values of about half that of the IC50 of monastrol in both cell lines. As with monastrol, flow cytometry analyses showed that the 20e and 20h compounds induced cell cycle arrest in the G2/M phase, and immunocytochemistry essays revealed the formation of monopolar spindles due to Eg5 inhibition without any toxicity to Caenorhabditis elegans.

| S-EPMC6072436 | biostudies-literature

Small-molecule and mutational analysis of allosteric Eg5 inhibition by monastrol.

Project description:BackgroundA recent crystal structure of monastrol in a ternary complex with the kinesin Eg5 motor domain highlights a novel, induced-fit drug binding site at atomic resolution. Mutational obliteration of the monastrol binding site results in a monastrol-resistant, but otherwise catalytically active Eg5 motor domain. However, considering the conformational changes at this site, it is unclear what specific interactions stabilize the interaction between monastrol and the Eg5 motor domain.ResultsTo study the molecular complementarity of the monastrol-Eg5 interaction, we used a combination of synthetic chemistry and targeted mutations in Eg5 to measure the contribution of specific contacts to inhibition of Eg5 in vitro and in cultured cells. Structure-activity data on chemical derivatives, sequence analysis of Eg5 homologs from different species, and the effect of mutations near the drug binding site were consistent with the crystal structure.ConclusionThe mechanism of monastrol revealed by our data rationalizes its specificity for Eg5 over other kinesins and highlights a potential mechanism of drug resistance for anti-cancer therapy targeting this site in Eg5.

| S-EPMC1448180 | biostudies-literature

ATPase mechanism of Eg5 in the absence of microtubules: insight into microtubule activation and allosteric inhibition by monastrol.

Project description:The ATPase mechanism of kinesin superfamily members in the absence of microtubules remains largely uncharacterized. We have adopted a strategy to purify monomeric human Eg5 (HsKSP/Kinesin-5) in the nucleotide-free state (apoEg5) in order to perform a detailed transient state kinetic analysis. We have used steady-state and presteady-state kinetics to define the minimal ATPase mechanism for apoEg5 in the absence and presence of the Eg5-specific inhibitor, monastrol. ATP and ADP binding both occur via a two-step process with the isomerization of the collision complex limiting each forward reaction. ATP hydrolysis and phosphate product release are rapid steps in the mechanism, and the observed rate of these steps is limited by the relatively slow isomerization of the Eg5-ATP collision complex. A conformational change coupled to ADP release is the rate-limiting step in the pathway. We propose that the microtubule amplifies and accelerates the structural transitions needed to form the ATP hydrolysis competent state and for rapid ADP release, thus stimulating ATP turnover and increasing enzymatic efficiency. Monastrol appears to bind weakly to the Eg5-ATP collision complex, but after tight ATP binding, the affinity for monastrol increases, thus inhibiting the conformational change required for ADP product release. Taken together, we hypothesize that loop L5 of Eg5 undergoes an "open" to "closed" structural transition that correlates with the rearrangements of the switch-1 and switch-2 regions at the active site during the ATPase cycle.

| S-EPMC2270472 | biostudies-literature

Impaired angiogenesis and tumor development by inhibition of the mitotic kinesin Eg5.

Project description:Kinesin motor proteins exert essential cellular functions in all eukaryotes. They control mitosis, migration and intracellular transport through interaction with microtubules. Small molecule inhibitors of the mitotic kinesin KiF11/Eg5 are a promising new class of anti-neoplastic agents currently evaluated in clinical cancer trials for solid tumors and hematological malignancies. Here we report induction of Eg5 and four other mitotic kinesins including KIF20A/Mklp2 upon stimulation of in vivo angiogenesis with vascular endothelial growth factor-A (VEGF-A). Expression analyses indicate up-regulation of several kinesin-encoding genes predominantly in lymphoblasts and endothelial cells. Chemical blockade of Eg5 inhibits endothelial cell proliferation and migration in vitro. Mitosis-independent vascular outgrowth in aortic ring cultures is strongly impaired after Eg5 or Mklp2 protein inhibition. In vivo, interfering with KIF11/Eg5 function causes developmental and vascular defects in zebrafish and chick embryos and potent inhibition of tumor angiogenesis in experimental tumor models. Besides blocking tumor cell proliferation, impairing endothelial function is a novel mechanism of action of kinesin inhibitors.

| S-EPMC3926828 | biostudies-other

Alzheimer amyloid beta inhibition of Eg5/kinesin 5 reduces neurotrophin and/or transmitter receptor function.

Project description:The mechanism by which amyloid beta (Aβ) causes neuronal dysfunction and/or death in Alzheimer's disease (AD) is unclear. Previously, we showed that Aβ inhibits several microtubule-dependent kinesin motors essential for mitosis and also present in mature neurons. Here, we show that inhibition of kinesin 5 (Eg5) by Aβ blocks neuronal function by reducing transport of neurotrophin and neurotransmitter receptors to the cell surface. Specifically, cell-surface NGF/NTR(p75) and NMDA receptors decline in cells treated with Aβ or the kinesin 5 inhibitor monastrol, or expressing APP. Aβ and monastrol also inhibit NGF-dependent neurite outgrowth from PC12 cells and glutamate-dependent Ca++ entry into primary neurons. Like Aβ, monastrol inhibits long-term potentiation, a cellular model of NMDA-dependent learning and memory, and kinesin 5 activity is absent from APP/PS transgenic mice brain or neurons treated with Aβ. These data imply that cognitive deficits in AD may derive in part from inhibition of neuronal Eg5 by Aβ, resulting in impaired neuronal function and/or survival through receptor mislocalization. Preventing inhibition of Eg5 or other motors by Aβ may represent a novel approach to AD therapy.

| S-EPMC4084549 | biostudies-literature

Real-time structural transitions are coupled to chemical steps in ATP hydrolysis by Eg5 kinesin.

Project description:At the biochemical level, motor proteins are enzymatic molecules that function by converting chemical energy into mechanical motion. The key element for energy transduction and a major unresolved question common for all motor proteins is the coordination between the chemical and conformational steps in ATP hydrolysis. Here we show time-lapse monitoring of an in vitro ATP hydrolysis reaction by the motor domain of a human Kinesin-5 protein (Eg5) using difference Fourier transform infrared spectroscopy and UV photolysis of caged ATP. In this first continuous observation of a biological reaction coordinate from substrate to product, direct spectral markers for two catalytic events are measured: proton abstraction from nucleophilic water by the catalytic base and formation of the inorganic phosphate leaving group. Simultaneous examination of conformational switching in Eg5, in parallel with catalytic steps, shows structural transitions in solution consistent with published crystal structures of the prehydrolytic and ADP-bound states. In addition, we detect structural modifications in the Eg5 motor domain during bond cleavage between the beta- and gamma-phosphates of ATP. This conclusion challenges mechanochemical models for motor proteins that utilize only two stages of the catalytic cycle to drive force and motion.

| S-EPMC2856982 | biostudies-literature

Half-site inhibition of dimeric kinesin head domains by monomeric tail domains.

Project description:The two heavy chains of kinesin-1 are dimerized through extensive coiled coil regions and fold into an inactive conformation through interaction of the C-terminal tail domains with the N-terminal motor (head) domains. Although this potentially allows a dimer of tail domains to interact symmetrically with a dimer of head domains, we report here that only one of the two available monomeric tail peptides is sufficient for tight binding and inhibition of a dimer of head domains. With a dimeric tail construct, the other tail peptide does not make tight contact with the head dimer and can bind a second head dimer to form a complex containing one tail dimer and two head dimers. The IAK domain and neighboring positively charged region of the tail is sufficient for tight half-site interaction with a dimer of heads. The interaction of tails with monomeric heads is weak, but a head dimer produced by the dimerization of the neck coil is not required because an artificial dimer of head domains also binds monomeric tail peptides with half-site stoichiometry in the complete absence of the native neck coil. The binding of tail peptides to head dimers is fast and readily reversible as determined by FRET between mant-ADP bound to the head dimer and a tail labeled with GFP. The association and dissociation rates are 81 microM(-1) s(-1) and 32 s(-1), respectively. This half-site interaction suggests that the second tail peptide in a folded kinesin-1 might be available to bind other molecules while kinesin-1 remained folded.

| S-EPMC3321547 | biostudies-literature

Kinesin molecular motor Eg5 functions during polypeptide synthesis.

Project description:The kinesin-related molecular motor Eg5 plays roles in cell division, promoting spindle assembly. We show that during interphase Eg5 is associated with ribosomes and is required for optimal nascent polypeptide synthesis. When Eg5 was inhibited, ribosomes no longer bound to microtubules in vitro, ribosome transit rates slowed, and polysomes accumulated in intact cells, suggesting defects in elongation or termination during polypeptide synthesis. These results demonstrate that the molecular motor Eg5 associates with ribosomes and enhances the efficiency of translation.

| S-EPMC3172266 | biostudies-literature

Structural and Thermodynamic Basis of the Enhanced Interaction between Kinesin Spindle Protein Eg5 and STLC-type Inhibitors.

Project description:For a better understanding of protein-inhibitor interactions, we report structural, thermodynamic, and biological analyses of the interactions between S-trityl-l-cysteine (STLC) derivatives and the motor domain of kinesin spindle protein Eg5. Binding of STLC-type inhibitors to Eg5 was enthalpically driven and entropically unfavorable. The introduction of a para-methoxy substituent in one phenyl ring of STLC enhances its inhibitory activity resulting from a larger enthalpy gain possibly due to the increased shape complementarity. The substituent fits to a recess in the binding pocket. To avoid steric hindrance, the substituted STLC is nudged toward the side opposite to the recess, which enhances the interaction of Eg5 with the remaining part of the inhibitor. Further introduction of an ethylene linkage between two phenyl rings enhances Eg5 inhibitory activity by reducing the loss of entropy in forming the complex. This study provides valuable examples of enhancing protein-inhibitor interactions without forming additional hydrogen bonds.

| S-EPMC6644766 | biostudies-literature

Kinesin-14 HSET may not oppose kinesin-5 Eg5 activity in RPE-1 cells.

Project description:Human retinal pigment epithelium RPE-1 cells are immortalized diploid wild-type cells. RPE-1 is increasingly used for studies of spindle assembly dynamics and chromosome segregation. Here, we imaged living RPE-1 cells using the spinning disk confocal microscope and report their complete spindle assembly dynamic parameters. Live-cell experiments enabled ascribing precise timing of function of the kinesin-5 Eg5 and kinesin-14 HSET throughout different phases of mitosis. Eg5 functions at prophase and metaphase, to assemble and maintain spindle bipolarity, respectively. Eg5 inhibition results in spindle collapse during prophase and metaphase, resulting in monoastral/monopolar spindles. HSET functions throughout mitosis to maintain spindle length. HSET degradation results in shorter spindles through all phases of mitosis. Double-inhibition of Eg5 and HSET produces only monoastral/monopolar spindles, indicating that Eg5 and HSET may not be antagonistic in wild-type RPE-1 cells, contrary to previous studies using cancer cells. In the context of spindle assembly, our results highlight potential important differences between RPE-1 and other cancer-derived cell lines.

| S-EPMC9393730 | biostudies-literature

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