Mechanistic Analysis of the Mitotic Kinesin Eg5

Cochran, Jared C.; Sontag, Christopher A.; Maliga, Zoltan; Kapoor, Tarun M.; Correia, John J.; Gilbert, Susan P.

doi:10.1074/jbc.m404203200

Cited by 87 publications

(144 citation statements)

References 86 publications

(74 reference statements)

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“…The apoEg5 protein preparations were tested for activity by measuring the steadystate ATPase kinetics as a function of microtubule concentration at saturating MgATP, and as a function of MgATP concentration at saturating microtubule concentration (Table 1). These experiments demonstrated that, under the same experimental conditions as previous studies (13), apoEg5 retained 95-99% of its microtubule-activated ATPase activity. In addition, pulsechase, acid-quench, and P i release experiments were performed using the purified apoEg5 in the presence of microtubules, and we observed full burst amplitude from each transient, demonstrating that the entire population of apoEg5 sites was fully active.…”

Section: Apoeg5 Was Active and Nucleotide-freesupporting

confidence: 80%

“…Figure 2 shows that the maximum observed rate of MgATP turnover by apoEg5 was 0.02 s −1 , similar to former Eg5 preparations (13), with a very tight K m,ATP at 0.17 μM. The K m,ATP was greater than 40-fold tighter in the absence of microtubules compared to microtubule-activated steady-state ATPase: 0.17 μM (apoEg5) versus 6.95 μM (Mt·Eg5) ( Table 1).…”

Section: Steady-state Atpase Reveals Tight Atp Binding Yet Inefficiesupporting

confidence: 72%

“…We determined the apoEg5 protein concentration by the Bio-Rad protein assay with IgG as the standard. This purification strategy yielded >99% pure protein with >95% basal and microtubule-activated ATPase activity under the same experimental conditions when compared to prior Eg5 purifications [ Table 1 ( 13)]. In addition, we performed pulse-chase, acid-quench, and P i product release experiments with apoEg5 in the presence of microtubules, and observed a burst stoichiometry near unity ( Figure 8B-D), suggesting that the entire population of apoEg5 enzyme binds and hydrolyzes ATP during the first ATP turnover.…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

“…Our previous Eg5-367 purification strategy yielded a population of motors that retained MgADP bound at the active site (13,22). The modifications (see Materials and Methods for details) were sufficient to isolate apoEg5 at >99% purity.…”

Section: Apoeg5 Was Active and Nucleotide-freementioning

confidence: 99%

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ATPase Mechanism of Eg5 in the Absence of Microtubules: Insight into Microtubule Activation and Allosteric Inhibition by Monastrol

Cochran

Gilbert

2005

Biochemistry

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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.Motor proteins from the myosin, kinesin, and dynein superfamilies are important molecular machines that utilize the energy of ATP turnover to generate force and perform various functions in eukaryotic cells. These enzymes coordinate movements of conserved structural elements located at the nucleotide binding site (P-loop, switch-1, switch-2) with structural elements that interact with the filament surface (actin-or microtubule-binding interface) (1)(2)(3)(4)(5)(6)(7)(8). The ATPase activity and enzymatic efficiency of these molecular motors are activated in the presence of their filament partner, which is thought to be mediated through acceleration of the rate of product release (reviewed in ref 9). However, the structural basis for this phenomenon is not well understood.The ATPase mechanisms of several different monomeric kinesins have been extensively studied in the presence of microtubules: conventional kinesin/Kinesin-1 (10-12), Eg5/ Kinesin-5 (13), and Ncd/Kar3/ . On the other hand, very little is known about the ATPase mechanism of kinesins in the absence of microtubules (17). Historically, kinesins have been purified with ADP bound to the nucleotide binding site (18), and attempts † This work was supported by Grant GM54141 from NIGMS, National Institutes of Health (NIH), and through Career Development Award K02-AR47841 from NIAMS, NIH, Department of Health and Human Services (to S.P.G.). to isolate a homogeneous, nucleotide-free population have been difficult due to the in...

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Section: Apoeg5 Was Active and Nucleotide-freesupporting

confidence: 80%

Section: Steady-state Atpase Reveals Tight Atp Binding Yet Inefficiesupporting

confidence: 72%

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

Section: Apoeg5 Was Active and Nucleotide-freementioning

confidence: 99%

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ATPase Mechanism of Eg5 in the Absence of Microtubules: Insight into Microtubule Activation and Allosteric Inhibition by Monastrol

Cochran

Gilbert

2005

Biochemistry

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“…Labeling stoichiometries of 1.7-1.9 were achieved for both constructs (data not shown). The MT-activated k cat for TMR-labeled V256C,V365C was 5.1 Ϯ 1.6 s Ϫ1 , whereas that for TMR-labeled A2C,V256C was 4.5 Ϯ 0.9 s Ϫ1 , both reasonably close to the corresponding value for wild type (33). Additionally, labeling with the FRET donor/acceptor pair AEDANS-DDPM was accomplished by incubating the motor domain construct with 0.2 mol eq of 1,5-IAEDANS for 24 h, followed by incubation with a 10-fold molar excess of DDPM.…”

Section: Methodssupporting

confidence: 48%

The Structural Basis of Force Generation by the Mitotic Motor Kinesin-5

Goulet

Behnke-Parks

Sindelar

et al. 2012

Journal of Biological Chemistry

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Background: Kinesin-5 motors are important for formation and maintenance of the bipolar mitotic spindle. Results: ATP binding triggers coupled conformational changes of kinesin-5 specific structural elements in the microtubulebound motor domain. Conclusion: Kinesin-5 mechanochemistry is tuned to its cellular functions. Significance: Subnanometer resolution structure determination of microtubule-bound kinesin-5s and kinetics experiments reveal the molecular basis of their motor properties and of drug inhibition.

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Synergistic Anticancer Effect of Peptide‐Docetaxel Nanoassembly Targeted to Tubulin: Toward Development of Dual Warhead Containing Nanomedicine

Mohapatra

Saha

Mondal

et al. 2016

Adv Healthcare Materials

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Microtubule dynamics play a crucial role in cancer cell division. Various drugs are developed to target microtubule. Although a few of them show potential in treatment of cancer, but success rate is limited due to their poor bioavailability and lack of specificity. Thus, development of highly bioavailable and target specific anticancer drug is extremely necessary. To address these key issues, here, a combination of approaches such as development of a dodecapeptide-docetaxel nanoassembly targeted to tubulin and MUC1 (mucin 1, cell surface associated glycoprotein) targeting oligonucleotide aptamer conjugated liposome for delivering peptide-docetaxel nanoassembly into the breast cancer cell have been demonstrated. These studies reveal that the peptide forms nanoassembly and entraps docetaxel drug. Further, the liposomal formulation of peptide-docetaxel exerts synergistic anticancer effect, activates key mitotic check point proteins, and inhibits bipolar spindle formation, metastatic cancer cell migration, and growth of tumor mimicking 3D multicellular spheroid.

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Mechanistic Analysis of the Mitotic Kinesin Eg5

Cited by 87 publications

References 86 publications

ATPase Mechanism of Eg5 in the Absence of Microtubules: Insight into Microtubule Activation and Allosteric Inhibition by Monastrol

ATPase Mechanism of Eg5 in the Absence of Microtubules: Insight into Microtubule Activation and Allosteric Inhibition by Monastrol

The Structural Basis of Force Generation by the Mitotic Motor Kinesin-5

Synergistic Anticancer Effect of Peptide‐Docetaxel Nanoassembly Targeted to Tubulin: Toward Development of Dual Warhead Containing Nanomedicine

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