Inhibition of a Mitotic Motor Protein: Where, How, and Conformational Consequences

Yan, Yong-Bin; Sardana, Vinod; Xu, Bei; Homnick, Carl F.; Halczenko, Wasyl; Buser, Carolyn A.; Schaber, Michael D.; Hartman, George D.; Huber, Hans E.; Kuo, Lawrence C.

doi:10.1016/j.jmb.2003.10.074

Cited by 201 publications

(267 citation statements)

References 22 publications

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“…Thus, we hypothesize that the movement of loop L5 is tightly correlated to Eg5's mechanochemical cycle. A recent study demonstrated that if loop L5 from Eg5 were replaced with a homologous loop from Kinesin-1, the ATPase in the absence of microtubules decreased ~2-fold, thus supporting our hypothesis (32).All Eg5 crystal structures solved to date contain ADP at the active site (21,31,32). Interestingly, the Eg5·ADP· inhibitor crystal structures display an altered conformation of loop L5 compared to the Eg5·ADP structure.…”

supporting

confidence: 82%

“…Experiments containing monastrol were performed using the more active S-enantiomer (19)(20)(21)(22).…”

Section: Experimental Conditionsmentioning

confidence: 99%

“…All Eg5 crystal structures solved to date contain ADP at the active site (21,31,32). Interestingly, the Eg5·ADP· inhibitor crystal structures display an altered conformation of loop L5 compared to the Eg5·ADP structure.…”

Section: Movement Of Loop L5 Correlates With Movement Of the Switch Rmentioning

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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supporting

confidence: 82%

“…Experiments containing monastrol were performed using the more active S-enantiomer (19)(20)(21)(22).…”

Section: Experimental Conditionsmentioning

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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“…Thus, the issue of target specificity is of great importance for inhibitors targeting Eg5. Structural analyses of the interaction between the Eg5 motor domain and various inhibitors such as monastrol, its analogues and other A c c e p t e d M a n u s c r i p t inhibitor scaffolds have been recently carried out [25][26][27][28][29]. These ligands bind in a unique pocket in the Eg5 motor domain formed by the secondary elements helix 2/loop L5 (Tyr125-Glu145) and helix 3 (Ile202-Leu227) (shown in Fig.…”

Section: Introductionmentioning

confidence: 99%

Mutations in the human kinesin Eg5 that confer resistance to monastrol and S-trityl-l-cysteine in tumor derived cell lines

Tcherniuk

Lis

Kozielski

et al. 2010

Biochemical Pharmacology

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. Mutations in the human kinesin Eg5 that confer resistance to monastrol and S-Trityl-L-Cysteine in tumor derived cell lines.. Biochemical Pharmacology, Elsevier, 2010, 79 (6) This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. confirms the target specificity of monastrol and STLC for Eg5. These data also suggest a possible mechanism by which drug resistance may occur in tumors treated with agents targeting Eg5.

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“…Structures solved using x-ray crystallography have provided residue-by-residue views of the human K5 MD, including highlighting potential conformational variations in the K5 NL and L5 (11,15,20,21). In parallel, spectroscopic measurements have provided dynamic data concerning the timing and regulation of conformational changes within the MD and have been correlated with K5 enzyme kinetics (12,14,22).…”

mentioning

confidence: 99%

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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Inhibition of a Mitotic Motor Protein: Where, How, and Conformational Consequences

Cited by 201 publications

References 22 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

Mutations in the human kinesin Eg5 that confer resistance to monastrol and S-trityl-l-cysteine in tumor derived cell lines

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

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