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J. Biol. Chem., Vol. 280, Issue 13, 12658-12667, April 1, 2005

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Monastrol Inhibition of the Mitotic Kinesin Eg5^*

Jared C. Cochran, Joseph E. Gatial, III, Tarun M. Kapoor, and Susan P. Gilbert¶

From the Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 and the Laboratory of Chemistry and Cell Biology, Rockefeller University, New York, New York 10021

Monastrol is a small, cell-permeable molecule that arrests cells in mitosis by specifically inhibiting Eg5, a member of the Kinesin-5 family. We have used steady-state and presteady-state kinetics as well as equilibrium binding approaches to define the mechanistic basis of S-monastrol inhibition of monomeric human Eg5/KSP. In the absence of microtubules (Mts), the basal ATPase activity is inhibited through slowed product release. In the presence of microtubules, the ATPase activity is also reduced with weakened binding of Eg5 to microtubules during steady-state ATP turnover. Monastrol-treated Eg5 also shows a decreased relative affinity for microtubules under equilibrium conditions. The Mt·Eg5 presteady-state kinetics of ATP binding and the subsequent ATP-dependent isomerization are unaffected during the first ATP turnover. However, monastrol appears to stabilize a conformation that allows for reversals at the ATP hydrolysis step. Monastrol promotes a dramatic decrease in the observed rate of Eg5 association with microtubules, and ADP release is slowed without trapping the Mt·Eg5·ADP intermediate. We propose that S-monastrol binding to Eg5 induces a stable conformational change in the motor domain that favors ATP re-synthesis after ATP hydrolysis. The aberrant interactions with the microtubule and the reversals at the ATP hydrolysis step alter the ability of Eg5 to generate force, thereby yielding a nonproductive Mt·Eg5 complex that cannot establish or maintain the bipolar spindle.

Received for publication, November 22, 2004 , and in revised form, January 18, 2005.

^* 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.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biological Sciences, University of Pittsburgh, 518 Langley Hall, 4249 Fifth Ave., Pittsburgh, PA 15260. Tel.: 412-624-5842; Fax: 412-624-4759; E-mail: spg1{at}pitt.edu.

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