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Dissecting kinesin motor function with small-molecule inhibitors

Record Type:	Electronic resources : Monograph/item
Title/Author:	Dissecting kinesin motor function with small-molecule inhibitors
Author:	Maliga, Zoltan.
Description:	133 p.
Notes:	Adviser: Timothy J. Mitchison.
Notes:	Source: Dissertation Abstracts International, Volume: 65-05, Section: B, page: 2295.
Contained By:	Dissertation Abstracts International65-05B.
Subject:	Biophysics, General.
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3131918
ISBN:	0496791583

Dissecting kinesin motor function with small-molecule inhibitors
Maliga, Zoltan.

Dissecting kinesin motor function with small-molecule inhibitors [electronic resource] - 133 p.

Adviser: Timothy J. Mitchison.

Thesis (Ph.D.)--Harvard University, 2004.

Kinesin motors comprise a diverse number of related proteins required for intracellular transport and cell division. Dissecting the role of kinesin motors is limited by the specific reagents, antibodies and RNAi, currently used to disrupt protein function. Specific small-molecule inhibitors of kinesin motor proteins can be useful mechanistic and biochemical probes to dissect motor function in complex biological systems. The first example of such a reagent, monastrol, is a cell-permeable drug that specifically blocks the motor activity of the mitotic kinesin Eg5, causing mitotic arrest and spindle collapse in vivo. In this study, we set out to investigate the mechanism by which monastrol inhibits the motor activity of the Eg5 mitotic kinesin using a combination of biochemistry and X-ray crystallography. Whereas previous kinesin inhibitors compete with ATP (Kapoor & Mitchison, 1999) or microtubule binding (Sakowicz et al., 1998; Hopkins et al, 2000), monastrol binds to the motor domain of Eg5, blocking the ATP hydrolysis cycle by a non-competitive mechanism. We solved the X-ray crystal structure of Eg5 in a ternary complex with monastrol and ADP, revealing a novel allosteric binding pocket that is targeted by monastrol. The crystal structure is consistent with the structure-activity relationship (SAR) of monastrol and mutational analysis of the drug binding site. The Eg5 motor domain adopts a neck-linker docked conformation that was previously associated with a rigid microtubule-bound state. Surprisingly, microtubule co-sedimentation experiments shows that Eg5 binds microtubules poorly in the presence of monastrol, indicating that spindle collapse may be a direct result of microtubule release in vivo. To identify novel and specific reagents to study kinesin function in mitosis, we performed a pure protein, high-throughput screen for inhibitors of microtubule-stimulated ATP hydrolysis by the motor domains of Eg5, CenpE, and MKLP1. We identified a novel class of specific Eg5 inhibitors that are more potent than monastrol and inhibit Eg5 by a similar mechanism.

ISBN: 0496791583Subjects--Topical Terms:

226901
Biophysics, General.

Dissecting kinesin motor function with small-molecule inhibitors
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502 $a Thesis (Ph.D.)--Harvard University, 2004.
520 # $a Kinesin motors comprise a diverse number of related proteins required for intracellular transport and cell division. Dissecting the role of kinesin motors is limited by the specific reagents, antibodies and RNAi, currently used to disrupt protein function. Specific small-molecule inhibitors of kinesin motor proteins can be useful mechanistic and biochemical probes to dissect motor function in complex biological systems. The first example of such a reagent, monastrol, is a cell-permeable drug that specifically blocks the motor activity of the mitotic kinesin Eg5, causing mitotic arrest and spindle collapse in vivo. In this study, we set out to investigate the mechanism by which monastrol inhibits the motor activity of the Eg5 mitotic kinesin using a combination of biochemistry and X-ray crystallography. Whereas previous kinesin inhibitors compete with ATP (Kapoor & Mitchison, 1999) or microtubule binding (Sakowicz et al., 1998; Hopkins et al, 2000), monastrol binds to the motor domain of Eg5, blocking the ATP hydrolysis cycle by a non-competitive mechanism. We solved the X-ray crystal structure of Eg5 in a ternary complex with monastrol and ADP, revealing a novel allosteric binding pocket that is targeted by monastrol. The crystal structure is consistent with the structure-activity relationship (SAR) of monastrol and mutational analysis of the drug binding site. The Eg5 motor domain adopts a neck-linker docked conformation that was previously associated with a rigid microtubule-bound state. Surprisingly, microtubule co-sedimentation experiments shows that Eg5 binds microtubules poorly in the presence of monastrol, indicating that spindle collapse may be a direct result of microtubule release in vivo. To identify novel and specific reagents to study kinesin function in mitosis, we performed a pure protein, high-throughput screen for inhibitors of microtubule-stimulated ATP hydrolysis by the motor domains of Eg5, CenpE, and MKLP1. We identified a novel class of specific Eg5 inhibitors that are more potent than monastrol and inhibit Eg5 by a similar mechanism.
520 # $a Our work provides a biochemical and structural basis for the specific interaction of monastrol with the Eg5 motor domain. In addition, our results suggest that monastrol-induced spindle collapse is due to microtubule release by Eg5.
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