Cochran, Jared Clinton
(2006)
Investigation of the Mechanochemical Cycle of the Mitotic Kinesin Eg5.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Cytoskeletal motor proteins utilize the energy from ATP turnover to perform work along their filament tracks. Eg5 is a microtubule-based motor of the Kinesin-5/BimC family that is essential for bipolar spindle formation during eukaryotic cell division. I have performed a detailed kinetic analysis to define the minimal Eg5 ATPase mechanism. Eg5 association with the microtubule, ADP product release, and ATP binding are rapid steps in the mechanism, while ATP hydrolysis, phosphate product release, and detachment from the microtubule occur relatively slowly. Phosphate release coupled to Mt•Eg5 dissociation represents the rate-limiting step. Therefore, Eg5 remains tightly associated with the microtubule during most of the ATPase cycle, thus adapting the motor for its function within the mitotic spindle.Monastrol is a small molecule that specifically inhibits Eg5. I performed experiments to define the mechanistic basis for Eg5 inhibition by monastrol. The ATPase activity of Eg5 is reduced with weakened binding to microtubules. Monastrol also stabilizes a subpopulation of "non-productive" complexes that slowly hydrolyze ATP. After ATP hydrolysis, phosphate product is rapidly released coupled to detachment from the microtubule. Therefore, monastrol inhibits Eg5 force generation to yield a motor that cannot function properly in the mitotic spindle.I have purified Eg5 in the nucleotide-free state to perform a detailed kinetic analysis in the absence of microtubules. 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. These studies provide insight into how the microtubule guides the structural transitions needed to form the ATP hydrolysis-competent state and for rapid ADP release. In addition, 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.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
17 March 2006 |
| Date Type: |
Completion |
| Defense Date: |
18 November 2005 |
| Approval Date: |
17 March 2006 |
| Submission Date: |
28 November 2005 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Biological Sciences |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Adenosine triphosphatase/chemistry/metabolism; Adenosine triphosphate/chemistry; Dose-Response Relationship/Drug; Eg5; enzyme kinetics; inhibition; kinesin; KSP; monastrol; Adenosine diphosphate/chemistry; BimC; Humans; Hydrolysis; Kinesin-5; KSP inhibitor; mitosis; mitotic spindle; nucleotide; protein conformation; protein binding |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-11282005-113458/, etd-11282005-113458 |
| Date Deposited: |
10 Nov 2011 20:06 |
| Last Modified: |
15 Nov 2016 13:52 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/9822 |
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