Loop L5 Acts as a Conformational Latch in the Mitotic Kinesin Eg5

Behnke-Parks, William M.; Vendôme, Jérémie; Maliga, Zoltan; Moores, Carolyn A.; Rosenfeld, Steven

doi:10.1016/j.bpj.2010.12.3096

Cited by 13 publications

(42 citation statements)

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“…The k cat for the MT-activated ATPase for this construct (8.9 Ϯ 0.7 s Ϫ1 ) is in the general range of values reported for wild type monomeric Eg5 constructs (5.5-8.9 s Ϫ1 ) (3,18). We labeled the V356C construct with the fluorescent donor 2Ј-deoxy-3Ј-mant-ADP (2ЈdmD) and fluorescent acceptor Oregon Green 488 maleimide (Molecular Probes) for FRET measurements as described previously (13).…”

Section: Methodssupporting

confidence: 63%

“…A logical candidate for such a structural element is L5. Point or deletion mutations of this loop uncouple nucleotide binding from NL docking (3,5), and cryo-EM reconstructions of Eg5-decorated MTs demonstrate that in rigor, the N-terminal portion of L5 is in a position to sterically block nucleotide binding (12).…”

Section: L5 Is a Kinetic Regulator Of The Catalytic Site And Nl-ourmentioning

confidence: 99%

“…Whereas this work has provided important mechanistic insights, both crystallography and cryo-EM provide "static" pictures of discrete conformational states that do not reveal the speed or the sequence by which these state changes occur. Likewise, whereas transient kinetic studies of kinesin motors, including Eg5, have provided important information on the sequence of state changes in the ATPase cycle (3,17,18,21), they do not necessarily reveal the nature of the structural transitions whose timing they monitor. We have therefore combined both approaches to generate a series of structural "snapshots" of the mitotic kinesin Eg5.…”

Section: Small Molecule Inhibitors Provide Insight Into the Physiologmentioning

confidence: 99%

“…To do this, we generated a series of single and double cysteine-containing Eg5 mutants that our previous work had shown behave enzymatically in a manner very similar to wild type Eg5 monomeric constructs (3,12).…”

Section: Small Molecule Inhibitors Provide Insight Into the Physiologmentioning

confidence: 99%

“…S1B), which coordinate the ␤-and ␥-phosphates, respectively, of bound ATP. Second, point mutations in L5 affect ATP and microtubule (MT) affinity (3,4), and molecular dynamics simulations of these mutations reveal that L5 modulates the degree of flexibility of switch I and the P-loop. Finally, deleting L5 enhances the mobility of spin-labeled ADP in the active site and uncouples nucleotide binding from NL docking (5).…”

mentioning

confidence: 99%

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“Snapshots” of Ispinesib-induced Conformational Changes in the Mitotic Kinesin Eg5

Kaan

Major

Tkocz

et al. 2013

Journal of Biological Chemistry

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Section: Methodssupporting

confidence: 63%

Section: L5 Is a Kinetic Regulator Of The Catalytic Site And Nl-ourmentioning

confidence: 99%

Section: Small Molecule Inhibitors Provide Insight Into the Physiologmentioning

confidence: 99%

Section: Small Molecule Inhibitors Provide Insight Into the Physiologmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

“Snapshots” of Ispinesib-induced Conformational Changes in the Mitotic Kinesin Eg5

Kaan

Major

Tkocz

et al. 2013

Journal of Biological Chemistry

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Src family kinase phosphorylation of the motor domain of the human kinesin‐5, Eg5

et al. 2017

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Spindle formation in mammalian cells requires precise spatial and temporal regulation of the kinesin-5, Eg5, which generates outward force to establish spindle bipolarity. Our results demonstrate that Eg5 is phosphorylated in cultured cells by Src family kinases (SFKs) at three sites in the motor head: Y125, Y211, and Y231. Mutation of these sites diminishes motor activity in vitro, and replacement of endogenous Eg5 with phosphomimetic Y211 in LLC-Pk1 cells results in monopolar spindles, consistent with loss of Eg5 activity. Cells treated with SFK inhibitors show defects in spindle formation, similar to those in cells expressing the non-phosphorylatable Y211 mutant, and distinct from inhibition of other mitotic kinases. We propose that this phosphoregulatory mechanism tunes Eg5 enzymatic activity for optimal spindle morphology.

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Mechanisms of Action of Eg5 Inhibitors

Cross

2015

Kinesins and Cancer

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The kinetic mechanism of Eg5 is broadly similar to the widely studied kinesin-1 mechanism, but with some distinct features (Waitzman and Rice 2013 ). As with all molecular motors, the most stable state (the ground state) is the apo state, in which the active site is empty and the motor binds strongly (stably) and stereospecifi cally to its microtubule (MT) track. The subsequent steps of MgATP turnover progressively destabilise MT binding. The initial binding of MgATP or MgAMPPNP (a non-hydrolysable analog) into the empty active site drives the motor into a new conformational state, but this new motor·MgADP state binds to MTs only slightly less stably than the apo (empty) state, so that the motor head can still hold substantial force. The subsequent catalytic steps of hydrolysis and Pi release generate a motor·MgADP state that tends to detach rapidly from the MT. Rebinding of this motor·MgADP state to MTs triggers MgADP release, regenerating the apo state. In the motor·MgADP state, spontaneous release of MgADP from the active site is extremely slow, ~0.005 s −1 , and rate-limiting for ATP turnover in the absence of MTs. The weak binding motor·MgADP state is a "slip-state" that slides along the MT almost without friction if force is applied [ 1 ]. Nonetheless, this motor·MgADP slip-state is crucial because it provides access to a transient strong binding motor·MgADP state from which MgADP release is very fast: around three orders of magnitude faster than in the absence of MTs. The MT binding surface of the motor head is on the opposite side of the central beta sheet backbone to the active site, so that much of the structural mechanism of MT activation is necessarily allosteric. As I discuss below, the binding of allosteric inhibitors to the Eg5 motor head adds further stability to the motor·MgADP state and thereby inhibits MT-activated MgADP release. The crucial point therefore is that inhibitors and MTs have

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Loop L5 Acts as a Conformational Latch in the Mitotic Kinesin Eg5

Cited by 13 publications

References 0 publications

“Snapshots” of Ispinesib-induced Conformational Changes in the Mitotic Kinesin Eg5

“Snapshots” of Ispinesib-induced Conformational Changes in the Mitotic Kinesin Eg5

Src family kinase phosphorylation of the motor domain of the human kinesin‐5, Eg5

Mechanisms of Action of Eg5 Inhibitors

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