Kinetic evidence for low chemical processivity in ncd and Eg5

Crevel, Isabelle; Lockhart, Andrew; Cross, Robert A.

doi:10.1006/jmbi.1997.1319

Cited by 84 publications

(85 citation statements)

References 33 publications

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“…The revised sequence has three amino acid changes from that of the published sequence as Tyr to Ser at position 9, Gly to Pro at position 12, and Met to Ile at position 393. All of the clones used for the work reported here contain the wild-type Pro 12 and Ile 393 . Clones derived from the modified 5Ј-primer above, however, contain Tyr at position 9 instead of Ser because the 5Ј PCR primer was originally designed to match the published sequence.…”

Section: Construction Of Expression Plasmids-thementioning

confidence: 99%

“…Initial kinetic characterization (11) established that the ATPase mechanism of Eg5 was similar to that of conventional kinesin except that it was slower, with ADP release being at least partially ratelimiting and with non-hydrolyzable ATP analogs producing tighter binding to MTs. Unlike conventional kinesin, however, there are indications that Eg5 dimers are not processive (12). Recent work has indicated how the kinetics and state of oligomerization vary with inclusion of increasing regions of the neck coil (7,8).…”

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confidence: 99%

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The Kinesin Family Member BimC Contains a Second Microtubule Binding Region Attached to the N terminus of the Motor Domain

Stock

Chu

Hackney

2003

Journal of Biological Chemistry

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The kinesin family member BimC has a highly positively charged domain of ϳ70 amino acids at the N terminus of the motor domain. Motor domain constructs of BimC were prepared with and without this extra domain to determine its influence. The level of microtubules needed for half saturation of the ATPase of BimC motor domain constructs is reduced by ϳ7000-fold at low ionic strength upon addition of this extra N-terminal extension. Although the change in microtubule affinity is less at higher salt, addition of the N-terminal domain still produces a 20-fold increase in affinity for microtubules in 200 mM potassium acetate. A fusion protein of the N-terminal domain and thioredoxin binds tightly to MTs at low salt, consistent with the increased affinity of motor domain constructs (which contain the N-terminal domain) being due to the additional binding of the N-terminal domain to the microtubule. Hydrodynamic analysis indicates that the N-terminal extension is in a highly extended conformation, suggesting that it may be intrinsically disordered. Fusion of the N-terminal extension of BimC onto the motor domain of conventional kinesin produces a similar large increase in microtubule affinity without significant reduction in k cat or velocity in an in vitro motility assay, suggesting that the N-terminal extension can act in a modular manner to increase the microtubule affinity of kinesin motor domains without a decrease in velocity.

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Section: Construction Of Expression Plasmids-thementioning

confidence: 99%

mentioning

confidence: 99%

The Kinesin Family Member BimC Contains a Second Microtubule Binding Region Attached to the N terminus of the Motor Domain

Stock

Chu

Hackney

2003

Journal of Biological Chemistry

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“…This postulated strain dependence of ADP trapping would provide a mechanism for coordinating neighbouring heads, because opening of the gate which traps ADP on the leading head is synchronized with closure of the equivalent gate on the trailing head. For non-processive kinesins (we believe ncd and eg5 are essentially non-processive (Crevel et al 1997;Stewart et al 1998)), this type of mechanochemical scheme can still apply, but tension signals are passed between heads via the microtubule. The key feature of the scheme, strain-sensitive ADP trapping, works equally well as a coordinating mechanism when the heads are not part of the same molecule.…”

Section: Towards a Mechanochemical Modelmentioning

confidence: 99%

The conformational cycle of kinesin

Cross

Crevel

Carter

et al. 2000

Phil. Trans. R. Soc. Lond. B

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The stepping mechanism of kinesin can be thought of as a programme of conformational changes. We brie£y review protein chemical, electron microscopic and transient kinetic evidence for conformational changes, and working from this evidence, outline a model for the mechanism. In the model, both kinesin heads initially trap Mg¢ADP. Microtubule binding releases ADP from one head only (the trailing head). Subsequent ATP binding and hydrolysis by the trailing head progressively accelerate attachment of the leading head, by positioning it closer to its next site. Once attached, the leading head releases its ADP and exerts a sustained pull on the trailing head. The rate of closure of the molecular gate which traps ADP on the trailing head governs its detachment rate. A speculative but crucial coordinating feature is that this rate is strain sensitive, slowing down under negative strain and accelerating under positive strain.

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“…In contrast, Eg5 is thought to work in small ensembles and need not be processive. A previous study used solution biochemical measurements to estimate the number of ATPase cycles per diffusion-limited encounter with the microtubule (known as 'chemical processivity') and reported that Xenopus laevis Eg5-437-GST dimers were less chemically processive 10 than either kinesin monomers (K340) or Drosophila melanogaster ncd (non-claret disjunctional, a Kinesin-14A family member) dimers (GSTMC5) 11 . Given that single-molecule experiments have directly measured the number of steps taken by single motors while bound to the microtuble (known as 'mechanical processivity') and shown that both kinesin monomers 12 and ncd 13 are mechanically nonprocessive, these chemical processivity data have been widely interpreted as evidence of nonprocessivity for Eg5 (refs 4 , 7, 14 ).…”

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Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro

et al. 2006

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Eg5, a member of the kinesin superfamily of microtubule-based motors, is essential for bipolar spindle assembly and maintenance during mitosis, yet little is known about the mechanisms by which it accomplishes these tasks. Here, we used an automated optical trapping apparatus in conjunction with a novel motility assay that employed chemically modified surfaces to probe the mechanochemistry of Eg5. Individual dimers, formed by a recombinant human construct Eg5-513-5His, stepped processively along microtubules in 8-nm increments, with short run lengths averaging approximately eight steps. By varying the applied load (with a force clamp) and the ATP concentration, we found that the velocity of Eg5 was slower and less sensitive to external load than that of conventional kinesin, possibly reflecting the distinct demands of spindle assembly as compared with vesicle transport. The Eg5-513-5His velocity data were described by a minimal, three-state model where a force-dependent transition follows nucleotide binding.Eg5, a Kinesin-5 (formerly known as BimC) family member, has an unusual homotetrameric protein structure that is formed by the anti-parallel arrangement of heavy chains, with pairs of heads situated at opposite ends of a common stalk 1 . In dividing cells, Eg5 is essential for the organization and maintenance of mitotic and meiotic spindles. Organized bipolar spindles contain microtubule arrays emanating from each of two focused poles, with interdigitated, antiparallel microtubules occupying the mid-zone. Immunodepletion of Eg5 leads to monoastral spindles with disorganized poles and to the disruption of preformed bipolar spindles 2 , and several small-molecule inhibitors of Eg5 have been identified 3, 4 .However, some clues about the function of Eg5 in spindle organization are emerging. Microtubule gliding-filament assays have shown that this motor is plus-end-directed and slow, with reported velocities ranging from 10-40 nm s −1 (refs 2 , 5-7 ). These velocities are similar 6 Correspondence should be addressed to S.M.B. (e-mail: sblock@stanford.edu). 5 These authors contributed equally to this work.Note: Supplementary Information is available on the Nature Cell Biology website. AUTHOR CONTRIBUTIONS P.M.F. and M.T.V. designed, performed and analysed the single-molecule experiments and co-wrote the manuscript. T.C.K. and S.P.G. generated the Eg5 constructs and conducted the biochemical measurements. S.M.B. and S.P.G. supplied guidance, designed research and edited the manuscript. COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests. Despite recent advances, fundamental questions persist, particularly with regard to Eg5 processivity (the average number of sucessive enzymatic cycles carried out while bound to the microtubule substrate). Conventional kinesin is processive and single motors can transport cargo over large distances. In contrast, Eg5 is thought to work in small ensembles and need not be processive. A previous study used solution biochemical measur...

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Kinetic evidence for low chemical processivity in ncd and Eg5

Cited by 84 publications

References 33 publications

The Kinesin Family Member BimC Contains a Second Microtubule Binding Region Attached to the N terminus of the Motor Domain

The Kinesin Family Member BimC Contains a Second Microtubule Binding Region Attached to the N terminus of the Motor Domain

The conformational cycle of kinesin

Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro

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