Examining kinesin processivity within a general gating framework

Andreasson, Johan O. L.; Milic, Bojan; Chen, Geng Yuan; Guydosh, Nicholas R.; Hancock, William O.; Block, Steven M.

doi:10.7554/elife.07403

Cited by 172 publications

(340 citation statements)

References 78 publications

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“…Our results are most consistent with a two-step model of the powerstroke in which ATP binding only partially docks the NL and hydrolysis completes docking to accelerate tethered head attachment (19,41,48). This model contrasts with the conventional model of ATP binding driving full NL docking (34,39).…”

Section: Discussionsupporting

confidence: 76%

“…S1), strongly suggesting that under physiological conditions, hydrolysis occurs before tethered head attachment. Milic and coworkers recently showed that processivity under assisting load is not increased in ATPγS but is increased by adding phosphate to the media, in support of ATP hydrolysis occurring before tethered head attachment (19,41). We extend that result by showing that in a zero-load assay the run length in ATPγS is not higher than the run length in ATP (Fig.…”

Section: Discussionsupporting

confidence: 72%

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Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

Mickolajczyk

Deffenbaugh

Arroyo

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

124

222

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To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the onehead-bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.kinesin | iSCAT | microscopy | structural kinetics | structure-function K inesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1-6).Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7-10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4,11,12), consistent with the motor walking in a handover-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7-10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head-bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads-bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4,11,(14)(15)(16)(17)(18)(19)(20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7,19,21,22) and sin...

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Section: Discussionsupporting

confidence: 76%

Section: Discussionsupporting

confidence: 72%

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

Mickolajczyk

Deffenbaugh

Arroyo

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

124

222

View full text Add to dashboard Cite

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“…The measured Mt binding and ADP release rates are similar to rates measured previously (21), but the tethered head in State 3 had a relatively low ADP affinity of 1.8 M. We found previously for kinesin-1 that extending the neck linker domain from wild-type 14 residues to 18 residues (which matches the native neck linker of kinesin-5; Ref. 40) results in the rate of tethered head ADP exchange increasing from 0.4 s Ϫ1 to 1.3 s Ϫ1 (18). The measured kinesin-5 tethered head ADP off-rate of 1.3 s Ϫ1 (based on mADP exchange with ADP in Fig.…”

Section: Discussionsupporting

confidence: 72%

“…1d), there is no evidence of rearhead gating, meaning that interhead strain does not appear to accelerate trailing head detachment. A recent optical trapping study using kinesin-1 with extended neck linkers suggested that the interhead tension present in wild-type kinesin is relieved when the neck linker is extended from 14 to 15 or more residues (18), in agreement with this lack of rear-head gating of kinesin-5 (which has an 18 residues neck linker). Based on the tethered head exchange results (ADP in Fig.…”

mentioning

confidence: 55%

“…For instance, a 4-piconewton assisting load that reduced kinesin-1 run lengths by roughly 10-fold only reduced kinesin-5 run length by a factor of 2 (18,19), and in mixed motor gliding assays kinesin-5 motors were able to efficiently slow the transport of microtubules driven by faster transport motors (20). This ability to resist loads is consistent with the proposed role of kinesin-5 as a "brake" that stabilizes microtubule bundles in axons; motor inhibition leads to faster axonal outgrowth and longer branches in culture (7).…”

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confidence: 99%

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The Kinesin-5 Chemomechanical Cycle Is Dominated by a Two-heads-bound State

Chen

Mickolajczyk

Hancock

2016

Journal of Biological Chemistry

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Single-molecule microscopy and stopped-flow kinetics assays were carried out to understand the microtubule polymerase activity of kinesin-5 (Eg5). Four lines of evidence argue that the motor primarily resides in a two-heads-bound (2HB) state. First, upon microtubule binding, dimeric Eg5 releases both bound ADPs. Second, microtubule dissociation in saturating ADP is 20-fold slower for the dimer than for the monomer. Third, ATPtriggered mant-ADP release is 5-fold faster than the stepping rate. Fourth, ATP binding is relatively fast when the motor is locked in a 2HB state. Shortening the neck-linker does not facilitate rear-head detachment, suggesting a minimal role for rearhead-gating. This 2HB state may enable Eg5 to stabilize incoming tubulin at the growing microtubule plus-end. The finding that slowly hydrolyzable ATP analogs trigger slower nucleotide release than ATP suggests that ATP hydrolysis in the bound head precedes stepping by the tethered head, leading to a mechanochemical cycle in which processivity is determined by the race between unbinding of the bound head and attachment of the tethered head.

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Processivity of dimeric kinesin‐1 molecular motors

et al. 2018

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Kinesin‐1 is a homodimeric motor protein that can move along microtubule filaments by hydrolyzing ATP with a high processivity. How the two motor domains are coordinated to achieve such high processivity is not clear. To address this issue, we computationally studied the run length of the dimer with our proposed model. The computational data quantitatively reproduced the puzzling experimental data, including the dramatically asymmetric character of the run length with respect to the direction of external load acting on the coiled‐coil stalk, the enhancement of the run length by addition of phosphate, and the contrary features of the run length for different types of kinesin‐1 with extensions of their neck linkers compared with those without extension of the neck linker. The computational data on other aspects of the movement dynamics such as velocity and durations of one‐head‐bound and two‐head‐bound states in a mechanochemical coupling cycle were also in quantitative agreement with the available experimental data. Moreover, predicted results are provided on dependence of the run length upon external load acting on one head of the dimer, which can be easily tested in the future using single‐molecule optical trapping assays.

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Examining kinesin processivity within a general gating framework

Cited by 172 publications

References 78 publications

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

The Kinesin-5 Chemomechanical Cycle Is Dominated by a Two-heads-bound State

Processivity of dimeric kinesin‐1 molecular motors

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