Abstract
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, ATP-triggered 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 rear-head-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.
Highlights
IntroductionA 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]
Achieving this goal requires first characterizing the chemomechanical cycle of kinesin-5 as it steps along the microtubule lattice, and this characterization in turn requires understanding family-specific properties of the individual heads
The goal of the present study was to uncover aspects of the kinesin-5 chemomechanical cycle that contribute to this end binding and polymerase activity
Summary
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]. The motor pauses for ϳ7 s at the plus-end of both taxol-stabilized and dynamic microtubules, a property that is assumed to relate to its polymerase activity
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