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

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, 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.

Eg5 Inhibitors Have Contrasting Effects on Microtubule Stability and Metaphase Spindle Integrity

“…At limiting ADP concentrations the MT unbinding rate is near zero (confirming that the apo-state binds tightly), and at saturating ADP concentrations the MT unbinding rate plateaus; the ADP concentration at which the rate is half-maximal represents the K D for ADP binding to the Eg5-MT species 40 (see Figure 3c inset). Under control conditions, Eg5 had a maximal MT-unbinding rate of 0.55 ± 0.02 s −1 and an ADP affinity of K D ADP = 86 ± 7 μM ADP (Figure 3c), in agreement with previous work 14 . In the presence of BRD9876, the effective nucleotide affinity dropped 25-fold (K D apparent = 2.2 ± 1.1 mM ADP), while the maximal off-rate remained at 0.52 ± 0.18 s −1 (Figure 3c).…”

“…To analyze the mean-square displacement (MSD), either a quadratic form ( MSD(Δt) = v 2 Δt 2 + 2DΔt + σ 2 ) or a linear form ( MSD(Δt) = 2DΔt + σ 2 ) was assigned based on the presence or the absence of stepping. In saturating ATP, the motor had a velocity of 66 ± 26 nm/s and a negligible diffusion coefficient of 594 ± 167 nm 2 /s, consistent with our previous findings that Eg5 dimers step processively in ATP 14 . In contrast, in saturating ADP the motor diffused along the microtubule with D = 3,845 ± 119 nm 2 /s.…”

“…As seen in Figure 1c, uniform populations of KHC moved MT at ~700 nm/s 18 , uniform populations of Eg5 moved MT at ~70 nm/s 14 , and a minor fraction of Eg5 (22%) was sufficient to slow velocity to the Eg5 speed, consistent with this motor’s documented “braking” ability 12,15,20,38 . In 100 μM STLC, the MT gliding activity of Eg5 was abolished at high fractions of Eg5, consistent with full motor inhibition.…”

“…Using stopped-flow, we confirmed that STLC and other L5 inhibitors slow the Eg5 MT-stimulated mADP release by greater than 20-fold (Figure S3) and we also observed that STLC and filanesib, which had the strongest mechanical inhibition in the mixed motor gliding assay (Figure 1d) most strongly inhibited the MT-stimulated mADP release rate. The finding that STLC leads to a higher diffusion constant in ADP than in ATP can be interpreted either that the STLC-mediated diffusion in ATP occurs in the ADP-Pi state 14 or that STLC-inhibited Eg5 spends approximately half of its cycle in a high affinity state in ATP.…”

Mechanical coupling of microtubule-dependent motor teams during peroxisome transport in Drosophila S2 cells

“…Dimeric Eg5(513) is characterized by its low processivity (∼70 nm) and speed (< 100 nm/s). Previous works also showed that the stall force of this motor [35,51] and the kinetic constants of disociation from and association to microtubules [52–54] are similar to those of kinesin-1.…”