ATPase Mechanism of Eg5 in the Absence of Microtubules:  Insight into Microtubule Activation and Allosteric Inhibition by Monastrol

Abstract

The ATPase mechanism of kinesin superfamily members in the absence of microtubules remains largely uncharacterized. We have adopted a strategy to purify monomeric human Eg5 (HsKSP/ Kinesin-5) in the nucleotide-free state (apoEg5) in order to perform a detailed transient state kinetic analysis. We have used steady-state and presteady-state kinetics to define the minimal ATPase mechanism for apoEg5 in the absence and presence of the Eg5-specific inhibitor, monastrol. ATP and ADP binding both occur via a two-step process with the isomerization of the collision complex limiting each forward reaction. ATP hydrolysis and phosphate product release are rapid steps in the mechanism, and the observed rate of these steps is limited by the relatively slow isomerization of the Eg5-ATP collision complex. A conformational change coupled to ADP release is the rate-limiting step in the pathway. We propose that the microtubule amplifies and accelerates the structural transitions needed to form the ATP hydrolysis competent state and for rapid ADP release, thus stimulating ATP turnover and increasing enzymatic efficiency. Monastrol appears to bind weakly to the Eg5-ATP collision complex, but after tight ATP binding, the affinity for monastrol increases, thus inhibiting the conformational change required for ADP product release. Taken together, we hypothesize that loop L5 of Eg5 undergoes an "open" to "closed" structural transition that correlates with the rearrangements of the switch-1 and switch-2 regions at the active site during the ATPase cycle.Motor proteins from the myosin, kinesin, and dynein superfamilies are important molecular machines that utilize the energy of ATP turnover to generate force and perform various functions in eukaryotic cells. These enzymes coordinate movements of conserved structural elements located at the nucleotide binding site (P-loop, switch-1, switch-2) with structural elements that interact with the filament surface (actin-or microtubule-binding interface) (1)(2)(3)(4)(5)(6)(7)(8). The ATPase activity and enzymatic efficiency of these molecular motors are activated in the presence of their filament partner, which is thought to be mediated through acceleration of the rate of product release (reviewed in ref 9). However, the structural basis for this phenomenon is not well understood.The ATPase mechanisms of several different monomeric kinesins have been extensively studied in the presence of microtubules: conventional kinesin/Kinesin-1 (10-12), Eg5/ Kinesin-5 (13), and Ncd/Kar3/ . On the other hand, very little is known about the ATPase mechanism of kinesins in the absence of microtubules (17). Historically, kinesins have been purified with ADP bound to the nucleotide binding site (18), and attempts † This work was supported by Grant GM54141 from NIGMS, National Institutes of Health (NIH), and through Career Development Award K02-AR47841 from NIAMS, NIH, Department of Health and Human Services (to S.P.G.). to isolate a homogeneous, nucleotide-free population have been difficult due to the in...

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

“…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).…”

“…We measured values for the MT-activated k cat and K 0.5,MT of mBBr-labeled Eg5 W127C of 5.1 Ϯ 0.5 s Ϫ1 and 0.26 Ϯ 0.12 M, which are very similar to the corresponding values for a monomeric wild type Eg5 construct (18).…”

“…The fluorescence of this tryptophan is quenched by the binding of small molecule inhibitors of Eg5 that have a strong near UV absorbance (18), and a previous study has used this effect to measure the kinetics of ispinesib binding to L5 (16). We repeated this experiment with our Cys-light, Eg5 V365C mutant to confirm that the Cys-light mutations have not appreciably altered the kinetics of binding.…”

“…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.…”

Allosteric Drug Discrimination Is Coupled to Mechanochemical Changes in the Kinesin-5 Motor Core

“…However, conformational transitions of the L5 loop are found not only in response to drug binding but also in normal motor function. The Eg5 conformer trapped by allosteric agents is proposed to be an intermediate state of its normal ATP hydrolysis cycle (25). The L5 loop is observed in the closed conformation in cryo-electron microscopy experiments with a Drosophila melanogaster homologue of Eg5 bound to microtubules using non-hydrolyzable substrate analogues (26) and in Eg5⅐AMPPNP crystals in the absence of allosteric agents (27).…”