Microtubule-based transport by the kinesin motors, powered by ATP hydrolysis, is essential for a wide range of vital processes in eukaryotes. We obtained insight into this process by developing atomic models for no-nucleotide and ATP states of the monomeric kinesin motor domain on microtubules from cryo-EM reconstructions at 5–6 Å resolution. By comparing these models with existing X-ray structures of ADP-bound kinesin, we infer a mechanistic scheme in which microtubule attachment, mediated by a universally conserved ‘linchpin’ residue in kinesin (N255), triggers a clamshell opening of the nucleotide cleft and accompanying release of ADP. Binding of ATP re-closes the cleft in a manner that tightly couples to translocation of cargo, via kinesin's ‘neck linker’ element. These structural transitions are reminiscent of the analogous nucleotide-exchange steps in the myosin and F1-ATPase motors and inform how the two heads of a kinesin dimer ‘gate’ each other to promote coordinated stepping along microtubules.DOI: http://dx.doi.org/10.7554/eLife.04686.001

Monastrol is a small, cell-permeable molecule that arrests cells in mitosis by specifically inhibiting Eg5, a member of the Kinesin-5 family. We have used steady-state and presteady-state kinetics as well as equilibrium binding approaches to define the mechanistic basis of S-monastrol inhibition of monomeric human Eg5/KSP. In the absence of microtubules (Mts), the basal ATPase activity is inhibited through slowed product release. In the presence of microtubules, the ATPase activity is also reduced with weakened binding of Eg5 to microtubules during steadystate ATP turnover. Monastrol-treated Eg5 also shows a decreased relative affinity for microtubules under equilibrium conditions. The Mt⅐Eg5 presteady-state kinetics of ATP binding and the subsequent ATP-dependent isomerization are unaffected during the first ATP turnover. However, monastrol appears to stabilize a conformation that allows for reversals at the ATP hydrolysis step. Monastrol promotes a dramatic decrease in the observed rate of Eg5 association with microtubules, and ADP release is slowed without trapping the Mt⅐Eg5⅐ADP intermediate. We propose that S-monastrol binding to Eg5 induces a stable conformational change in the motor domain that favors ATP re-synthesis after ATP hydrolysis. The aberrant interactions with the microtubule and the reversals at the ATP hydrolysis step alter the ability of Eg5 to generate force, thereby yielding a nonproductive Mt⅐Eg5 complex that cannot establish or maintain the bipolar spindle.Accurate segregation of replicated chromosomes during cell division depends on the correct assembly and proper maintenance of the bipolar spindle (reviewed in Refs. 1-15). Several members of the kinesin superfamily localize to the mitotic spindle, and inhibition of one or more of these motors leads to drastic spindle abnormalities and loss of normal chromosome segregation (reviewed in Refs. 3,[16][17][18]. Monastrol is a reversible, cell-permeable, small molecule that selectively inhibits the plus-end-directed Kinesin-5 family member, Eg5 (19 -23), a microtubule-based motor protein that is required for the formation and maintenance of the bipolar spindle (24 -26). On the other hand, monastrol does not bind to or inhibit the ATPase activity of other well studied kinesin superfamily members (22,27). Monastrol treatment of dividing cells results in spindle collapse and cell cycle arrest with a monoastral spindle, which is similar to the phenotype observed when Eg5 is inhibited by anti-Eg5 antibodies (20,24,25,28). Recently, Eg5 inhibitors with nanomolar affinity have been identified (29 -31).Previous studies with monastrol have revealed an inducedfit, allosteric binding site outside the nucleotide binding pocket of the protein (21-23, 31). By comparing the monastrol-treated Eg5 crystal structure (Eg5 S ⅐ADP) 1 (23) to the Eg5⅐ADP structure (32), monastrol appears to induce dramatic conformational changes throughout the catalytic core, including transformation of the insertion loop (L5) of helix ␣2 and the neck-linker/ switch II cluster wit...

Eg5 is a slow, plus-end-directed microtubule-based motor of the BimC kinesin family that is essential for bipolar spindle formation during eukaryotic cell division. We have analyzed two human Eg5/KSP motors, Eg5-367 and Eg5-437, and both are monomeric based on results from sedimentation velocity and sedimentation equilibrium centrifugation as well as analytical gel filtration. The steady-state parameters were: for Eg5-367: Eukaryotic cell division requires proper assembly and maintenance of the bipolar spindle, an intricate protein complex composed of a dynamic array of microtubules and microtubulebased motor proteins (reviewed in Refs.

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...

The flexible tubulin C-terminal tails (CTTs) have recently been implicated in the walking mechanism of dynein and kinesin. To address their role in the case of conventional kinesin, we examined the structure of kinesin-microtubule (MT) complexes before and after CTT cleavage by subtilisin. Our results show that the CTTs directly modulate the motor-tubulin interface and the binding properties of motors. CTT cleavage increases motor binding stability, and kinesin appears to adopt a binding conformation close to the nucleotide-free configuration under most nucleotide conditions. Moreover, C-terminal cleavage results in trapping a transient motor-ADP-MT intermediate. Using SH3-tagged dimeric and monomeric constructs, we could also show that the position of the kinesin neck is not affected by the C-terminal segments of tubulin. Overall, our study reveals that the tubulin C-termini define the stability of the MT-kinesin complex in a nucleotidedependent manner, and highlights the involvement of tubulin in the regulation of weak and strong kinesin binding states.

Structural maintenance of chromosomes (SMC) complexes shape the genomes of virtually all organisms, but how they function remains incompletely understood. Recent studies in bacteria and eukaryotes have led to a unifying model in which these ring-shaped ATPases act along contiguous DNA segments, processively enlarging DNA loops. In support of this model, single-molecule imaging experiments indicate that Saccharomyces cerevisiae condensin complexes can extrude DNA loops in an ATP-hydrolysis-dependent manner in vitro. Here, using time-resolved high-throughput chromosome conformation capture (Hi-C), we investigate the interplay between ATPase activity of the Bacillus subtilis SMC complex and loop formation in vivo. We show that point mutants in the SMC nucleotide-binding domain that impair but do not eliminate ATPase activity not only exhibit delays in de novo loop formation but also have reduced rates of processive loop enlargement. These data provide in vivo evidence that SMC complexes function as loop extruders.

SUMMARY Segregation of nonexchange chromosomes during Drosophila melanogaster meiosis requires the proper function of NOD, a nonmotile Kinesin-10. We have determined the X-ray crystal structure of the NOD catalytic domain in the ADP1- and AMPPNP-bound states. These structures reveal an alternate conformation of the microtubule binding region as well as a nucleotide-sensitive relay of hydrogen bonds at the active site. Additionally, a cryo-electron microscopy reconstruction of the nucleotide-free microtubule-NOD complex shows an atypical binding orientation. Thermodynamic studies show NOD binds tightly to microtubules in the nucleotide-free state, yet other nucleotide states, including AMPPNP, are weakened. Our presteady-state kinetic analysis demonstrates that NOD interaction with microtubules occurs slowly with weak activation of ADP product release. Upon rapid substrate binding, NOD detaches from the microtubule prior to the rate-limiting step of ATP hydrolysis, which is also atypical for a kinesin. We propose a model for NOD’s microtubule plus-end tracking that drives chromosome movement.

Kinesin-5 family members including human Eg5/KSP contribute to the plus-end-directed force necessary for the assembly and maintenance of the bipolar mitotic spindle. We have used monomeric Eg5-367 in the nucleotide-free state to evaluate the role of microtubules at each step in the ATPase cycle. The pre-steady-state kinetic results show that the microtubule-Eg5 complex binds MgATP tightly, followed by rapid ATP hydrolysis with a subsequent slow step that limits steady-state turnover. We show that microtubules accelerate the kinetics of each step in the ATPase pathway, suggesting that microtubules amplify the nucleotide-dependent structural transitions required for force generation. The experimentally determined rate constants for phosphate product release and Eg5 detachment from the microtubule were similar, suggesting that these two steps are coupled with one occurring at the slow rate after ATP hydrolysis followed by the second step occurring more rapidly. The rate of this slow step correlates well with the steady-state k cat , indicative that it is the rate-limiting step of the mechanism.Kinesins are cytoskeletal motor proteins that utilize the energy from the ATPase cycle to perform mechanical work along microtubules. Conversion of the nucleotide state at the motor active site triggers conformational changes in the motor, which dictates the affinity of the motor for its filament and ultimately results in force generation. Although the specific structural events linking the ATP hydrolysis cycle to molecular motion are unknown, a mechanism for the pathway of conformational change that is responsible for amplifying small movements at the nucleotide binding site to large-scale changes in distant regions of the motor is now emerging.Kinesins share an ~350 amino acid motor domain that contains the nucleotide binding site and the microtubule binding region (reviewed in refs 1 and 2 and also see the kinesin homepage: http://www.proweb.org/kinesin/). A third region that is conserved within kinesin subfamilies is the ~14-20 amino acid sequence adjacent to the motor domain called the neck linker. Together, the motor domain and the neck linker make up the core domain, and numerous studies have investigated the ATPase mechanism of different kinesin monomers in the presence of microtubules including Kinesin-1/conventional kinesin (3-8), Kinesin-5/Eg5/BimC (9-11), and Kinesin-14/Ncd/Kar3/(12-15). The structural differences that arise between various kinesin subfamilies have fine-tuned the rate and equilibrium constants that govern the overall mechanochemical cycles. Therefore, each kinesin motor elicits a different work output that is † This work was supported by NIH Grant GM54141 from NIGMS and NIH K02-AR47841 Career Development Award from NIAMS to S.P.G. * Corresponding author. . E-mail: spg1@pitt.edu. ‡ Present address: Department of Chemistry, Dartmouth College, Hanover, NH 03755.1 Abbreviations: Eg5-367, human Eg5/KSP motor domain containing the N-terminal 367 residues followed by a C-terminal His6 tag; Eg5-43...