Microtubule cross-linking triggers the directional motility of kinesin-5

Although assembly of the mitotic spindle is known to be a precisely controlled process, regulation of the key motor proteins involved remains poorly understood. In eukaryotes, homotetrameric kinesin-5 motors are required for bipolar spindle formation. Eg5, the vertebrate kinesin-5, has two modes of motion: an adenosine triphosphate (ATP)-dependent directional mode and a diffusive mode that does not require ATP hydrolysis. We use single-molecule experiments to examine how the switching between these modes is controlled. We find that Eg5 diffuses along individual microtubules without detectable directional bias at close to physiological ionic strength. Eg5's motility becomes directional when bound between two microtubules. Such activation through binding cargo, which, for Eg5, is a second microtubule, is analogous to known mechanisms for other kinesins. In the spindle, this might allow Eg5 to diffuse on single microtubules without hydrolyzing ATP until the motor is activated by binding to another microtubule. This mechanism would increase energy and filament cross-linking efficiency.     

The Effect of Monastrol on the Processive Motility of a Dimeric Kinesin-5 Head/Kinesin-1 Stalk Chimera

Controlled activity of several kinesin motors is required for the proper assembly of the mitotic spindle. Eg5, a homotetrameric bipolar kinesin-5 from Xenopus laevis, can cross-link and slide anti-parallel microtubules apart by a motility mechanism comprising diffusional and directional modes. How this mechanism is regulated, possibly by the tail domains of the opposing motors, is poorly understood. In order to explore the basic unregulated kinesin-5 motor activity, we generated a stably dimeric kinesin-5 construct, Eg5Kin, consisting of the motor domain and neck linker of Eg5 and the neck coiled coil of Drosophila melanogaster kinesin-1 (DmKHC). In single-molecule motility assays, we found this chimera to be highly processive. In addition, we studied the effect of the kinesin-5-specific inhibitor monastrol using single-molecule fluorescence assays. We found that monastrol reduced the length of processive runs, but strikingly did not affect velocity. Quantitative analysis of monastrol dose dependence suggests that two bound monastrol molecules are required to be bound to an Eg5Kin dimer to terminate a run.     

ATP-competitive inhibitors of the mitotic kinesin KSP that function via an allosteric mechanism

The mitotic kinesin KSP (kinesin spindle protein, or Eg5) has an essential role in centrosome separation and formation of the bipolar mitotic spindle. Its exclusive involvement in the mitotic spindle of proliferating cells presents an opportunity for developing new anticancer agents with reduced side effects relative to antimitotics that target tubulin. Ispinesib is an allosteric small-molecule KSP inhibitor in phase 2 clinical trials. Mutations that attenuate ispinesib binding to KSP have been identified, which highlights the need for inhibitors that target different binding sites. We describe a new class of selective KSP inhibitors that are active against ispinesib-resistant forms of KSP. These ATP-competitive KSP inhibitors do not bind in the nucleotide binding pocket. Cumulative data from generation of resistant cells, site-directed mutagenesis and photo-affinity labeling suggest that they compete with ATP binding via a novel allosteric mechanism.     

The loop 5 element structurally and kinetically coordinates dimers of the human kinesin-5, Eg5

Eg5 is a homotetrameric kinesin-5 motor protein that generates outward force on the overlapping, antiparallel microtubules (MTs) of the mitotic spindle. Upon binding an MT, an Eg5 dimer releases one ADP molecule, undergoes a slow (∼0.5 s⁻¹) isomerization, and finally releases a second ADP, adopting a tightly MT-bound, nucleotide-free (APO) conformation. This conformation precedes ATP binding and stepping. Here, we use mutagenesis, steady-state and pre-steady-state kinetics, motility assays, and electron paramagnetic resonance spectroscopy to examine Eg5 monomers and dimers as they bind MTs and initiate stepping. We demonstrate that a critical element of Eg5, loop 5 (L5), accelerates ADP release during the initial MT-binding event. Furthermore, our electron paramagnetic resonance data show that L5 mediates the slow isomerization by preventing Eg5 dimer heads from binding the MT until they release ADP. Finally, we find that Eg5 having a seven-residue deletion within L5 can still hydrolyze ATP and move along MTs, suggesting that L5 is not required to accelerate subsequent steps of the motor along the MT. Taken together, these properties of L5 explain the kinetic effects of L5-directed inhibition on Eg5 activity and may direct further interventions targeting Eg5 activity.     

Discovery and biochemical characterization of selective ATP competitive inhibitors of the human mitotic kinesin KSP

The kinesin spindle protein (KSP, also known as Eg5) is essential for the proper separation of spindle poles during mitosis, and inhibition results in mitotic arrest and the formation of characteristic monoaster spindles. Several distinct classes of KSP inhibitors have been described previously in the public and patent literature. However, most appear to share a common induced-fit allosteric binding site, suggesting a common mechanism of inhibition. In a high-throughput screen for inhibitors of KSP, a novel class of thiazole-containing inhibitors was identified. Unlike the previously described allosteric KSP inhibitors, the thiazoles described here show ATP competitive kinetic behavior, consistent with binding within the nucleotide binding pocket. Although they bind to a pocket that is highly conserved across kinesins, these molecules exhibit significant selectivity for KSP over other kinesins and other ATP-utilizing enzymes. Several of these compounds are active in cells and produce a phenotype similar to that observed with previously published allosteric inhibitors of KSP.     

The Structural Basis of Force Generation by the Mitotic Motor Kinesin-5

Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved distinct properties for its spindle-based functions. In this study, we report subnanometer resolution cryoelectron microscopy reconstructions of microtubule-bound human kinesin-5 before and after nucleotide binding and combine this information with studies of the kinetics of nucleotide-induced neck linker and cover strand movement. These studies reveal coupled, nucleotide-dependent conformational changes that explain many of this motor''s properties. We find that ATP binding induces a ratchet-like docking of the neck linker and simultaneous, parallel docking of the N-terminal cover strand. Loop L5, the binding site for allosteric inhibitors of kinesin-5, also undergoes a dramatic reorientation when ATP binds, suggesting that it is directly involved in controlling nucleotide binding. Our structures indicate that allosteric inhibitors of human kinesin-5, which are being developed as anti-cancer therapeutics, bind to a motor conformation that occurs in the course of normal function. However, due to evolutionarily defined sequence variations in L5, this conformation is not adopted by invertebrate kinesin-5s, explaining their resistance to drug inhibition. Together, our data reveal the precision with which the molecular mechanism of kinesin-5 motors has evolved for force generation.     

Getting in sync with dimeric Eg5. Initiation and regulation of the processive run

Eg5/KSP is the kinesin-related motor protein that generates the major plus-end directed force for mitotic spindle assembly and dynamics. Recent work using a dimeric form of Eg5 has found it to be a processive motor; however, its mechanochemical cycle is different from that of conventional Kinesin-1. Dimeric Eg5 appears to undergo a conformational change shortly after collision with the microtubule that primes the motor for its characteristically short processive runs. To better understand this conformational change as well as head-head communication during processive stepping, equilibrium and transient kinetic approaches have been used. By contrast to the mechanism of Kinesin-1, microtubule association triggers ADP release from both motor domains of Eg5. One motor domain releases ADP rapidly, whereas ADP release from the other occurs after a slow conformational change at approximately 1 s(-1). Therefore, dimeric Eg5 begins its processive run with both motor domains associated with the microtubule and in the nucleotide-free state. During processive stepping however, ATP binding and potentially ATP hydrolysis signals rearward head advancement 16 nm forward to the next microtubule-binding site. This alternating cycle of processive stepping is proposed to terminate after a few steps because the head-head communication does not sufficiently control the timing to prevent both motor domains from entering the ADP-bound state simultaneously.     

The role of Kinesin neck linker and neck in velocity regulation

Despite the high level of similarity in structural organisation of their motor domains and, consequently, in the mechanism of motility generation, kinesin-5 moves about 25-fold slower than conventional kinesin (kinesin-1). To elucidate the structural motifs contributing to velocity regulation, we expressed a set of Eg5- and KIF5A-based chimeric proteins with interchanged native neck linker and neck elements. Among them, the chimera consisting of the Eg5 catalytic core (residues 1-357) fused to the KIF5A linker and neck (residues 326-450) displayed increased velocity compared to the Eg5 control protein. This is the first evidence that the velocity of the slow-moving motor Eg5 can be elevated by insertion of neck linker and neck elements taken from a fast-moving motor. Whereas the complementary chimera composed of the KIF5A core (1-325) and the Eg5 linker and neck (358-513) was found to be immotile, insertion of the first half-KIF5A linker into this chimera restored motility. Our results indicate that the neck linker and the neck are involved not only in motility generation in general and in determination of movement direction, but also in velocity regulation.     

A structural model for monastrol inhibition of dimeric kinesin Eg5

Eg5 or KSP is a homotetrameric Kinesin-5 involved in centrosome separation and assembly of the bipolar mitotic spindle. Analytical gel filtration of purified protein and cryo-electron microscopy (cryo-EM) of unidirectional shadowed microtubule-Eg5 complexes have been used to identify the stable dimer Eg5-513. The motility assays show that Eg5-513 promotes robust plus-end-directed microtubule gliding at a rate similar to that of homotetrameric Eg5 in vitro. Eg5-513 exhibits slow ATP turnover, high affinity for ATP, and a weakened affinity for microtubules when compared to monomeric Eg5. We show here that the Eg5-513 dimer binds microtubules with both heads to two adjacent tubulin heterodimers along the same microtubule protofilament. Under all nucleotide conditions tested, there were no visible structural changes in the monomeric Eg5-microtubule complexes with monastrol treatment. In contrast, there was a substantial monastrol effect on dimeric Eg5-513, which reduced microtubule lattice decoration. Comparisons between the X-ray structures of Eg5-ADP and Eg5-ADP-monastrol with rat kinesin-ADP after docking them into cryo-EM 3-D scaffolds revealed structural evidence for the weaker microtubule-Eg5 interaction in the presence of monastrol.     

Force and Premature Binding of ADP Can Regulate the Processivity of Individual Eg5 Dimers

Using a high-resolution optical trapping instrument, we directly observed the processive motions of individual Eg5 dimers over a range of external loads and ATP, ADP, and phosphate concentrations. To constrain possible models for dissociation from the microtubule, we measured Eg5 run lengths and also compared the duration of the last step of a processive run to all previous step durations. We found that the application of large longitudinal forces in either hindering or assisting directions could induce Eg5-microtubule dissociation. At a constant moderate force, maintained with a force clamp, the premature binding of ADP strongly promoted microtubule release by Eg5, whereas the addition of ATP or phosphate had little effect on dissociation. These results imply that run length is determined not only by the load, but also by the concentration and type of nucleotides present, and therefore that the biochemical cycles of the two motor domains of the Eg5 dimer are coordinated to promote processive stepping.     

Disparity in allosteric interactions of monastrol with Eg5 in the presence of ADP and ATP: a difference FT-IR investigation

Eg5 is a kinesin-like motor protein required for mitotic progression in higher eukaryotes. It is thought to cross-link antiparallel microtubules, and provides a force required for the formation of a bipolar spindle. Monastrol causes the catastrophic collapse of the mitotic spindle through the allosteric inhibition of Eg5. Utilizing a truncated Eg5 protein, we employ difference infrared spectroscopy to probe structural changes that occur in the motor protein with monastrol in the presence of either ADP or ATP. Difference FT-IR spectra of Eg5-monastrol-nucleotide complexes demonstrate that there are triggered conformational changes corresponding to an interconversion of secondary structural elements in the motor upon interaction with nucleotides. Notably, conformational changes elicited in the presence of ADP are different from those in the presence of ATP. In Eg5-monastrol complexes, exchange of ADP is associated with a decrease in random structure and an increase in alpha-helical content. In contrast, formation of the Eg5-monastrol-ATP complex is associated with a decrease in alpha-helical content and a concomitant increase in beta-sheet content. One or more carboxylic acid residues in Eg5 undergo unique changes when ATP, but not ADP, interacts with the motor domain in the presence of monastrol. This first direct dissection of inhibitor-protein interactions, using these methods, demonstrates a clear disparity in the structural consequences of monastrol in the presence of ADP versus ATP.     

Molecular dissection of the inhibitor binding pocket of mitotic kinesin Eg5 reveals mutants that confer resistance to antimitotic agents

The mitotic kinesin Eg5 plays an essential role in establishing the bipolar spindle. Recently, several antimitotic inhibitors have been shown to share a common binding region on Eg5. Considering the importance of Eg5 as a potential drug target for cancer chemotherapy it is essential to understand the molecular mechanism, by which these agents block Eg5 activity, and to determine the "key residues" crucial for inhibition. Eleven residues in the inhibitor binding pocket were mutated and the effects were monitored by kinetic analysis and mass spectrometry. Mutants R119A, D130A, P131A, I136A, V210A, Y211A and L214A abolish the inhibitory effect of monastrol. Results for W127A and R221A are less striking, but inhibitor constants are still considerably modified compared to wild-type Eg5. Only one residue, Leu214, was found to be essential for inhibition by STLC. W127A, D130A, V210A lead to increased K(i)(app) values, but binding of STLC is still tight. R119A, P131A, Y211A and R221A convert STLC into a classical rather than a tight-binding inhibitor with increased inhibitor constants. These results demonstrate that monastrol and STLC interact with different amino acids within the same binding region, suggesting that this site is highly flexible to accommodate different types of inhibitors. The drug specificity is due to multiple interactions not only with loop L5, but also with residues located in helices alpha2 and alpha3. These results suggest that tumour cells might develop resistance to Eg5 inhibitors, by expressing Eg5 point mutants that retain the enzyme activity, but prevent inhibition, a feature that is observed for certain tubulin inhibitors.     

Loop 5-directed compounds inhibit chimeric kinesin-5 motors: implications for conserved allosteric mechanisms

The human Eg5 (HsEg5) protein is unique in its sensitivity to allosteric agents even among phylogenetic kin. For example, S-trityl-l-cysteine (STC) and monastrol are HsEg5 inhibitors that bind to a surface pocket created by the L5 loop, but neither compound inhibits the Drosophila Kinesin-5 homologue (Klp61F). Herein we ask whether or not drug sensitivity can be designed into Klp61F. Two chimeric Klp61F motor domains were engineered, bacterially expressed, and purified to test this idea. We report that effector binding can elicit a robust allosteric response comparable with HsEg5 in both motor domain chimeras. Furthermore, isothermal titration calorimetry confirms that the Klp61F chimeras have de novo binding affinities for both STC and monastrol. These data show that the mechanism of intramolecular communication between the three ligand binding sites is conserved in the Kinesin-5 family, and reconstitution of a drug binding cassette within the L5 pocket is sufficient to restore allosteric inhibition. However, the two compounds were not equivalent in their allosteric inhibition. This surprising disparity in the response between the chimeras to monastrol and STC suggests that there is more than one allosteric communication network for these effectors.     

The Conserved L5 Loop Establishes the Pre-Powerstroke Conformation of the Kinesin-5 Motor, Eg5

Kinesin superfamily motor proteins contain a structurally conserved loop near the ATP binding site, termed L5. The function of L5 is unknown, although several drug inhibitors of the mitotic kinesin Eg5 bind to L5. We used electron paramagnetic resonance spectroscopy (EPR) to investigate the function of L5 in Eg5. We site-specifically attached EPR probes to ADP, L5, and the neck linker element that docks along the enzymatic head to drive forward motility on microtubules (MTs). Nucleotide-dependent spectral mobility shifts occurred in all of these structural elements, suggesting that they undergo coupled conformational changes. These spectral shifts were altered by deletion of L5 or addition of S-trityl-l-cysteine (STLC), an allosteric inhibitor that binds to L5. In particular, EPR probes attached to the neck linker of MT-bound Eg5 shifted to a more immobilized component in the nucleotide-free state relative to the ADP-bound state, consistent with the neck linker docking upon ADP release. In contrast, after L5 deletion or STLC addition, EPR spectra were highly immobilized in all nucleotide states. We conclude that L5 undergoes a conformational change that enables Eg5 to bind to MTs in a pre-powerstroke state. Deletion or inhibition of L5 with the small-molecule inhibitor STLC blocks this pre-powerstroke state, forcing the Eg5 neck linker to dock regardless of the nucleotide state.     

Arp2/3 phosphorylation kickstarts cells

The kinesin-13 motor protein family members drive the removal of tubulin from microtubules (MTs) to promote MT turnover. A point mutation of the kinesin-13 family member mitotic centromere-associated kinesin/Kif2C (E491A) isolates the tubulin-removal conformation of the motor, and appears distinct from all previously described kinesin-13 conformations derived from nucleotide analogues. The E491A mutant removes tubulin dimers from stabilized MTs stoichiometrically in adenosine triphosphate (ATP) but is unable to efficiently release from detached tubulin dimers to recycle catalytically. Only in adenosine diphosphate (ADP) can the mutant catalytically remove tubulin dimers from stabilized MTs because the affinity of the mutant for detached tubulin dimers in ADP is low relative to lattice-bound tubulin. Thus, the motor can regenerate for further cycles of disassembly. Using the mutant, we show that release of tubulin by kinesin-13 motors occurs at the transition state for ATP hydrolysis, which illustrates a significant divergence in their coupling to ATP turnover relative to motile kinesins.     

Kinesin’s Neck-Linker Determines its Ability to Navigate Obstacles on the Microtubule Surface

The neck-linker is a structurally conserved region among most members of the kinesin superfamily of molecular motor proteins that is critical for kinesin’s processive transport of intracellular cargo along the microtubule surface. Variation in the neck-linker length has been shown to directly modulate processivity in different kinesin families; for example, kinesin-1, with a shorter neck-linker, is more processive than kinesin-2. Although small differences in processivity are likely obscured in vivo by the coupling of most cargo to multiple motors, longer and more flexible neck-linkers may allow different kinesins to navigate more efficiently around the many obstacles, including microtubule-associated proteins (MAPs), that are found on the microtubule surface within cells. We hypothesize that, due to its longer neck-linker, kinesin-2 can more easily navigate obstacles (e.g., MAPs) on the microtubule surface than kinesin-1. We used total internal reflection fluorescence microscopy to observe single-molecule motility from different kinesin-1 and kinesin-2 neck-linker chimeras stepping along microtubules in the absence or presence of two Tau isoforms, 3RS-Tau and 4RL-Tau, both of which are MAPs that are known to differentially affect kinesin-1 motility. Our results demonstrate that unlike kinesin-1, kinesin-2 is insensitive to the presence of either Tau isoform, and appears to have the ability to switch protofilaments while stepping along the microtubule when challenged by an obstacle, such as Tau. Thus, although kinesin-1 may be more processive, the longer neck-linker length of kinesin-2 allows it to be better optimized to navigate the complex microtubule landscape. These results provide new insight, to our knowledge, into how kinesin-1 and kinesin-2 may work together for the efficient delivery of cargo in cells.     

Effects of kinesin-5 inhibition on dendritic architecture and microtubule organization

Kinesin-5 is a slow homotetrameric motor protein best known for its essential role in the mitotic spindle, where it limits the rate at which faster motors can move microtubules. In neurons, experimental suppression of kinesin-5 causes the axon to grow faster by increasing the mobility of microtubules in the axonal shaft and the invasion of microtubules into the growth cone. Does kinesin-5 act differently in dendrites, given that they have a population of minus end–distal microtubules not present in axons? Using rodent primary neurons in culture, we found that inhibition of kinesin-5 during various windows of time produces changes in dendritic morphology and microtubule organization. Specifically, dendrites became shorter and thinner and contained a greater proportion of minus end–distal microtubules, suggesting that kinesin-5 acting normally restrains the number of minus end–distal microtubules that are transported into dendrites. Additional data indicate that, in neurons, CDK5 is the kinase responsible for phosphorylating kinesin-5 at Thr-926, which is important for kinesin-5 to associate with microtubules. We also found that kinesin-5 associates preferentially with microtubules rich in tyrosinated tubulin. This is consistent with an observed accumulation of kinesin-5 on dendritic microtubules, as they are known to be less detyrosinated than axonal microtubules.     

A kinetic dissection of the fast and superprocessive kinesin-3 KIF1A reveals a predominant one-head-bound state during its chemomechanical cycle

The kinesin-3 family contains the fastest and most processive motors of the three neuronal transport kinesin families, yet the sequence of states and rates of kinetic transitions that comprise the chemomechanical cycle and give rise to their unique properties are poorly understood. We used stopped-flow fluorescence spectroscopy and single-molecule motility assays to delineate the chemomechanical cycle of the kinesin-3, KIF1A. Our bacterially expressed KIF1A construct, dimerized via a kinesin-1 coiled-coil, exhibits fast velocity and superprocessivity behavior similar to WT KIF1A. We established that the KIF1A forward step is triggered by hydrolysis of ATP and not by ATP binding, meaning that KIF1A follows the same chemomechanical cycle as established for kinesin-1 and -2. The ATP-triggered half-site release rate of KIF1A was similar to the stepping rate, indicating that during stepping, rear-head detachment is an order of magnitude faster than in kinesin-1 and kinesin-2. Thus, KIF1A spends the majority of its hydrolysis cycle in a one-head-bound state. Both the ADP off-rate and the ATP on-rate at physiological ATP concentration were fast, eliminating these steps as possible rate-limiting transitions. Based on the measured run length and the relatively slow off-rate in ADP, we conclude that attachment of the tethered head is the rate-limiting transition in the KIF1A stepping cycle. Thus, KIF1A''s activity can be explained by a fast rear-head detachment rate, a rate-limiting step of tethered-head attachment that follows ATP hydrolysis, and a relatively strong electrostatic interaction with the microtubule in the weakly bound post-hydrolysis state.     

Diffusion and directed movement: in vitro motile properties of fission yeast kinesin-14 Pkl1

Fission yeast Pkl1 is a kinesin-14A family member that is known to be localized at the cellular spindle and is capable of hydrolyzing ATP. However, its motility has not been detected. Here, we show that Pkl1 is a slow, minus end-directed microtubule motor with a maximum velocity of 33+/-9 nm/s. The Km,MT value of steady-state ATPase activity of Pkl1 was as low as 6.4+/-1.1 nM, which is 20-30 times smaller than that of kinesin-1 and another kinesin-14A family member, Ncd, indicating a high affinity of Pkl1 for microtubules. However, the duty ratio of 0.05 indicates that Pkl1 spends only a small fraction of the ATPase cycle strongly associated with a microtubule. By using total internal reflection fluorescence microscopy, we demonstrated that single molecules of Pkl1 were not highly processive but only exhibited biased one-dimensional diffusion along microtubules, whereas several molecules of Pkl1, probably fewer than 10 molecules, cooperatively moved along microtubules and substantially reduced the diffusive component in the movement. Our results suggest that Pkl1 molecules work in groups to move and generate forces in a cooperative manner for their mitotic functions.     

Subunits interactions in kinesin motors

Kinesins form a large and diverse superfamily of proteins involved in numerous important cellular processes. The majority of them are molecular motors moving along microtubules. Conversion of chemical energy into mechanical work is accomplished in a sequence of events involving both biochemical and conformational alternation of the motor structure called the mechanochemical cycle. Different members of the kinesin superfamily can either perform their function in large groups or act as single molecules. Conventional kinesin, a member of the kinesin-1 subfamily, exemplifies the second type of motor which requires tight coordination of the mechanochemical cycle in two identical subunits to accomplish processive movement toward the microtubule plus end. Recent results strongly support an asymmetric hand-over-hand model of "walking" for this protein. Conformational strain between two subunits at the stage of the cycle where both heads are attached to the microtubule seems to be a major factor in intersubunit coordination, although molecular and kinetic details of this phenomenon are not yet deciphered. We discuss also current knowledge concerning intersubunit coordination in other kinesin subfamilies. Members of the kinesin-3 class use at least three different mechanisms of movement and can translocate in monomeric or dimeric forms. It is not known to what extent intersubunit coordination takes place in Ncd, a dimeric member of the kinesin-14 subfamily which, unlike conventional kinesin, exercises a power-stroke toward the microtubule minus end. Eg5, a member of the kinesin-5 subfamily is a homotetrameric protein with two kinesin-1-like dimeric halves controlled by their relative orientation on two microtubules. It seems that diversity of subunit organization, quaternary structures and cellular functions in the kinesin superfamily are reflected also by the divergent extent and mechanism of intersubunit coordination during kinesin movement along microtubules.