Eg5 Motor Domain Research Articles

Intra-Motor Domain Coupling is a Strong Driver of Eg5 Motor Activity

The tetrameric kinesin family member Eg5 walks a pair of motor domains along each of two antiparallel microtubules to help set up the mitotic spindle. Previous work has identified a strong coupling between the conformations of two structurally distant elements of the Eg5 motor domain: loop 5 (L5) and the motility-generating neck-linker. However, this work was performed in isolated monomers and how applicable these results are to higher-order assemblies of Eg5 remains unknown. Using electron paramagnetic resonance (EPR) spectroscopy, we have determined the structural relationships between the neck-linkers, L5s and nucleotide binding pockets of isolated Eg5 dimers. Intra-motor domain coupling appears to be a much stronger driver of conformation than inter-motor domain coordination_as was observed in the monomer, the major determinant of neck-linker conformation is the nucleotide state. The docking of the neck-linker upon ejection of ADP is conserved from the monomer to the dimer, and this conformational change is dependent upon the presence of an intact L5. We are currently investigating the effects of disrupting neck-linker-motor domain coupling on dimer motility. Our work forms a basis for future studies of motor domain coordination and provides context for the impact of external regulators on Eg5 activity.

Structural Basis for Inhibition of Eg5 by Dihydropyrimidines: Stereoselectivity of Antimitotic Inhibitors Enastron, Dimethylenastron and Fluorastrol

Human kinesin Eg5, which plays an essential role in mitosis by establishing the bipolar spindle, has proven to be an interesting drug target for the development of cancer chemotherapeutics. Here, we report the crystal structures of the Eg5 motor domain complexed with enastron, dimethylenastron, and fluorastrol. By comparing these structures to that of monastrol and mon-97, we identified the main reasons for increased potency of these new inhibitors, namely the better fit of the ligand to the allosteric binding site and the addition of fluorine atoms. We also noticed preferential binding of the S-enantiomer of enastron and dimethylenastron to Eg5, while the R-enantiomer of fluorastrol binds preferentially to Eg5. In addition, we performed a multidrug resistance (MDR) study in cell lines overexpressing P-glycoprotein (Pgp). We showed that one of these inhibitors may have the potential to overcome susceptibility to this efflux pump and hence overcome common resistance associated with tubulin-targeting drugs.

Nucleophosmin/B23 Inhibits Eg5-mediated Microtubule Depolymerization by Inactivating Its ATPase Activity

Nucleophosmin/B23, an abundant nucleolar protein, plays multiple roles in cell growth and proliferation, and yet, little has been studied about its function in regulating dynamics of microtubules. Here, we report that B23 directly interacts with Eg5, a member of the kinesin family, in the cytosol. The DNA/RNA binding domain of B23 and the motor domain of Eg5 were found to be involved in their interaction. Both in vivo and in vitro evidences showed that B23 acts as an upstream regulator of Eg5 in promoting microtubule polymerization. Moreover, we further demonstrated that B23 regulates microtubule dynamics by directly inhibiting Eg5 ATPase activity.

Real-time Structural Transitions Are Coupled to Chemical Steps in ATP Hydrolysis by Eg5 Kinesin

At the biochemical level, motor proteins are enzymatic molecules that function by converting chemical energy into mechanical motion. The key element for energy transduction and a major unresolved question common for all motor proteins is the coordination between the chemical and conformational steps in ATP hydrolysis. Here we show time-lapse monitoring of an in vitro ATP hydrolysis reaction by the motor domain of a human Kinesin-5 protein (Eg5) using difference Fourier transform infrared spectroscopy and UV photolysis of caged ATP. In this first continuous observation of a biological reaction coordinate from substrate to product, direct spectral markers for two catalytic events are measured: proton abstraction from nucleophilic water by the catalytic base and formation of the inorganic phosphate leaving group. Simultaneous examination of conformational switching in Eg5, in parallel with catalytic steps, shows structural transitions in solution consistent with published crystal structures of the prehydrolytic and ADP-bound states. In addition, we detect structural modifications in the Eg5 motor domain during bond cleavage between the beta- and gamma-phosphates of ATP. This conclusion challenges mechanochemical models for motor proteins that utilize only two stages of the catalytic cycle to drive force and motion.

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.

Multivariate Data Analyses for Classifying Allosteric Inhibition in Human Eg5 Kinesin

Like other motor proteins, the human Eg5 kinesin couples ATP hydrolysis to large conformational changes distal from the nucleotide site, thereby driving movement along microtubules. The Eg5 motor domain uniquely possesses an allosteric L5 loop, responsible for sensitivity to small-molecule inhibitors. Prior studies support a common kinetic mode of inhibition by monastrol, S-trityl-L-cysteine, and ispinesib. However, missing is the role of individual residues within the L5 loop in allosteric communication and why there are differing efficacies in drug inhibition. Here we demonstrate an integrated approach to build, test, and refine a model of how the L5 loop alters the conformation of the motor domain in solution. Over 30 perturbations of the L5 loop, either by sequence variation or drug binding, were analyzed using kinetic data, vibrational spectroscopy, and multivariate analysis. Principal component analysis organized mutant kinesins into two populations of Eg5 conformers, distinguished by changes mainly in 310 helices and unordered regions. The presence of inhibitors also resulted in coincident, steady-state structural changes in this kinesin. We surmise that the above conformational changes are localized to the L5 loop. Unexpectedly, conformational changes were not restricted to the drug-binding pocket alone: we have directly measured long-distance changes to the beta-sheet core of the kinesin protein, a requirement for allostery that is quantifiable in this analysis. Such tools can ultimately permit prediction of pleiotropic changes in structure and consequently protein function and drug efficacy.

Tetrameric Chimera DK4mer is a Tool to Study Mechanisms Of Kinesin-5 Regulation. A Tetrameric Chimera of a Kinesin 1 and a Kinesin 5 is a Fast Microtubule Sliding Motor

The homo-tetrameric motor protein Eg5 from X. laevis drives relative sliding of anti-parallel microtubules by the processive action of its two opposing sets of dimeric motor. As shown by Kwok et al. (2006, Nat. Chem. Biol. 2:480) and Kapitein et al. (2008, J. Cell Biol. 182:421), tetrameric motors move slowly (∼20nm/s), but processively on a single microtubule alternating between diffusional and directional episodes, while motors moving between two microtubules move in a highly directional and processive fashion.In order to obtain a tetrameric model system with more clearly defined properties and motile phases, we have constructed a tetrameric chimera by replacing Eg5-motor domain and neck-linker by the homologous regions of D. melanogaster Kinesin 1 (DK4mer).In surface-gliding assays, Dk4mer showed fast motility (553±31nm/s), irrespective of a C-terminal his- or GFP-his-tag. Comparison to DmKHC shows a similar k0.5, ATP of ∼0.06mM, suggesting that the GFP-tagged version is suitable for single-molecule fluorescence studies. Single GFP-tagged DK4mer motors moved processively along the MT at speeds comparable to those seen in surface-gliding assays (499±3nm/s). We observe clearly distinguished directional and diffusional episodes and an overall run length of ∼9μm on average. We further performed relative sliding assays using DK4mer and observe the expected trimodal distribution of velocities at v=0, v=v1 and v=2v1, v1 being 500nm/s, clearly showing that DK4mer is capable of sliding microtubules apart simultaneously using both pairs of motor domains.The DK4mer is thus an excellent model system to study regulatory aspects of Kinesin-5 due to its high speed, its long processivity and its clear separation of diffusive and directional motility and its fast and efficient relative sliding of microtubules.(∗authors contributed equally)

Cryo-electron tomography of microtubule–kinesin motor complexes

Microtubules complexed with molecular motors of the kinesin family or non-motor microtubule associated proteins (MAPs) such as tau or EB1 have been the subject of cryo-electron microcopy based 3-D studies for several years. Most of these studies that targeted complexes with intact microtubules have been carried out by helical 3-D reconstruction, while few were analyzed by single particle approaches or from 2-D crystalline arrays. Helical reconstruction of microtubule-MAP or motor complexes has been extremely successful but by definition, all helical 3-D reconstruction attempts require perfectly helical assemblies, which presents a serious limitation and confines the attempts to 15- or 16-protofilament microtubules, microtubule configurations that are very rare in nature. The rise of cryo-electron tomography within the last few years has now opened a new avenue towards solving 3-D structures of microtubule-MAP complexes that do not form helical assemblies, most importantly for the subject here, all microtubules that exhibit a lattice seam. In addition, not all motor domains or MAPs decorate the microtubule surface regularly enough to match the underlying microtubule lattice, or they adopt conformations that deviate from helical symmetry. Here we demonstrate the power and limitation of cryo-electron tomography using two kinesin motor domains, the monomeric Eg5 motor domain, and the heterodimeric Kar3Vik1 motor. We show here that tomography does not exclude the possibility of post-tomographic averaging when identical sub-volumes can be extracted from tomograms and in both cases we were able to reconstruct 3-D maps of conformations that are not possible to obtain using helical or other averaging-based methods.

An allosteric transition trapped in an intermediate state of a new kinesin–inhibitor complex

Human kinesin Eg5 plays an essential role in mitosis by separating duplicated centrosomes and establishing the bipolar spindle. Eg5 is an interesting drug target for the development of cancer chemotherapy, with seven inhibitors already in clinical trials. In the present paper, we report the crystal structure of the Eg5 motor domain complexed with a potent antimitotic inhibitor STLC (S-trityl-L-cysteine) to 2.0 A (1 A=0.1 nm) resolution. The Eg5-STLC complex crystallizes in space group P3(2) with three molecules per asymmetric unit. Two of the molecules reveal the final inhibitor-bound state of Eg5, whereby loop L5 has swung downwards to close the inhibitor-binding pocket, helix alpha4 has rotated by approx. 15 degrees and the neck-linker has adopted a docked conformation. The third molecule, however, revealed an unprecedented intermediate state, whereby local changes at the inhibitor-binding pocket have not propagated to structural changes at the switch II cluster and neck-linker. This provides structural evidence for the sequence of drug-induced conformational changes.

Mutations in the human kinesin Eg5 that confer resistance to monastrol and S-trityl- l-cysteine in tumor derived cell lines

The kinesin Eg5 plays an essential role in bipolar spindle formation. A variety of structurally diverse inhibitors of the human kinesin Eg5, including monastrol and STLC, share the same binding pocket on Eg5, composed by helix α2/loop L5, and helix α3 of the Eg5 motor domain. Previous biochemical analysis in the inhibitor binding pocket of Eg5 identified key residues in the inhibitor binding pocket of Eg5 that in the presence of either monastrol or STLC exhibited ATPase activities similar to the untreated wild type Eg5. Here we evaluated the ability of full-length human Eg5 carrying point mutations in the drug binding pocket to confer resistance in HeLa and U2OS cells to either monastrol or STLC, as measured by the formation of bipolar spindles. Both transfected cells expressing wild type Eg5 and untransfected cells were equally sensitive to both inhibitors. Expression of Eg5 single point mutants R119A, D130A, L132A, I136A, L214A and E215A conferred significant resistance to monastrol. Certain mutations inducing monastrol resistance such as R119A, D130A and L214A also conferred significant resistance to STLC. For the first time at a cellular level, the propensity of selected Eg5 point mutants to confer drug resistance confirms the target specificity of monastrol and STLC for Eg5. These data also suggest a possible mechanism by which drug resistance may occur in tumors treated with agents targeting Eg5.

Assessing compound binding to the Eg5 motor domain using a thermal shift assay

Eg5 is a kinesin whose inhibition leads to cycle arrest during mitosis, making it a potential therapeutic target in cancers. Circular dichroism and isothermal titration calorimetry of our pyrrolotriazine-4-one series of inhibitors with Eg5 motor domain revealed enhanced binding in the presence of adenosine 5′-diphosphate (ADP). Using this information, we studied the interaction of this series with ADP–Eg5 complexes using a thermal shift assay. We measured up to a 7 °C increase in the thermal melting ( T m) of Eg5 for an inhibitor that produced IC 50 values of 60 and 130 nM in microtubule-dependent adenosine triphosphatase (ATPase) and cell-based cytotoxicity assays, respectively. In general, the inhibitor potency of the pyrrolotriazine-4-one series in in vitro biological assays correlated with the magnitude of the thermal stability enhancement of ADP–Eg5. The thermal shift assay also confirmed direct binding of Eg5 inhibitors identified in a high-throughput screen and demonstrated that the thermal shift assay is applicable to a range of chemotypes and can be useful in evaluating both potent (nM) and relatively weakly binding (μM) leads. Overall, the thermal shift assay was found to be an excellent biophysical method for evaluating direct binding of a large number of compounds to Eg5, and it complemented the catalytic assay screens by providing an alternative determination of inhibitor potency.

Conformational specificity in allosteric signaling: high‐throughput measurement of modular secondary structural changes within human Eg5 kinesin

The motor domain of human Eg5 kinesin (HsEg5) displays an allosteric pocket at the extracellular surface; binding of monastrol or S‐trityl‐L‐cysteine (STC) to this allosteric site, created by the L5 loop, and subsequent distal conformational changes impairs ATP hydrolysis and cellular function. We examined the roles of E116 and E118 in the L5 loop in allosteric signaling. First, there is a positional correlation on the outcome of amino acid substitutions, irrespective of its nature. Single‐site substitution of E118 increased basal ATP hydrolysis rates of Eg5, whereas substitution of E116 resulted in lower ATPase rates. Thus, sequence variation at residues 116 and 118 of kinesin L5 loop can drive upregulation and downregulation of ATP hydrolysis, respectively. From high‐throughput measurements of the secondary structure composition of proteins in solution, vibrational signatures from upregulated proteins were distinct from the net changes measured from downregulated proteins. Moreover, spectral changes found for E116 variants were similar to those of wildtype Eg5 with monastrol or STC. Therefore, allosteric inhibition by amino acid substitution or by small molecules both result in convergent steady‐state changes to the secondary structure of the Eg5 motor domain in solution, and these rapid methods will provide insight into how long‐range structural changes impact motor function.

Crystal structure of HsEg5 in complex with S‐trityl‐L‐cysteine

Eg5, a motor kinesin involved in the formation of the bipolar mitotic spindle, is essential for the successful completion of mitosis. Monastrol and S‐trityl‐L‐cysteine (STC) are two well‐characterized inhibitors of Eg5, with STC being the more potent inhibitor. We have determined the 2.5 Åresolution crystal structure of the Eg5 motor domain in complex with STC and Mg ?ADP. STC interacts with Eg5 via a pocket formed by helices a2, a3 and loop L5, and induces conformational changes within the Eg5 necklinker and switch I and II regions similar to those seen with bound monastrol. However, there are two distinctions between the STC‐Eg5 and monastrol‐Eg5 structures: the nature of the interactions between the allosteric ligand and Eg5 and solvent accessibility of residues in the allosteric binding site. Monastrol contacts with Eg5 are largely mediated by nonpolar drug surfaces. During STC binding, contact between the motor domain and polar surfaces of the drug is increased in relation to that of monastrol by over 20 Å2. STC binding occludes ~80 Å2 more of the Eg5 surface from solvent access than does monastrol. Site‐directed mutagenesis and coupled biochemical assays monitoring ATP hydrolysis were used to test the hypothesis that several residues altered in solvent accessibility upon STC binding to Eg5 were critical to the increased efficacy of inhibition by this small molecule.Supported by Louisiana Board of Regents Grant

Crystal Structure of HsEg5 in Complex with S-trityl-L-cysteine

Eg5 is a motor kinesin involved in the formation of the bipolar mitotic spindle which is essential for the completion of mitosis. The discovery of a class of allosteric Eg5 inhibitors has raised the possibility of a novel approach for the treatment of cancer. Monastrol and S-trityl-L-cysteine (STC) are two well-characterized inhibitors of Eg5 known to prevent ADP release by the motor domain, with the latter compound being a more potent inhibitor of Eg5 than the former. We have determined the 2.5 Å resolution crystal structure of the Eg5 motor domain in complex with STC and Mg•ADP. STC interacts with Eg5 via a pocket formed by helices α2, α3 and loop L5, and induces conformational changes within the Eg5 motor domain similar to those seen with bound monastrol. The necklinker is positioned in the “docked” conformation seen in the monastrol-bound Eg5 structures, and the switch I and II regions also adopt conformations similar to those observed for bound monastrol. Moreover, STC contacts with Eg5 differ from those seen in monastrol-bound crystal structures. Monastrol contacts with Eg5 are largely mediated by nonpolar surfaces of the drug. During STC binding, contact between the motor domain and polar surfaces of the drug is increased in relation to that of monastrol by over 20 Å2. STC binding occludes approximately 80 Å2 more of the Eg5 surface from solvent access than does monastrol. Site-directed mutagenesis and coupled biochemical assays monitoring ATP hydrolysis were used to examine whether these residues involved in polar interactions between STC and Eg5, and residues altered in solvent accessibility upon STC binding to Eg5, were critical in the increased efficacy of Eg5 inhibition by this small molecule.

Truncated form of tenascin-X, XB-S, interacts with mitotic motor kinesin Eg5

XB-S is a protein with an amino-terminal-truncated form of tenascin-X (TNXB). However, the precise roles of XB-S in vivo are unknown. In this study, to determine the role of XB-S in vivo, we screened XB-S-binding proteins. FLAG-tagged XB-S was transiently introduced into 293T cells. Then its associated proteins were purified by immunoprecipitation using an anti-FLAG antibody and its components were identified by mass spectrometric analyses. Mitotic motor kinesin Eg5 was identified in the immunoprecipitates. XB-S and Eg5 proteins were co-localized in the cytoplasm in interphase and mitosis, but XB-S did not localize on mitotic spindle microtubules, on which Eg5 prominently localized in mitosis. As for Eg5 binding to XB-S, glutathione S-transferase-fused XB-S expressed in vitro directly bound to full-length Eg5 translated in reticulocyte lysate, and the XB-S-binding region was located in the motor domain of Eg5. Furthermore, during cell cycle progression XB-S showed a similar expression profile to that of Eg5. These results suggest possible involvement of XB-S in the function of Eg5.

Structure of Human Eg5 in Complex with a New Monastrol-based Inhibitor Bound in the R Configuration

Drugs that target mitotic spindle proteins have been proven useful for tackling tumor growth. Eg5, a kinesin-5 family member, represents a potential target, since its inhibition leads to prolonged mitotic arrest through the activation of the mitotic checkpoint and apoptotic cell death. Monastrol, a specific dihydropyrimidine inhibitor of Eg5, shows stereo-specificity, since predominantly the (S)-, but not the (R)-, enantiomer has been shown to be the biologically active compound in vitro and in cell-based assays. Here, we solved the crystal structure (2.7A) of the complex between human Eg5 and a new keto derivative of monastrol (named mon-97), a potent antimitotic inhibitor. Surprisingly, we identified the (R)-enantiomer bound in the active site, and not, as for monastrol, the (S)-enantiomer. The absolute configuration of this more active (R)-enantiomer has been unambiguously determined via chemical correlation and x-ray analysis. Unexpectedly, both the R- and the S-forms inhibit Eg5 ATPase activity with IC(50) values of 110 and 520 nM (basal assays) and 150 nm and 650 nm (microtubule-stimulated assays), respectively. However, the difference was large enough for the protein to select the (R)- over the (S)-enantiomer. Taken together, these results show that in this new monastrol family, both (R)- and (S)-enantiomers can be active as Eg5 inhibitors. This considerably broadens the alternatives for rational drug design.

S-Trityl-L-cysteine Is a Reversible, Tight Binding Inhibitor of the Human Kinesin Eg5 That Specifically Blocks Mitotic Progression

Human Eg5, responsible for the formation of the bipolar mitotic spindle, has been identified recently as one of the targets of S-trityl-L-cysteine, a potent tumor growth inhibitor in the NCI 60 tumor cell line screen. Here we show that in cell-based assays S-trityl-L-cysteine does not prevent cell cycle progression at the S or G(2) phases but inhibits both separation of the duplicated centrosomes and bipolar spindle formation, thereby blocking cells specifically in the M phase of the cell cycle with monoastral spindles. Following removal of S-trityl-L-cysteine, mitotically arrested cells exit mitosis normally. In vitro, S-trityl-L-cysteine targets the catalytic domain of Eg5 and inhibits Eg5 basal and microtubule-activated ATPase activity as well as mant-ADP release. S-trityl-L-cysteine is a tight binding inhibitor (estimation of K(i,app) <150 nm at 300 mm NaCl and 600 nm at 25 mm KCl). S-trityl-L-cysteine binds more tightly than monastrol because it has both an approximately 8-fold faster association rate and approximately 4-fold slower release rate (6.1 microM(-1) s(-1) and 3.6 s(-1) for S-trityl-L-cysteine versus 0.78 microM(-1) s(-1) and 15 s(-1) for monastrol). S-trityl-L-cysteine inhibits Eg5-driven microtubule sliding velocity in a reversible fashion with an IC(50) of 500 nm. The S and D-enantiomers of S-tritylcysteine are nearly equally potent, indicating that there is no significant stereospecificity. Among nine different human kinesins tested, S-trityl-L-cysteine is specific for Eg5. The results presented here together with the proven effect on human tumor cell line growth make S-trityl-L-cysteine a very attractive starting point for the development of more potent mitotic inhibitors.

A Pathway of Structural Changes Produced by Monastrol Binding to Eg5

Monastrol is a small molecule inhibitor that is specific for Eg5, a member of the kinesin 5 family of mitotic motors. Crystallographic models of Eg5 in the presence and absence of monastrol revealed that drug binding produces a variety of structural changes in the motor, including in loop L5 and the neck linker. What is not clear from static crystallographic models, however, is the sequence of structural changes produced by drug binding. Furthermore, because crystallographic structures can be influenced by the packing forces in the crystal, it also remains unclear whether these drug-induced changes occur in solution, at physiologically active concentrations of monastrol or of other drugs that target this site. We have addressed these issues by using a series of spectroscopic probes to monitor the structural consequences of drug binding. Our results demonstrated that the crystallographic model of an Eg5-ADP-monastrol ternary complex is consistent with several solution-based spectroscopic probes. Furthermore, the kinetics of these spectroscopic signal changes allowed us to determine the temporal sequence of drug-induced structural transitions. These results suggested that L5 may be an element in the pathway that links the state of the nucleotide-binding site to the neck linker in kinesin motors.

Small-molecule and mutational analysis of allosteric Eg5 inhibition by monastrol

BackgroundA recent crystal structure of monastrol in a ternary complex with the kinesin Eg5 motor domain highlights a novel, induced-fit drug binding site at atomic resolution. Mutational obliteration of the monastrol binding site results in a monastrol-resistant, but otherwise catalytically active Eg5 motor domain. However, considering the conformational changes at this site, it is unclear what specific interactions stabilize the interaction between monastrol and the Eg5 motor domain.ResultsTo study the molecular complementarity of the monastrol-Eg5 interaction, we used a combination of synthetic chemistry and targeted mutations in Eg5 to measure the contribution of specific contacts to inhibition of Eg5 in vitro and in cultured cells. Structure-activity data on chemical derivatives, sequence analysis of Eg5 homologs from different species, and the effect of mutations near the drug binding site were consistent with the crystal structure.ConclusionThe mechanism of monastrol revealed by our data rationalizes its specificity for Eg5 over other kinesins and highlights a potential mechanism of drug resistance for anti-cancer therapy targeting this site in Eg5.

Docking and Rolling, a Model of How the Mitotic Motor Eg5 Works

Whereas kinesin I is designed to transport cargoes long distances in isolation, a closely related kinesin motor, Eg5, is designed to generate a sustained opposing force necessary for proper mitotic spindle formation. Do the very different roles for these evolutionarily related motors translate into differences in how they generate movement? We have addressed this question by examining when in the ATPase cycle the Eg5 motor domain and neck linker move through the use of a series of novel spectroscopic probes utilizing fluorescence resonance energy transfer, and we have compared our results to kinesin I. Our results are consistent with a model in which movement in Eg5 occurs in two sequential steps, an ATP-dependent docking of the neck linker, followed by a rotation or "rolling" of the entire motor domain on the microtubule surface that occurs with ATP hydrolysis. These two forms of movement are consistent with the functions of a motor designed to generate sustained opposing force, and hence, our findings support the argument that the mechanochemical features of a molecular motor are shaped more by the demands placed on it than by its particular family of origin.