Susan P. GilbertChun Ju Chen, Ken Porche, Ivan Rayment and  Kinesin-14 Kar3Vik1 PowerstrokeThe ATPase Pathway That Drives theCell Biology:

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The ATPase Pathway That Drives the Kinesin-14 Kar3Vik1Powerstroke*□S

Received for publication, June 26, 2012, and in revised form, August 21, 2012 Published, JBC Papers in Press, September 12, 2012, DOI 10.1074/jbc.M112.395590

Chun Ju Chen‡, Ken Porche‡, Ivan Rayment§, and Susan P. Gilbert‡1

From the ‡Department of Biology and the Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute,Troy, New York 12180 and §Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

Background: Kar3Vik1 binds side-by-side microtubule protofilaments and utilizes a minus-end-directed powerstroke.Results:Microtubule collision occurs through Vik1 followed by Kar3 binding and ADP release, which destabilize Vik1and generate the intermediate poised for ATP binding.Conclusion: The transient Kar3Vik1 two-head-bound state intermediate was identified.Significance: This study provides new insights into force generation by kinesin-14 motors.

Kar3, a Saccharomyces cerevisiaemicrotubule minus-end-di-rected kinesin-14, dimerizes with either Vik1 or Cik1. TheC-terminal globular domain of Vik1 exhibits the structure of akinesinmotor domain andbindsmicrotubules independently ofKar3 but lacks a nucleotide binding site. The only known func-tion of Kar3Vik1 is to cross-link parallel microtubules at thespindle poles during mitosis. In contrast, Kar3Cik1 depolymer-izes microtubules during mating but cross-links antiparallelmicrotubules in the spindle overlap zone during mitosis. Arecent study showed thatKar3Vik1binds across adjacentmicro-tubule protofilaments and uses a minus-end-directed power-stroke to drive ATP-dependent motility. The presteady-stateexperiments presented here extend this study and establish anATPase model for the powerstroke mechanism. The resultsincorporated into the model indicate that Kar3Vik1 collideswith the microtubule at 2.4 �M�1 s�1 through Vik1, promotingmicrotubule binding by Kar3 followed by ADP release at 14 s�1.The tight binding of Kar3 to the microtubule destabilizes theVik1 interaction with the microtubule, positioning Kar3Vik1for the start of the powerstroke. Rapid ATP binding to Kar3 isassociated with rotation of the coiled-coil stalk, and the post-powerstroke ATP hydrolysis at 26 s�1 is independent of Vik1,providing further evidence that Vik1 rotates with the coiled coilduring the powerstroke. Detachment of Kar3Vik1 from themicrotubule at 6 s�1 completes the cycle and allows themotor toreturn to its initial conformation. The results also reveal keydifferences in the ATPase cycles of Kar3Vik1 and Kar3Cik1,supporting the fact that these two motors have distinctive bio-logical functions.

Kinesin-14 represents a subfamily of kinesins that are non-processive, promote microtubule (MT)2 minus-end-directedforce generation, and contain C-terminal motor domains that

are dimerized through an N-terminal coiled coil (1–7). Unlikethe well known N-terminal motor domain kinesins that use anasymmetric hand-over-hand mechanism for MT plus-end-di-rected processive stepping (8–11), kinesin-14s use an MTminus-end-directed rotation or bending of the coiled-coil stalkto generate force (12–14). This rotation coupled to ATP turn-over is designated the powerstroke and is used to slide oneMTrelative to another (15–19). During mitosis, kinesin-14s areknown to cross-link parallelMTs at the spindle poles but cross-link antiparallel interpolar MTs in the spindle overlap zone(20–24). Furthermore, at the spindle poles and the spindleoverlap zone, MT plus-end-directed kinesin-5 is present andprovides an outward force to counterbalance the inward forceof kinesin-14 to achieve spindle integrity yet allowMT dynam-ics and other motor and protein interactions required for chro-mosome segregation (25–28).Saccharomyces cerevisiae kinesin-14 Kar3 is an intriguing

model to study kinesin-14 mechanochemistry and head-headcommunication because Kar3 forms heterodimers with eitherCik1 or Vik1, both of which lack a nucleotide binding site, andKar3Cik1 and Kar3Vik1 have different physiological roles dur-ing the yeast life cycle (21, 22, 29–32). Moreover, the C-termi-nal domain of Vik1 exhibits the structural fold of a kinesinmotor domain, and it can bind MTs with high affinity. How-ever, the absence of a nucleotide binding site indicates thatnucleotide cannot modulate the interactions of Vik1 with theMT lattice (33).Recent studies using equilibrium binding, cryo-EM, x-ray

crystallography, and site-directed cross-links at the base of thecoiled coil with analysis by in vitromotility assays clearly dem-onstrated that both Kar3 and Vik1 interact with the MT formotility (14). Furthermore, an unprecedented mode of MTbinding was revealed for Kar3Vik1 by high resolution unidirec-tional metal shadowing, which strongly emphasizes the surfacefeatures when viewed by EM. The images of the shadowedMT�Kar3Vik1 complexes in the presence of ADP, used tomimic the beginning of the cycle when Kar3Vik1 collides withthe MT, revealed that Kar3 and Vik1 bound side-by-side MTprotofilaments rather than binding along a single protofilamentin a head-to-tail fashion (14). Because the complexes wereformed with ADP, the results suggested that the initial event of

* This work was supported, in whole or in part, by National Institutes of HealthGrants GM54141 (to S. P. G.) and GM086351 (to I. R.).

□S This article contains supplemental Fig. S1.1 To whom correspondence should be addressed: Dept. of Biology, Rensse-

laer Polytechnic Inst., 110 8th St., Troy, NY 12180. Tel.: 518-276-4415;E-mail: sgilbert@rpi.edu.

2 The abbreviations used are: MT, microtubule; AMPPNP, adenosine 5�-(�,�-imido)triphosphate; mant, 2�(3�)-O-(N-methylanthraniloyl).

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 44, pp. 36673–36682, October 26, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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MT collision occurred through Vik1; however, this collisioncomplex was not observed through cryo-EM. Docking of theKar3Vik1 x-ray structure into the cryo-EM maps captured anucleotide-free prepowerstroke state in which one head wasbound to the MT, presumably Kar3, and the second head wasdetached from the MT with the coiled-coil stalk pointingtoward the MT plus-end. In contrast, the ATP state, generatedby AMPPNP, showed a new position for the coiled-coil stalk,rotated �90° and pointed toward the MT minus-end. In theAMPPNP state, the Vik1 head appeared detached from theMTand in close contact to the coiled coil, suggesting that Vik1rotates with the coiled coil upon uptake of ATP (14, 34).In the study presented here, we pursued a mechanistic anal-

ysis using presteady-state methodologies to define the ATPasecycle and the sequence of events as the heads interact with theMT to generate force. Although our previous structural studies(14, 33) captured the stable intermediates, this analysis identi-fies the transient intermediates and associates specific chemicalsteps of ATP turnover with structural transitions required togenerate motility. Although the results reveal many similaritiesbetween the ATPase pathways for Kar3Cik1, Kar3Vik1, andDrosophila melanogaster Ncd, suggesting a conserved mecha-nism for kinesin-14s, there are distinct differences betweenKarVik1 and Kar3Cik1 that may be related to their differentbiological roles. In addition, the results provide insights tounderstand intermolecular communication mediated throughthe neck coiled coil for Ncd, Kar3Vik1, and Kar3Cik1.

EXPERIMENTAL PROCEDURES

Buffer and Experimental Conditions—The experiments wereperformed at 25 °C inATPase buffer (20mMHepes, pH7.2withKOH, 5 mMmagnesium acetate, 0.1 mM EDTA, 0.1 mM EGTA,50 mM potassium acetate, 1 mM dithiothreitol, 5% sucrose). Onthe morning of each experiment, the tubulin was cold depo-lymerized, clarified, and polymerized with 1 mM GTP at 34 °C.The MTs were stabilized with 40 �M paclitaxel, and the MTconcentrations are reported as paclitaxel-stabilized tubulinpolymer. All reported concentrations of nucleotides and nucle-otide analogs also include the equivalent concentration ofmag-nesium acetate. Errors are reported as S.E.Kar3Vik1—The GCN4-KarVik1 heterodimeric kinesin-14

used in this study was characterized previously (14). The trun-cated KAR3 gene was cloned into pET-24d (Novagen; kanamy-cin selection). When expressed, this plasmid yields amino acidresidues MSVKELEDKVEELLSKNYHLENEVARLKKLV-Lys353–Lys729 with a predicted Mr of 46,151. The truncatedVIK1 gene was cloned into pKLC37, a plasmid modified frompET-16b (Novagen; ampicillin selection). Vik1 is expressed asMSYYHHHHHHDYDIPTSENLYFQGASMSVKELEDKVEEL-LSKNHLENEVARLKKLV-Ser341–Thr647 with a predicted Mrof 42,724. The sequenceMSVKELEDKVEELLSKNYHLENEV-ARLKKLV of Kar3 and Vik1 represents the GCN4 leucine zip-per domain for dimerization (35). Kar3 and Vik1 were co-ex-pressed in Escherichia coli BL21-CodonPlus(DE)-RIL asdescribed previously (33, 36), yielding a Kar3Vik1 heterodimerthat formed through the GCN4 leucine zipper with a predictedMr of 88,875.

For the experiments, Kar3Vik1 was clarified (BeckmanCoulterTLA 100 rotor, 3 min, 90,000 rpm, 4 °C), and the motor concen-tration was determined by the Bio-Rad protein assay with IgG asthe standard. Concentration is reported as the Kar3Vik1 het-erodimer with one nucleotide binding site per Kar3Vik1.Formation of the Nucleotide-free MT�Kar3Vik1 Complex—

Nucleotide-free Kar3Vik1 was generated as described previouslyfor Kar3Cik1 (36) and used to measure the kinetics of ATP bind-ing, ATP hydrolysis, and ATP-promoted MT�Kar3Vik1 dissocia-tion. Briefly, the MT�Kar3Vik1 complex was preformed in thepresence of 100 �M MgADP followed by an apyrase treatment(0.02unit/ml; gradeVII, Sigma-Aldrich) for90min.Thisapproachassumes thatVik1will bind to theMTin thepresenceofADPwiththe Kar3�ADP head detached from the MT or weakly bound buttethered to theMT through Vik1. The apyrase VII isoform selec-tively convertsADP toAMP,whichhas a veryweak affinity for theKar3 active site such that theMT�Kar3Vik1 complex is essentiallynucleotide-free. The concentration of apyrase is sufficiently lowthat apyrase does not compete with Kar3Vik1 for ATP in theexperiments.Kinetics of MantATP Binding—The nucleotide-free MT�

Kar3Vik1 complex was rapidly mixed with mantATP in anSF-2003 KinTek Corp. stopped-flow instrument (see Fig. 1),and the fluorescence enhancementwasmonitored as a functionof time with excitation at 360 nm and detection at 450 nm via aSemrock 409-nm long pass filter (36, 37). The exponentialincrease in fluorescence is correlated with mantATP bindingbecause the fluorescence of mant nucleotides is enhanced bythe hydrophobic environment of the active site. Each averagedtransient was fit to a single exponential function plus a linearphase. The observed rates of the initial exponential phase of thetransients were plotted as a function of increasing mantATPconcentration. These data were fit to a linear function,

kobs � k�1 �mantATP� � k�1 (Eq. 1)

where kobs is the observed rate of the initial exponential phase ofthe fluorescence enhancement, k�1 defines the second-orderrate constant for mantATP binding, and k�1 represents theobserved rate constant of mantATP dissociation as obtainedfrom the y intercept. The equilibriumdissociation constant wasdetermined by Kd � k�1/k�1 (Scheme 1).

MT�E � ATP ^1

MT�E�ATP-|01�

MT�E*�ATP^2

MT�E�ADP�Pi^3

MT � Pi � E�ADP

MT � E�ADP ^4

MT�E�ADP^5

MT�E � ADP

SCHEME 1

Pulse-Chase Kinetics of ATP Binding—The nucleotide-freeMT�Kar3Vik1 complex was rapidly mixed in the KinTek Corp.chemical quench-flow instrument with ATP plus trace[�-32P]ATP and 200 mM KCl (syringe concentration) for timesranging from 5 to 500 ms (see Fig. 2 and Refs. 36 and 37). Thereaction was subsequently chased with 30 mM nonradiolabeledATP (syringe concentration) and allowed to proceed for 6 s(�10 ATP turnovers). The reaction was expelled from the

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instrument into a 1.5-ml tube containing 100 �l of 22 N formicacid to terminate the reaction and release nucleotide from theactive site. This experimental design quantifies [�-32P]ATP sta-bly bound at the active site that proceeds through ATP hydrol-ysis. Thus, the pulse-chase experimentmeasures the kinetics ofATP binding. The products [�-32P]ADP � Pi were separatedfrom [�-32P]ATP by thin-layer chromatography for quantifica-tion. The concentration of [�-32P]ADP product formed wasplotted as a function of time. Each transient was fit to the burstequation.

Product � A0�1 � exp�kbt� � kslowt (Eq. 2)

where A0 is the amplitude of the exponential burst phase rep-resenting the concentration of ATP stably bound at the activesite that proceeded through ATP hydrolysis, kb is the observedrate of the exponential burst phase, and t is the time in seconds.The kslow is the rate of the linear phase (�M�s�1) and whendivided by the Kar3Vik1 concentration approximates thesteady-state turnover. The observed rates of the exponentialburst phase were plotted as a function of ATP concentration,and the data were fit to Equation 3,

kobs � �K1k�1��ATP�/K1�ATP� � 1� (Eq. 3)

whereK1 defines the equilibrium association constant for com-plex formation (Kd � 1/K1) and k�1� represents the first-orderrate constant for the ATP-promoted isomerization (Scheme 1).Acid Quench Kinetics of ATP Hydrolysis—The nucleotide-

free MT�Kar3Vik1 complex was rapidly mixed in the KinTekCorp. chemical quench-flow instrument with ATP, trace[�-32P]ATP, and 200mMKCl (syringe concentration) for timesranging from 5 to 500 ms (see Fig. 3 and Refs. 36 and 37). Thereactionwas subsequently quenchedwith 10 N formic acid. Theproducts [�-32P]ADP � Pi were separated from [�-32P]ATP bythin-layer chromatography for quantification. The concentra-tion of [�-32P]ADP product formedwas plotted as a function oftime, and each transient was fit to Equation 2 where A0 is theamplitude of the exponential burst phase representing the for-mation of [�-32P]ADP�Pi at the Kar3 active site during the firstATP turnover, kb is the observed rate of the initial exponentialphase that corresponds to the rate of product formation at theactive site during the first ATP turnover, t is the time in sec-onds, and kslow is the rate of the linear phase (�M�s�1), whichcorresponds to steady-state turnover at 100 mM KCl. Theobserved rates (Fig. 3B) and amplitudes of the exponential burstphase (Fig. 3C) were plotted as a function of MgATP concen-tration, and each data set was fit to a hyperbolic function. Themaximum rate constant obtained from the hyperbolic fit of thedata in Fig. 3B provides the rate constant ofATPhydrolysis, k�2(Scheme 1).MT�Kar3Vik1 Dissociation Kinetics—The nucleotide-free

MT�Kar3Vik1 complex was rapidly mixed with ATP, AMP-PNP, ADP, and buffer in the stopped-flow instrument, and thechange in turbidity at 340 nm was monitored as a function oftime (see Fig. 4 and Refs. 36 and 37). The decrease in the tur-bidity signal is associated with dissociation of the complexbecause of the decrease in mass of the MTs when Kar3Vik1detaches and because the motors in solution do not contribute

significantly to the turbidity signal. The observed rates of theinitial exponential phase of the ATP transients were plotted asan increase in ATP concentration, and the hyperbolic fit of thedata provided k�3, the rate constant of ATP-promotedKar3Vik1 detachment from the MT (Scheme 1). To testwhether ATP hydrolysis was required for MT�Kar3Vik1 disso-ciation, the experiment was repeated with buffer, ADP, and thenonhydrolyzable ATP analog AMPPNP.MT Kar3Vik1 Association Kinetics—Kar3Vik1 was rapidly

mixedwithMTs in the stopped-flow instrument, and the turbiditysignal at 340 nm was monitored as a function of time (see Fig. 5,supplemental Fig. S1, and Refs. 36 and 37). The initial exponentialrates of the transients were plotted as a function of increasingMTconcentration, and the data were fit to a linear function,

kobs � k�4�tubulin� � k�4 (Eq. 4)

where kobs is the observed rate for the initial exponential phaseof the averaged transients, k�4 defines the second-order rateconstant for MT association, and k�4 corresponds to theobserved rate constant of motor dissociation as determined bythe y intercept (Scheme 1).Kinetics of MantADP Release after MT Collision—Kar3Vik1

at 5 �Mwas incubated with 30 �MmantADP, and this complexwas subsequently mixed in the stopped-flow instrument withMTs plus 2 mM ATP (see Fig. 6 and Refs. 36 and 37). The fluo-rescence change was monitored over time with excitation at360 nm and detection at 450 nm via a Semrock 409-nm longpass filter (36, 37). The transients were fit to a double exponen-tial function, and the observed rate of the initial exponentialphase was plotted as a function of increasing concentrations ofMTs. The hyperbolic fit of the data provided themaximum rateconstant, k�5, of mantADP release (Scheme 1).

RESULTS

We previously proposed a model for Kar3Vik1 MT interac-tions based on a powerstroke mechanism for nonprocessivemovement (14, 33). This model (Scheme 1 and Fig. 7) was usedto design the experiments presented here and to test hypothe-ses that resulted from the earlier studies. Table 1 presents theconstants determined experimentally for Kar3Vik1 in compar-ison with the constants for Kar3Cik1 reported previously (36).ATPBinding Is Rapid and Promotes the Kar3Vik1 Coiled-coil

Stalk Rotation—Substrate binding for Kar3Vik1 was deter-mined by two approaches: fluorescent mantATP binding usingthe stopped-flow instrument (Fig. 1) and pulse-chase kineticswith [�-32P]ATP using the rapid quench-flow instrument (Fig.2). Fig. 1A shows representative transients ofmantATP bindingto the nucleotide-freeMT�Kar3Vik1 complex, and the observedrates of the initial exponential phase were plotted as a functionof increasing mantATP concentration (Fig. 1B). The fit of thedata provided a second-order rate constant of mantATP bind-ing of 0.8 �M�1 s�1 with the y intercept, indicating a dissocia-tion rate of 7.4 s�1.

In the second approach, the kinetics of ATP binding weremeasured using [�-32P]ATP, and representative transients areshown in Fig. 2A. The initial exponential phase corresponds tothe first ATP turnover with the following linear phase repre-

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senting subsequent ATP turnovers. The data show that therewas an increase in both the observed rate and amplitude of theinitial exponential phase as a function of ATP concentration.These rates for each transient were plotted as a function of ATPconcentration, and the observation that the rate becomes satu-rated rather than remaining linear is indicative of a rate-limit-ing ATP-promoted isomerization that occurs at the active siteto generate theMT�Kar3Vik1*�ATP intermediate that is poisedfor ATP hydrolysis (Scheme 1 and Table 1). Themaximum rateconstant (k�1�) of this conformational change is 55 s�1. Becausethe nonhydrolyzable ATP analog AMPPNP promotes rotation ofthe Kar3Vik1 coiled-coil stalk (14), we propose that the 55 s�1

isomerization measured here represents the rate constant of theATP-promoted stalk rotation to generate the postpowerstrokeintermediate that is poised for ATP hydrolysis.Fig. 2C shows the amplitude of each transient plotted as a func-

tion of increasing ATP concentration, and the fit of the data pro-vides the maximum amplitude of 1 ADP�Pi per Kar3Vik1. Theamplitude represents the concentration of ATP bound at theactive site that proceeded throughATP hydrolysis during the firstATPturnover.BecauseKar3Vik1onlyhasa singleKar3ATPbind-ing site, an amplitude of �1 ADP�Pi per Kar3Vik1 was expectedand indicates thatATPbinding is sufficiently tight that everyATPon the active sites proceeds through ATP hydrolysis.ATP Hydrolysis Occurs after the Powerstroke—The acid

quench experimental design was used to measure the timecourse of ATP hydrolysis. The representative transients shownin Fig. 3A reveal a presteady-state burst of ADP�Pi product for-

mation at the active site during the first ATP turnover, indicat-ing that ATP binding and hydrolysis are fast followed by a sub-sequent slow step. At high ATP concentrations, ATP bindingoccurs faster than ATP hydrolysis. Therefore, ATP binding nolonger limits ATPhydrolysis, and the observed rate of the expo-nential phase predicts the rate of ATP hydrolysis. The rates ofthe initial exponential burst phase were plotted as a function ofincreasing ATP concentration (Fig. 3B), and the hyperbolic fitof the data provided a rate constant of ATP hydrolysis, k�2, of26 s�1 with an apparent Kd,ATP of 36 �M. These constants arecomparable with those measured for Kar3Cik1, 26 s�1 with aKd,ATP of 34 �M (Table 1 and Ref. 36), suggesting that ATPhydrolysis is intrinsic to Kar3 and independent of Vik1 or Cik1or alternatively that the presence of Vik1 or Cik1 affects ATPhydrolysis to the same extent. Because the rate constant is 70s�1 for the Kar3 motor domain (38, 39), we conclude that ATPhydrolysis is independent of Vik1 or Cik1, but heterodimeriza-tion through the coiled coil does have an effect.Fig. 3C presents the burst amplitude data for the presteady-

state transients. Note that the fit of the data provided a maxi-mum burst amplitude of 0.91 ADP�Pi formed on the active siteduring the first turnover for each Kar3Vik1 in the experiment.The approximately full burst amplitude observed for Kar3Vik1is in sharp contrast to the amplitude observed for Kar3Cik1 of0.56 ADP�Pi per Kar3Cik1 (Table 1). In the case of Kar3Cik1,the modeling of the kinetic data indicated that the loweredburst amplitudewasdue to reversals atATPhydrolysis, suggestingthat phosphate remained at the active site for resynthesis of ATP

TABLE 1Comparison of ATPase parameters for the MT�Kar3Vik1 and MT�Kar3Cik1 motors

Kar3Vik1 Kar3Cik1a

MantATP binding k�1 � 0.8 � 0.04 �M�1 s�1 k�1 � 2.1 � 0.06 �M�1 s�1

k�1 � 7.4 � 0.9 s�1 k�1 � 16.6 � 1.0 s�1

ATP binding (pulse-chase) K1k�1� � 18.6 � 0.03 �M�1 s�1 K1k�1� � 8.4 � 0.02 �M�1 s�1

k�1 not observed k�1 not observedK1 � 0.34 � 0.03 �M�1 K1 � 0.12 � 0.01 �M�1

k�1� � 54.9 � 1.0 s�1 k�1� � 69 � 1.4 s�1

Kd,ATP � 2.9 �M Kd,ATP � 8.3 �MA0 � 1.07 � 0.02 per KarVik1 A0 � 0.94 � 0.04 per KarCik1Kd,ATP � 25.2 � 1.3 �M Kd,ATP � 28.2 � 4.6 �M

ATP hydrolysis k�2 � 25.9 � 1.4 s�1 k�2 � 26 � 0.8 s�1

Kd,ATP � 34.4 � 5.1 �M Kd,ATP � 35.9 � 4.6 �MA0 � 0.91 � 0.03 per Kar3Vik1 A0 � 0.56 � 0.02 per Kar3Cik1Kd,ATP � 16 � 1.6 �M Kd,ATP � 9.8 � 2.5 �M

MT�Kar3Vik1 dissociation k�3 � 6.2 � 0.1 s�1 k�3 � 11.5 � 0.3 s�1

K1⁄2,ATP � 3.7 � 0.3 �M K1⁄2,ATP � 17.6 � 2.5 �M

MT Kar3Vik1 association k�4 � 2.4 � 0.1 �M�1 s�1 k�4 � 5.1 � 0.3 �M�1 s�1

k�4 not observed k�4 not observedk�4 � 4.0 � 0.32 �M�1 s�1 (ADP) k�4 � 4.9 � 0.1 �M�1 s�1 (ADP)k�4 � 29.1 � 1.5 s�1 k�4 not observed

MantADP releaseFast phase k�5 � 14.4 � 0.3 s�1 k�5 � 110 � 7.7 s�1

K1⁄2,MTs � 7.7 � 0.5 �M K1⁄2,MTs � 37.9 � 5.3 �M

Slow phase kobs � 1–3 s�1 kmax � 5.0 � 0.2 s�1

K1⁄2,MTs � 5.6 � 1.0 �M

Steady-state parameters kcat � 6.7 � 0.1 s�1 b kcat � 5.0 � 0.1 s�1

Km,ATP � 65.2 � 4.1 �Mb Km,ATP � 27.1 � 2.9 �MK1⁄2,MTs � 0.7 � 0.05 �Mb K1⁄2,MTs � 0.4 � 0.02 �M

Motility rate 5.87 � 0.05 �m/minb 2.69 � 0.08 �m/mina From Ref. 36.b From Ref. 14.

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(36). The approximately full burst amplitude result for Kar3Vik1indicates that phosphate release is sufficiently fast or that there isan isomerization at the active site that is sufficiently fast to preventresynthesis of ATP during the first turnover. Note that the burstamplitude for dimericNcdwas also�1ADP�Pi perNcd active siteas determined for Kar3Vik1 (40).MT�Kar3Vik1 Complex Dissociation Occurs after ATP

Hydrolysis—For most kinesins including Ncd and Kar3Cik1,ATP hydrolysis is required for the motor to detach from theMT (36, 41–43). To test the hypothesis that ATP hydrolysismust occur to generate the weak MT binding state, the pre-formed nucleotide-free MT�Kar3Vik1 complex was rapidlymixed in the stopped-flow instrument with the nonhydroly-zableATP analogAMPPNP, buffer, ATP, orADP, and turbiditywas monitored. Fig. 4A shows an ATP- and ADP-promotedexponential decrease in the turbidity signal as a function of timethat is correlatedwithKar3Vik1motor detachment from theMT.In contrast, the amplitudes associated with the buffer and AMP-PNP transients are extremely small, indicating that ATP hydroly-sis was required for the Kar3Vik1motors to detach from theMT.For the ATP transient, there was a short lag at the start of theATP-promoted turbidity decrease that is consistent with the timerequired for ATP binding and ATP hydrolysis to occur. In con-trast, the ADP-promoted dissociation transient shows an imme-diate decrease in turbidity without a lag, suggesting thatKar3Vik1�ADP is the species that detaches from theMT.

The dissociation experiments were repeated with increasingconcentrations of ATP. The observed rates for the initial expo-nential phase of each transient were plotted as a function ofATP concentrationwith the hyperbolic fit of the data providinga rate constant of MT�Kar3Vik1 complex dissociation, k�3, of6.2 s�1 (Table 1). Because ATP binding and ATP hydrolysisoccur as fast steps, the results are consistent with the conclu-sion that Kar3Vik1 detachment from the MT at 6.2 s�1 is rate-limiting for steady-state ATP turnover (kcat � 6.7 s�1; Table 1)and occurs after the ATP-promoted powerstroke.

FIGURE 1. MantATP binding kinetics. The nucleotide-free MT�Kar3Vik1 com-plex was rapidly mixed in the stopped-flow instrument with increasing con-centrations of mantATP. Final concentrations were 5 �M Kar3Vik1 and 12.5 �M

MTs for 7.5– 40 �M mant-ATP and 2.5 �M Kar3Vik1 and 12.5 �M MTs for 2.5–7.5�M mantATP. A, representative transients at 5, 10, and 15 �M mantATP (bot-tom to top) show an exponential increase in fluorescence as a function oftime. Each transient was fit to a single exponential function plus a linear term.B, the observed rates of the initial exponential phase of the transients wereplotted as a function of mantATP concentration, and the fit of the data pro-vided a second-order rate constant of mantATP binding, k�1 of 0.8 � 0.04�M

�1 s�1 and k�1 of 7.4 � 0.9 s�1.

FIGURE 2. Pulse-chase kinetics of ATP binding. The nucleotide-freeMT�Kar3Vik1 complex was rapidly mixed in the chemical quench-flow instru-ment with increasing concentrations of [�-32P]ATP plus KCl and chased withunlabeled ATP. Final concentrations were 10 �M Kar3Vik1 and 30 �M MTs for25–125 �M [�-32P]ATP, 5 �M Kar3Vik1 and 30 �M MTs for 5– 40 �M [�-32P]ATP,and 100 mM KCl. A, representative transients display an ATP concentration-dependent presteady-state burst of [�-32P]ADP�Pi product formation fol-lowed by a linear phase of product formation. Each transient was fit to Equa-tion 2. B, the observed exponential rates of the presteady-state burst phasewere plotted as a function of ATP concentration and fit to Equation 3, provid-ing a K1k�1� of 18.6 � 0.03 �M

�1 s�1, K1 of 0.34 � 0.03 �M�1, k�1� of 54.9 � 1.0

s�1, and an apparent Kd,ATP of 2.9 �M. C, the amplitude of each burst phase (asthe fraction of Kar3Vik1 active sites) was plotted as a function of ATP concen-tration and fit to a hyperbolic function, providing a maximum burst ampli-tude of 1.07 � 0.02 per Kar3Vik1 heterodimer and apparent Kd,ATP of 25.2 �1.3 �M. B and C include data from multiple experiments.

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MTKar3Vik1 Collision Occurs through Vik1—Kar3Vik1 wasrapidly mixed with MTs in the stopped-flow instrument, andthe exponential increase in the turbidity signal was correlatedwith formation of the MT�Kar3Vik1 complex (Fig. 5A). Theobserved rate of the initial fast phase increased linearly as afunction of increasingMT concentration, and the fit of the dataprovided an apparent second-order rate constant, k�4, of 2.4�M�1 s�1 (Fig. 5B and Table 1). The observed rates of the sec-ond exponential phase varied from1 to 3 s�1 (Fig. 5B). The ratesof the second phase were not dependent upon theMT concen-tration and were too slow to account for steady-state turnoverat 6.7 s�1. Therefore, the second phase was assumed to be an

isomerization that led to an increase in turbidity but was notpart of the ATPase pathway.The second experiment used to evaluate the collision of

Kar3Vik1with theMTwas tomeasure the kinetics ofmantADPrelease because MT association dramatically activates ADPrelease from the active site of kinesins (38, 40, 44–47). AKar3Vik1�mantADP intermediate was generated by incubatingthe Kar3Vik1 with mantADP at a 1:6 ratio to exchange ADP atthe active site with the fluorescent analog mantADP. ThisKar3Vik1�mantADP complex was then rapidly mixed in thestopped-flow instrument withMTs plus 1mMATP. The bipha-sic transients shown in Fig. 6A do exhibit a short lag, and thefluorescence decrease is correlated with mantADP leaving thehydrophobic environment of the Kar3 active site. The observedrates of the initial fast phase, when plotted as a function of MTconcentration, provided amaximumrate constant ofmantADPrelease of 14 s�1 and a K1⁄2,MTs of 7.7 �M (Fig. 6B and Table 1).The observed rates of the second slow phase were 1–3 s�1 andwere independent ofMT concentration (Fig. 6B), suggesting anoff-pathway isomerization.When this experiment was performed for Kar3Cik1, there

was no lag at the start of the transients, and the initial fast phaseof mantADP release was 110 s�1 (Table 1 and Ref. 36). Further-more, the second phase for Kar3Cik1 showed MT concentra-tion dependence, and the hyperbolic fit of the data provided a

FIGURE 3. Acid quench kinetics of ATP hydrolysis. The nucleotide-freeMT�Kar3Vik1 complex was rapidly mixed in the rapid quench instrument withincreasing concentrations of [�-32P]ATP plus KCl. Final concentrations were 5�M Kar3Vik1 and 30 �M MTs for 5– 80 �M [�-32P]ATP, 10 �M Kar3Vik1 and 30�M MTs for 10 –200 �M [�-32P]ATP, and 100 mM KCl. A, representative tran-sients display a time-dependent presteady-state burst of [�-32P]ADP�Pi prod-uct formation followed by a linear phase of subsequent ATP turnovers. Eachtransient was fit to Equation 2. B, the observed exponential rates of the prest-eady-state burst phase were plotted as a function of ATP concentration, andthe hyperbolic fit provided a rate constant of ATP hydrolysis, k�2, of 25.9 � 1.4s�1 and an apparent Kd,ATP of 34.4 � 5.1 �M. C, the amplitude of each expo-nential burst phase (as the fraction of Kar3Vik1 active sites) was plotted as afunction of ATP concentration and fit to a hyperbola, providing a maximumburst amplitude of 0.91 � 0.03 per Kar3Vik1 heterodimer and a Kd,ATP of 16 �1.6 �M. B and C include data from multiple experiments.

FIGURE 4. ATP-promoted dissociation kinetics of the MT�Kar3Vik1 com-plex. A, the nucleotide-free MT�Kar3Vik1 complex was preformed and rapidlymixed in the stopped-flow instrument with either a nonhydrolyzable ATPanalog, AMPPNP (orange), buffer (black), ATP (red), or MgADP (blue). Final con-centrations were 2.5 �M Kar3Vik1; 5.5 �M MTs; 1000 �M ATP, ADP, or AMPPNP;and 100 mM KCl. B, the dissociation experiment was performed with increasingconcentrations of ATP plus KCl. Final concentrations were 3.5 �M Kar3Vik1 and7.5�M MTs for 3.5–200�M ATP, 2.5�M Kar3Vik1 and 6�M MTs for 2.5–100�M ATP,and 100 mM KCl. The observed exponential rates of the turbidity decrease of eachtransient were plotted as a function of ATP concentration and fit to a hyperbola,providing a k�3 of 6.2 � 0.1 s�1 and a K1⁄2,ATP of 3.7 � 0.3 �M.

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kmax of 5 s�1 with aK1⁄2,MTs of 5.6 �M. Because of the absence ofa lag, the interpretation was that the 110 s�1 rate constant rep-resented MT collision by Kar3 for �1⁄3 of the population, andthe 5 s�1 rate constant represented �2⁄3 of the population ofmotors that collided with the MT through Cik1 followed byKar3 MT association and mantADP release. Because steady-state ATP turnover for Kar3Cik1 was also 5 s�1, the authorsproposed that the release ofmantADPat 5 s�1 captured the rateof the structural transition for Kar3 binding to the MT afterCik1 MT collision (36).When the mantADP release kinetics for Kar3Vik1 are inter-

preted in the context of the kinetics for Kar3Cik1, it is obviousthat the Kar3Vik1 14 s�1 rate constant cannot represent the

intrinsic rate constant for mantADP release from Kar3 if thishead were to collide with the MT first independently of Vik1because it would be observed at 110 s�1. Rather, the mantADPrelease kinetics for Kar3Vik1 indicate that the initial collision isthrough Vik1 followed by Kar3 binding and mantADP releaseat 14 s�1. The observation that the K1⁄2,MTs for Kar3Vik1 (7.7�M) is similar to the second phaseK1⁄2,MTs for Kar3Cik1 (5.6�M)supports this interpretation. These results also suggest thatthere is an asymmetry in the Kar3Vik1�ADP species that pro-motes MT collision through Vik1.The Two-head-bound State Is Transient—Rank et al. (14)

revealed a novel MT binding pattern in which Kar3 and Vik1bound to adjacentMT protofilaments. This binding configura-tion requires that Kar3 and Vik1 be in opposite orientations onthe MT lattice because the coiled coil does not unwind signifi-cantly and predicts that the E2 state with both heads bound tothe MT prior to ADP release is transient (Fig. 7). This bindingpattern suggests that the intermediate with both heads boundwould not be stable because of the distance between MT bind-ing sites and the structural transition that occurs with tightbinding of Kar3 (48). In initial MT association experiments, weassumed that collision would occur through either Kar3 orVik1, and to bias binding through Vik1, ADP was added to theMT syringe. Fig. 5C shows that the kinetics were biphasic, and

FIGURE 5. MT Kar3Vik1 association kinetics. Kar3Vik1 with ADP tightlybound at the Kar3 active site was rapidly mixed in the stopped-flow instru-ment with increasing concentrations of MTs, and the increase in turbidity wasmonitored. A, representative transients are shown at 1.5, 2, and 2.5 �M MTs,and individual transients were fit to a double exponential function. Final con-centrations were 2.5 �M Kar3Vik1 for 2.5–12.5 �M MTs and 1.25 �M Kar3Vik1for 1.25–2.5 MTs. B, the observed rates of the initial exponential phase of eachtransient were plotted as a function of MT concentration, and the linear fit ofthe data provided a k�4 of 2.4 � 0.1 �M

�1 s�1. The observed rates of thesecond slow phase (red Œ) were 1–3 s�1 and were not MT concentration-de-pendent. C, Comparison of the association kinetics in the presence or absenceof 1 mM ADP. Final concentrations: 2.5 �M Kar3Vik1, 5 �M MTs, � 1 mM ADP.

FIGURE 6. MantADP release kinetics. The Kar3Vik1�mantADP complex wasrapidly mixed in the stopped-flow instrument with increasing concentrationsof MTs plus ATP, and the decrease in fluorescence was monitored. Final con-centrations were 2.5 �M Kar3Vik1 for 2.5–50 �M MTs, 1 �M Kar3Vik1 for 1–2.5MTs, 15 �M mantADP, and 1 mM ATP. A, representative transients at 2.5, 5, and15 �M MTs are shown (top to bottom), and each was fit to a double exponen-tial function. B, the observed rates of the initial fast exponential phase of thetransients were plotted as a function of MT concentration. The fit of the datato a hyperbola provided a kmax of 14.4 � 0.3 s�1 and a K1⁄2,MTs of 7.7 � 0.5 �M.The observed rates of the second slow phase (red f) showed no concentra-tion dependence and varied from 1 to 3 s�1.

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the initial exponential phase of theADP transientwas similar tothat of the transient in the absence ofADP.However, the ampli-tude associated with the ADP transient was about 3 timessmaller than that of the transient in the absence of ADP. Theseresults suggest that although MT collision occurred throughVik1, binding of Kar3 to theMTdestabilized Vik1 such that thehigh concentration of ADP resulted in complete detachment ofa significant population of the Kar3Vik1motors. This interpre-tation was reinforced when the association kinetics were meas-ured as a function of MT concentration in 1 mM MgADP (sup-plemental Fig. S1). The results showed that Kar3Vik1 collideswith MTs at 4 �M�1 s�1; however, high ADP promoted a sig-nificant off-rate of 30 s�1. This is in sharp contrast to the data inFig. 3B where there is no evidence of an off-rate in the absenceof added ADP.The MT cosedimentation analysis of Kar3Vik1 demon-

strated that Kar3Vik1 partitioned with the MTs in 1 mM

MgADP and showed saturatedMT binding at 1 Kar3Vik1 per 2��-tubulin subunits. However, at conditions in which the MTcomplex was formed with nucleotide-free Kar3Vik1, the stoi-chiometry became �1:1 (14). Furthermore, the cryo-EMresults showed a stoichiometry at 1:1 for nucleotide-freeKar3Vik1, and the binding was cooperative and dominated bythe tight binding of the Kar3 motor domain. The results fromeach of these experiments are consistent with the interpreta-tion that the E2 species (Fig. 7) is transient, and the tight bind-ing by Kar3 after ADP release (E3) destabilizes the Vik1 MTcomplex due to the strain between theKar3 andVik1 heads andis mediated by the coiled coil.

DISCUSSION

Proposed Model of the Kar3Vik1 Powerstroke Mechanism—Themodel that has emerged from the structural andmechanis-tic studies is presented in Fig. 7. In solution, Kar3Vik1 has ADPtightly bound at the active site (E0). Kar3Vik1�ADP collideswith theMT, and we propose that the productive binding stateis through Vik1 followed by Kar3�ADP association and ADPrelease (E0–E3). Release of ADP tightens the MT affinity ofKar3 such that this conformational change destabilizes theinteraction of Vik1 with the MT (E2–E3) and forms the E3intermediate poised forATPbinding. ATPbinding at Kar3 pro-motes rotation of the coiled-coil stalk with Vik1 in associationwith the coiled coil, resulting in the large conformationalchange for force production (E3–E4). ATP hydrolysis occursindependently of Vik1, and phosphate release coupled toKar3Vik1 detachment from the MT completes the cycle (E4–E5). This powerstroke mechanism requires both Vik1- andKar3-coordinated interactions with theMTmediated by head-head communication via the coiled coil. We propose that tightbinding of Kar3 to theMT and the interactions of Vik1 with thecoiled coil at E4 prevent slippage such that Vik1 cannot rebindto the MT and the coiled-coil stalk cannot revert back to theprepowerstroke state at E3. Furthermore, this mechanisticcapability is intrinsic to the MT�Kar3Vik1 complex withoutrequiring an MT-Kar3Vik1-MT cross-linked configuration,

FIGURE 7. ATPase Model for Kar3Vik1 Force Generation. The cycle beginsas Kar3Vik1�ADP collides with the MT and productive MT binding occurs byVik1 followed by Kar3�ADP association and ADP release (E0 –E3). The releaseof ADP tightens the MT affinity of Kar3 such that this conformational changedestabilizes the interaction of Vik1 with the MT (E2–E3) and forms the E3intermediate poised for ATP binding. ATP binding at Kar3 promotes rotationof the coiled-coil stalk toward the MT minus-end with Vik1 in association withthe coiled coil, resulting in the large conformational change for force produc-tion (E3–E4). ATP hydrolysis occurs independently of Vik1, and phosphaterelease coupled to Kar3Vik1 detachment from the MT completes the cycle(E4 –E5). Once detached from the MT, Kar3Vik1 returns to the E0 state foranother round of the ATPase cycle. This model assumes that Kar3Vik1 is non-processive with one powerstroke per ATP turnover, requiring the coordi-

nated interactions of both Vik1 and Kar3 with the MT. Vik1MHD, Vik1 motorhomology domain; Kar3MD, Kar3 motor domain.

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suggesting that theVik1 associationwith the coiled coil plays animportant role in preventing reversals at E4.The structural studies published previously can only capture

the more stable MT�Kar3Vik1 intermediates. Cryoelectrontomography (34) and cryo-EM with three-dimensional helicalanalysis (14) identified the E3 prepowerstroke intermediate andthe postpowerstroke E4 intermediate. Although most struc-tures of kinesin have revealed the solution state, the x-raycrystal structure for Kar3Vik1 (Protein Data Bank code 4ETP)identified the E3 prepowerstroke intermediate (14) but not thesolution state. The results presented here provide evidence forthe more transient intermediates and the structural transitionsthat occur.We propose that the Kar3Vik1 collides with theMTthrough Vik1, which promotes Kar3 binding and ADP release(Figs. 5 and 6). Both E1 and E2 are transient states that were notcaptured by the structural studies. The kinetics of ATP binding(Fig. 2) identified the transition from E3 to E4 at 55 s�1, and theresults from Fig. 3 revealed that ATP hydrolysis was intrinsic toKar3 and independent of Vik1. The dissociation kinetics (Fig. 4)confirmed that Kar3Vik1 detached from the MT after ATPhydrolysis and therefore from the postpowerstroke state. Theresults also indicate that it is after detachment from the MTthat Kar3Vik1 returns to the E0 state whose conformationbiases it for productiveMT binding through Vik1 at the start ofthe next ATP cycle.Kinesin-14s: Kar3Vik1, Kar3Cik1, and Ncd—Both the struc-

tural and mechanistic results indicate that the ATPase mecha-nisms for Kar3Vik1 and Ncd are very similar even though Ncdis a homodimer and the nucleotide state can modulate theinteractions of both heads with the MT (12–14, 33, 40, 42, 49,50). Both Ncd and Kar3Vik1 show cooperative binding to theMT lattice such that in a population of MTs one MT will becompletely saturated by Kar3Vik1 or Ncd, but other nearbyMTs show no evidence of MT binding (12, 14, 33). This coop-erative binding behavior is inherent to the dimer state becausewhen similar experiments are performed with the Kar3 motordomain the monomers bind stochastically to the MT (33, 51).Asymmetry for Ncd is generated through asymmetric affinityfor ADP where one head holds ADP tightly and the other holdsADP weakly when Ncd is in solution and detached from theMT, and Foster et al. (42) proposed that it was this asymmetrythat biased the head that boundADPweakly to collide and formthe productive MT association complex. The Ncd prepower-stroke intermediate captured by cryo-EM is very similar to theMT�Kar3Vik1 prepowerstroke intermediate, and ATP bindingmimicked by AMPPNP for both Kar3Vik1 and Ncd results inthe postpowerstroke intermediate (12–14, 49). Cryo-EM forboth Ncd and Kar3Vik1 showed the detached head in associa-tion with the coiled coil, providing additional evidence that thishead rotates with the coiled coil to generate the postpower-stroke intermediate (12–14). ATP hydrolysis for both Ncd (40)and Kar3Vik1 showed full burst amplitude, indicating thatreversals at this step to resynthesize ATP do not occur, andMTdetachment for both occurred from the postpowerstroke state.Therefore, based on these results, the evidence supports a con-servedmechanism for Kar3Vik1 andNcd for cross-linking par-allel MTs as would occur at the spindle poles.

In contrast, Kar3Cik1 does not exhibit the cooperative bind-ing observed for Ncd and Kar3Vik1 (51). Instead, Cik1 targetsKar3 to the MT plus-end, and Kar3Cik1 promotes MT short-ening in vitro at conditions where the MTs are not highly sta-bilized by paclitaxel. Kar3Cik1-promoted MT shortening doesrequire ATP turnover, and the single exponential kinetics sug-gest that a single ATP turnover promotes release of a single��-tubulin subunit from theMTplus-end. In contrast, becauseof the cooperative binding, Ncd and Kar3Vik1 did not exhibitthe same characteristics of MT plus-end targeting for MTshortening as observed for Kar3Cik1 (33). For Kar3Cik1, theburst amplitude for the ATP hydrolysis kinetics was reduced,and computational modeling suggested that the reduced burstamplitude was due to reversals of ATP hydrolysis to resynthe-size ATP (Table 1 and Ref. 36). The MT�Kar3Cik1 dissociationkinetics promoted by ADP were quite different whereby therewas no evidence that ADP promoted Kar3Cik1 detachmentfrom theMT. Chen et al. (36) proposed that ADP, when boundto Kar3, led to the formation of the MT�Kar3Cik1�ADP inter-mediate in which the Cik1 head was tightly bound to the MTand the Kar3�ADP head was weakly bound or detached buttethered to theMT throughCik1. These differences would sug-gest that the E2 state may be more stable for Kar3Cik1 thanKar3Vik1. The last major difference was in the kinetics ofMT�motor association followed by mantADP release. Thekinetics for Kar3Vik1 indicated that there was an asymmetry inthe dimer such that Kar3Vik1 was biased to bind through Vik1for productive MT�Kar3Vik1 complex formation. In contrast,the kinetics for Kar3Cik1 revealed that �1⁄3 of the populationcollided with the MT initially by Kar3 but that the productivecollision complex occurred through Cik1 followed by Kar3MTbinding (36). Despite these distinctive differences, the mecha-nistic studies indicate that Kar3Cik1, at least at conditions forMT cross-linking, will also use an ATP-promoted powerstrokemechanism with ATP hydrolysis intrinsic to Kar3 and occur-ring as the postpowerstroke intermediate.In summary, the results presented here show that there is a

conservedmechanism for kinesin-14Ncd andKar3Vik1with evi-dence suggesting that Kar3Cik1 will show similarities. The keyquestions ahead are directed at Kar3Cik1 to determine whether italso exhibits an ATP-promoted powerstroke, whether a power-strokemechanism is used during Kar3Cik1-promotedMT short-ening, and the structural differences in Cik1 that change the MTinteractions for specific cellular functions.

Acknowledgments—We thank Soheila Vaezeslami (University ofWisconsin) for work on the GCN4-Kar3 and GCN4-Vik1 constructs,and we acknowledge Harjinder Sardar (Rensselaer) and KatherineRank (University of Wisconsin) for thoughtful discussions.

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Kinesin-14 Kar3Vik1 Microtubule Interactions

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