JMB—MS 596 Cust. Ref. No. CAM 037/95 [SGML]

J. Mol. Biol. (1995) 249, 763–771

Kinesin and ncd Bind Through a Single Head toMicrotubules and Compete for a Shared MTBinding Site

Andrew Lockhart, Isabelle M.-T. C. Crevel and Robert A. Cross*

Kinesin and non claret disjunctional are closely related molecular motors thatMolecular Motors Groupmove in opposite directions along microtubules. We have used recombinantMarie Curie Research

Institute, The Chart, Oxted single-headed and double-headed constructs of both rat kinesin heavy chainand non claret disjunctional to investigate the interactions of these motorSurrey RH8 0TL, UKproteins with microtubules. At saturation the stoichiometry of binding fornon claret disjunctional and kinesin to microtubules is one molecule (singleor double-headed) per tubulin heterodimer. In the absence of addednucleotide, addition of increasing amounts of one motor results in thecompetitive displacement of the other motor from the microtubules. Thiseffect is apparent also in the presence of the nucleotide analogue5'-adenylimidodiphosphate, which tightens the binding of both kinesin andnon claret disjunctional. Competition for binding sites occurs also underconditions of steady-state ATP turnover. We conclude that despite theiropposite directionality, kinesin and non claret disjunctional compete foroverlapping binding sites on the MT surface.

Since the binding of the second head of a double-headed motor is stericallyblocked, the data imply also that both kinesin and non claret disjunctionalmay translocate via a processive (alternating heads) mechanism with aminimum step size of 08 nm.

Keywords: kinesin; non claret disjunctional; microtubules; molecular*Corresponding author motors

Introduction

Kinesin and non claret disjunctional (ncd) aremechanoenzymes able to convert the energy derivedfrom ATP hydrolysis into movement along micro-tubule (MT) tracks. Both proteins are modular, eachpossessing twin motor domains (containing theATPase activity and MT binding sites) and twincargo-binding domains, linked by a central region ofrod-like alpha-helical coiled-coil. The two motorsdiffer, however, in the ordering of the modules:kinesin possessing N-terminal motor domains, ncdC-terminal motor domains. Kinesin and ncd displaya strong sequence homology (041% amino acididentity) within their motor domains yet translocatealong MTs in opposite directions: kinesin towardsthe plus end (Vale et al., 1985) and ncd towardsthe minus end (Walker et al., 1990) of MTs. The

mechanism of directional reversal is unknown and ofgreat interest.

In principle, directional reversal could be achievedkinetically, by reversing a force generating confor-mational change. This would require adjustment ofthe coupling of the active-site chemistry to motorconformation (Taylor, 1993), whereas the availableevidence suggests that the manner in which the twoproteins hydrolyse ATP appears to be very similar;ADP release is rate-limiting in the absence of MTsand the interaction of the motors with MTs stronglyaccelerates ADP release (Hackney, 1988; Lockhart &Cross, 1994). This switches both proteins into strongbinding (force holding) states, suggesting that ADPrelease is associated with force generation in bothcases. Accordingly we have searched for structuraldifferences in the binding of ncd and kinesin to MTs.

The interaction of kinesin with MTs has beenstudied in some detail. Hackney has presentedevidence that only one of the two heads of a dimerickinesin can interact with each tubulin heterodimer inthe MT lattice (Huang et al., 1994) and only releasesone if its tightly bound ADP molecules upon binding

Abbreviations used: ncd, non claret disjunctional; MT,microtubule; MAP, microtubule-associated protein;mantATP, methylanthraniloyl-ATP; GST, glutathioneS-transferase; AMP-PNP, 5'-adenylimidodiphosphate.

0022–2836/95/240763–09 $08.00/0 7 1995 Academic Press Limited

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to the MT (Hackney, 1994a). On the basis of theseresults a processive (‘‘head over head’’) mechanismfor movement based on the alternating participationof each motor domain was proposed. Initialcross-linking experiments indicated that the kinesinhead can form cross-links to b-tubulin althoughthese data did not exclude the possibility that theprotein contacts the a-tubulin subunit as well (Song& Mandlekow, 1993). More recent cross-linkingexperiments indicate that kinesin interacts with botha and b-tubulin subunits (Walker, 1995). Electronmicroscopy has confirmed that there is one kinesinhead binding site per tubulin heterodimer and thatthe bulk of the mass of the head contacts one subunitof the heterodimer, although the resolution isinsufficient to determine if there is a minor contactwith the other tubulin subunit (Kikkawa et al., 1994).Observations of the movements of kinesin-coatedbeads along MTs are consistent with a stepsizeequivalent to the 8 nm interdimer spacing of tubulin(Svoboda et al., 1993) and parallel with the longprotofilament axis of 13-protofilament MTs (Rayet al., 1993). Other studies with subtilisin-digestedMTs indicate that the binding site for kinesin isoutside the C-terminal region of tubulin (Marya et al.,1994) and that kinesin and the microtubule-associated proteins (MAPs), MAP2c and tau, do notcompete for MT binding sites, although MAP2 caninterfere with kinesin-dependent MT motility(Rodionov et al., 1990; Heins et al., 1991).

Much less is known about the interaction of the ncdmotor domain with MTs. The binding stoichiometryat saturation with ncd has been reported to be onedouble-headed molecule per tubulin heterodimer(Lockhart & Cross, 1994) and cross-linking studiesindicate interaction with both a and b-tubulin(Walker, 1995). ncd has been reported to generatetorque as it moves along MTs, suggesting that it doesnot move along a single protofilament axis butfollows a helical track around the MT (Walker et al.,1990). Similar results have been reported for kinesinbut only when using MTs containing abnormalnumbers of protofilaments arranged in shallowhelical paths (Ray et al., 1993).

We have employed a number of single anddouble-headed forms of kinesin and ncd, which wehave characterised biochemically and employed toaddress how these force-producing proteins interactwith each other on their MT tracks. We report that inMT pelleting assays and steady-state kinetic assays,kinesin and ncd are able to displace each other fromthe MTs, indicating that these proteins stronglycompete for MT binding sites.

Results

Characterisation of recombinant proteins

In all, four recombinant proteins were used in theexperiments (Figure 1). Single-headed forms of ncd(ND327) and kinesin (KD340) were engineered tocontain just their minimal motor domains (ATP andMT binding sites) and a double-headed form of

Figure 1. Representations of recombinant kinesin andncd proteins used in the study. The motor domains containthe ATPase activity and MT binding sites of the proteins.

kinesin (KD401) was engineered to contain all of themotor domain plus 71 residues of its stalk. GST-MC5is a double-headed form of ncd and contains residues294 to 700 of ncd fused to glutathione S-transferase(GST), its production and characterisation have beendescribed (Chandra et al., 1993; Lockhart & Cross,1994). All of these proteins when expressedrecombinantly are soluble and can be purified tohomogeneity in large quantities making themsuitable for biochemical analysis.

The physical properties of the proteins wereanalysed using gel-filtration chromatography andrate zonal centrifugation to determine the Stokesradii and sedimentation coefficients of KD340, KD401and ND327 (Table 1). The data were used to calculatethe native molecular mass of the proteins and were

Table 1. Summary of the physical properties of therecombinant kinesin (KD340 and KD401) and ncd(ND327) proteins

Rs Calculated Polypeptide Association(nm) s20, w Mr (kDa) Mr (kDa) state

ND327 2.95 3.67 48.2 42.3 MonomerKD340 2.90 3.25 43.3 37.8 MonomerKD401 4.1 5.31 78.0 45.0 Dimer

The Stokes radii (Rs) were determined by gel filtrationchromatography and the sedimentation coefficients (sw,20) weredetermined by rate zonal centrifugation. The calculated Mr wasdetermined as described by Freifelder (1982) and the polypeptideMr from the proteins primary sequence.

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Table 2. Summary of the kinetic properties ofrecombinant kinesin and ncd proteins

Basal ATPaserate of ADP MT-activated ATPaserelease (s−1) Vmax (s−1) Kact (mM)

GST-MC5 0.0036 1.1 5.7ND327 0.0011 6.7 3.0KD401 0.0050 20.6 0.8KD340 0.0020 39.3 4.1

The rates of ADP release were obtained from mantATPturnovers and the MT-activated ATPase values were obtainedusing a pyruvate kinase/lactate dehydrogenase linked assaysystem as described (Lockhart & Cross, 1994). The data shown arean average of two experiments.

Figure 2. Binding isotherms for (a) kinesin: (q) KD401,(w) KD340 and (b) ncd: (r) GST-MC5 and (+) ND327to 5.6 mM MTs in the absence of added nucleotide.The dissociation constants (Kd) and the binding stoichio-metries were obtained by least squares fitting of the datato rectangular hyperbolae.

compared with the molecular mass calculated fromthe predicted primary sequence. The results areconsistent with monomeric ND327 and KD340 anddimeric KD401. GST-MC5 has been reported to existas a mixture of dimers and tetramers in solution(Chandra et al., 1993) but we have found itsbehaviour both kinetically and in MT binding assaysto be identical with the ncd dimer MC5 (GST-MC5without the GST fusion; Lockhart & Cross, 1994).

The rate-limiting step of the ATPase cycle for bothkinesin and ncd in the absence of MTs is ADP release(Hackney, 1988; Lockhart & Cross, 1994). The rates ofADP release by these proteins were investigatedusing a fluorescent ATP analogue, methylanthra-niloyl-ATP (mantATP) as described (Lockhart &Cross, 1994). The rates of ADP release aresummarised in Table 2 and show that all of theproteins have a low basal ATPase. For each proteinthe rates of mantATP binding and mantADP releasewere similar, consistent with the proteins beingpurified with ADP prebound in their active sites(Lockhart & Cross, 1994). In all cases the fluorescenttraces of the mant turnovers fitted well to singleexponentials indicating that the protein preparationswere kinetically homogenous (data not shown).

The ability of MTs to activate ADP release wasinvestigated using a steady-state linked assay asdescribed (Lockhart & Cross, 1994). All of theproteins demonstrated a strong MT-activated ATPasewith both the single-headed constructs showing asignificantly higher Vmax than their double-headedcounterparts (Table 2). The amounts of MTs requiredfor half maximal activation (Kact) for both thesingle-headed proteins ND327 and KD340 aresimilar, whilst the Kact values for double-headedmotors, KD401 and GST-MC5, are different: KD401possessing a lower Kact than KD340 and GST-MC5having a slightly higher Kact than ND327.

All of the constructs were tested for their abilityto move MTs in in vitro motility assays. OnlyGST-MC5, as previously reported, was able toproduce movement (Chandra et al., 1993). Theseresults are consistent with other studies (Stewartet al., 1993), which reported a progressive inhibitionof motility for both ncd and kinesin as the stalkregion was shortened. This inhibition of motilityprobably reflects steric limitations in the geometry ofmotility assays.

Microtubule pelleting assays: stoichiometry ofbinding of kinesin and ncd to MTs

The interaction of the motor proteins with MTs canbe followed quantitatively in solution using MTpelleting assays. In such experiments the motor/track is separated from any unbound motor by ultra-centrifugation and the pelleted samples analysed bySDS-PAGE.

In the absence of added nucleotide the binding ofsingle-headed (KD340) and double-headed kinesin(KD401) saturated at a stoichiometry of one motormolecule per tubulin heterodimer and the Kd valuesfor KD340 and KD401 were 02.6 mM and 01.2 mMrespectively (Figure 2(a)). The binding of ND327 andGST-MC5 both saturated at a stoichiometry of onemotor molecule (single or double-headed) pertubulin heterodimer and had Kd values of 03.8 mMand 00.7 mM, respectively. The Kd reported forGST-MC5 in this study is threefold less than thevalue reported previously (Lockhart & Cross, 1994)and we believe this difference is due to our removalin the present experiments of contaminating traces ofguanine nucleotides by washing the MTs. Over the

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course of the study it became apparent that thebinding of both motor proteins to the MTs wassensitive to any exogenous GTP/GDP carried overfrom the MT polymerisation step and due to the factthat both kinesin (Gilbert & Johnson, 1993) and ncd(data not shown) are able to efficiently hydrolyseGTP instead of ATP. Pelleting and resuspending theMTs in buffer minus GTP before use alleviated thiseffect.

The increased MT affinity (decreased Kd values) ofKD401 and GST-MC5 relative to their single-headedcounterparts may simply be due to the increasedlocal concentration of heads which results fromthe tethering of unbound heads by bound heads.Alternatively, there may be subtle differences in thestructure of single heads relative to the heads ofdouble-headed molecules.

MT pelleting assays: competition assays usingKD401 and GST-MC5 +/– AMP-PNP

The pelleting assays were performed by incubat-ing increasing amounts of one motor (0.5 to 5 mM)with a fixed amount of MTs (2.5 mM) and adding tothis mixture a fixed amount of the other motor(5 mM). The resulting MT pellets were then analysedfor the relative amounts of bound motor. Figure 3(a)illustrates the effect of adding 5 mM GST-MC5 to MTsincubated with increasing amounts of KD401. As theamount of KD401 in the assay is increased more of themotor binds to the MTs and this results in less ofthe added GST-MC5 binding to the MTs. Analysis ofthe gel (Figure 4(a)) indicates that the total amountof motor protein pelleting remains fixed (02.5 mM)and is at the expected stoichiometry if only onehead of either motor can bind at a time to the MTs.Repeating the experiment with increasing amountsof GST-MC5 and fixed amounts of KD401 reproducesthe result: as more GST-MC5 is added less KD401 isable to bind to the MTs (data not shown).

The effect of the non-hydrolysable nucleotideanalogue 5'-adenylimidodiphosphate (AMP-PNP)on the assay was also investigated. Figure 3(b)illustrates the effect of adding 5 mM GST-MC5 toincreasing amounts of KD401 in the presence of1 mM AMP-PNP. The addition of AMP-PNP resultsin less GST-MC5 binding and more KD401 bindingto the MTs and at each concentration an approxi-mately equal amount of each protein bound to theMTs (Figure 4(b)). This was true also when KD401was added to increasing GST-MC5 and is in contrastto the assays performed without AMP-PNP whereGST-MC5 binds in excess over kinesin (Figure 4(c)).AMP-PNP is thus able to tighten the binding of bothkinesin and ncd to MTs (i.e. decreases their Kd valuesrelative to the no added nucleotide results reportedabove). The constant level of saturation binding,together with the symmetrical binding curves of thetwo motors, irrespective of their order of additionand of the added nucleotide, indicates that theinhibition constant for kinesin antagonising thebinding of ncd is equal to that of ncd antagonising

Figure 3. Competition between kinesin and ncd for MTbinding sites. (a) SDS-PAGE of competition experimentperformed in the absence of added nucleotide. MT pelletsshow the effect of adding GST-MC5 (5 mM) to MTs (2.5 mM)preincubated with increasing amounts of KD401 (0.5,1, 2, 2.5, 3.75 and 5 mM). (b) SDS-PAGE of competitionexperiment performed in the presence of 1 mM AMP-PNP.MT pellets show the effect of adding GST-MC5 (5 mM) toMTs (2.5 mM) preincubated with increasing amounts ofKD401 (0.5, 1, 2, 2.5, 3.75 and 5 mM).

kinesin, in other words that there is classicalcompetitive binding.

Steady-state kinetic competition assays

Because MT-activated KD340 can turn over ATPmuch faster than the ncd constructs it was possibleto set the conditions used in the steady-state assayssuch that the rate of ATP hydrolysis by the smallamount of KD340 used in the assays was around fourtimes faster than that of ncd. This ensured thatchanges in hydrolysis rate upon the binding ordisplacement of KD340 were easily sensed by theassay. The concentration of ncd proteins used in theassays were set so they were at their maximal rates.Further addition of ncd did not increase the rates ofATP hydrolysis indicating that under the conditionsof the steady-state assay ncd binding sites weresaturated (data not shown).

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Figure 4. Competition between kinesin and ncd for MTbinding sites. Analysis showing the amount of motorbound in MT pellet versus the amount of kinesin added in(a) the absence of added nucleotide and (b) the presence of1 mM AMP-PNP. (w) The amount of KD401 in the MTpellet; (q) the amount of GST-MC5 in the MT pellet; and(r) the total amount of motor in the MT pellet.

Figure 5. Steady-state kinetic competition assaysbetween KD340 and ND327. MT-activated ATPasemeasurements was measured using a pyruvate kinase/lactate dehydrogenase linked assay system as described(Lockhart & Cross, 1994). (a) The continuous black lineshows unprocessed data from one of the experiments. Theabsorbance of NADH was monitored at 340 nm using aShimadzu UV-1201 spectrophotometer. A stable baselinewas first recorded, ND327 was added to the reactionmixture (final concentration 10 mM) and the reactionfollowed for around 150 seconds. KD340 was then added(final concentration 0.5 mM) and the reaction followed fora further 150 seconds. The two phases of the reaction wereanalysed using KFIT (a kind gift from Dr N. Millar) tomeasure the steady-state rates of ADP production byND327 alone and ND327 + KD340 combined. The steady-state rate of KD340 alone added to the MTs was determinedseparately. The broken line overdrawn on the second halfof the trace illustrates the expected trace if ND327 andKD340 were able to be independently activated by MTs. (b)Histogram showing average calculated rates of ADPproduction (n = 3). Hatched bars indicate experimentalfindings, whilst the open bar indicates the expectedcombined rate if ND327 and ND340 are not competing forMT binding sites.

Initially the two single-headed proteins, ND327and KD340, were used in the steady-state assays. Asmall amount of MTs (1 mM) was mixed with atenfold excess of ND327 and the resulting activatedrate of ATP hydrolysis measured (Figure 5(a)). Asmall amount of KD340 (0.5 mM) was added to thisreaction and the combined rates of ATP hydrolysismeasured. The combined rates of hydrolysismeasured in these experiments were significantlyless than the sum of the ND327 and KD340 ratesmeasured separately (Figure 5(b), two-tailed t-test,p < 0.1%), indicating that KD340 was unable tointeract significantly with the MTs under theseconditions and that the two motors were competingstrongly for MT binding sites.

The kinetic assays were also performed using thedouble-headed GST-MC5 instead of ND327. Theexperiment was performed two ways. Firstly, MTs(1 mM) were incubated with KD340 (2 mM) and the

rate of ATP hydrolysis recorded, GST-MC5 (10 mM)was then added to the reaction and the new rate ofATP hydrolysis recorded. The rates of hydrolysismeasured in these experiments were again signifi-cantly less than the combined rates of the two motorsalone and were dominated by the slower GST-MC5

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Figure 6. Steady-state kinetic competition assaysbetween KD340 and GST-MC5. Histogram showingaverage calculated rates of ADP production (n = 2).Hatched bars indicate experimental findings, while theopen bar indicates the combined rate if GST-MC5 andND340 are not competing for MT binding sites.

next binding site. Such a mechanism would allow the08 nm step to contain contributions from bothheads. In the kinesin case, the trailing head woulddirect the leading head towards the plus end of theMTs, and in the ncd case, towards the minus end. Onthe basis of the current data we can conclude that atleast, simple steric hindrance causes the bound headto constrain the behaviour of the free head, for bothkinesin and ncd.

The mechanistic implications of this depend onthe MT lattice and on the fidelity of protofilamenttracking by the motor. Double-headed kinesin tracksprotofilaments with remarkable fidelity (Ray et al.,1993), moving along the MT axis of MTs with straightprotofilaments, and helically on MTs with helicalprotofilaments. The fidelity appears to require bothheads, because single-headed kinesin loses fidelity,moving much more slowly and making manyexcursions sideways across the MT surface (Berlineret al., 1995). ncd has been reported to move helicallyon MTs (Walker et al., 1990), but it is unclear whetherthis requires helical protofilaments, as only aproportion of the MTs in the assays rotated.

Given that double-headed kinesin faithfullyfollows the protofilaments axis, the current data con-siderably restrict the number of feasible mechanismsby which it might walk. If, as we suspect, ncd alsofollows protofilaments, then the following commentswill apply equally to ncd. Our titration data indicatethat the spatial distribution of kinesin/ncd headtight binding sites corresponds to that of the tubulinheterodimer lattice, although it might well bedisplaced relative to the underlying heterodimerlattice (Figure 7). To emphasise this point, the bindingsites in Figure 7 are shown straddling a andb-tubulin, and straddling protofilaments. How mighta double-headed motor step between these sites? Ifthe double-headed motor faithfully follows proto-filaments, then the two heads of the motor mustindividually track a protofilament. In the walkingmodels (Figure 7(b) and (c)) the two heads trackneighbouring protofilaments and in the tightropemodel (Figure 7(d)) they both track the same proto-filament. We cannot distinguish between thesemodels at present although it has been shown thatkinesin does not move on the antiparallel proto-filaments of zinc tubulin macrotubes (Ray et al.,1994), suggesting (but not proving) that movementon a single protofilament is unfeasible.

The interaction between kinesin and ncd on theMT lattice was investigated using MT pelletingassays and under conditions of steady-state ATPhydrolysis. The data indicate that kinesin and ncdheads target overlapping sites on the MT surface.Competition was apparent with no added nucleotideand with 1 mM AMP-PNP in the MT pelleting assaysand under conditions where the motors were activelyhydrolysing ATP. It is unclear in which nucleotidestate AMP-PNP traps either kinesin or ncd, but thebinding assay results suggest that it is much tighterthan the no added nucleotide state and may be morelike a ‘‘rigor’’ strong binding state. That theseoppositely directed kinesin superfamily motors

rate (Figure 6). When the experiment was performedin reverse with the KD340 (2 mM) added to theGST-MC5 (10 mM) and MTs (1 mM) the combinedrate of ATP hydrolysis was again substantially lessthan the sum of the two motors alone demonstratingthat KD340 could not significantly interact with theMTs.

Discussion

The data reported above indicate that the bindingof both double-headed and single-headed kinesinand ncd proteins saturates at a stoichiometry of onemolecule per tubulin heterodimer and that kinesinand ncd heads target overlapping, possibly identicalsites on the MT surface. How do these data informour view of the mechanism of these motors?

Binding of both double-headed and single-headedmotors saturates at a stoichiometry of one moleculeper tubulin heterodimer. This implies that for ncd aswell as kinesin (Huang et al., 1994; Harrison et al.,1993) only one head of a twin-headed molecule maybind at a time, consistent with a mechanism for bothmotors in which alternate heads bind tightly to themicrotubule. Related evidence that kinesin walks inthis way was reported by Hackney (1994a), whoshowed that MTs activated the release of only0.5 mol ADP per head of a recombinant two-headedkinesin, but displaced ADP stoichiometrically froma single-headed recombinant kinesin. Difficulty wasreported in making a direct titration of the MTbinding stoichiometry of single-headed kinesinbecause of problems with unspecific aggregation(Huang & Hackney, 1994). We were able to make anunambiguous titration of kinesin (possibly owing toour use of rat kinesin rather than Drosophila), and ofncd, allowing us to rule out the possibility that bothheads of a double-headed motor can bind simul-taneously and tightly. This behaviour provides anobvious candidate guidance mechanism (Schnapp,1995), whereby the tightly bound trailing head of apair guides the leading head with high fidelity to its

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Figure 7. A representation ofpotential stepping mechanisms formotor movement. (a) The spatialdistribution of motor binding sitesoverlaid on the MT lattice (shownwith a B lattice). The binding sites areshown as open circles and eachtubulin heterodimer is representedby one shaded and one open sphere.(b), (c) and (d) The potential ways adouble-headed motor could movealong protofilaments. The verticallines drawn alongside each latticeshow the axial distances betweeneach chosen binding site (i.e. thestep size). In (b) and (c) theheads (labelled 1 and 2) straddle one

protofilament. Model (b) results in a uniform step size of 08 nm and model (c) results in steps of alternating size,but which are on average 08 nm. In (d) both the heads follow the same protofilament resulting in a uniform stepsizeof 08 nm.

compete for MT binding sites is clearly a biologicallyimportant result, but it is necessary to consider if theresult is inevitable: is there space on the MT surfacefor two kinesin head-sized molecules to bind atmutually distinct sites? Recent electron microscopyon kinesin-decorated MTs indicate that the bulk ofthe mass of the kinesin head superimposes on onetubulin subunit (Kikkawa et al., 1994). Additionalevidence that there is considerable free space comesfrom the finding that kinesin, MAP1 and MAP2 donot compete for MT binding sites (Rodionov et al.,1990). Tau protein also binds to MTs without an-tagonising kinesin binding (Heins et al., 1991). Thisevidence suggests that kinesin and ncd heads couldin principle bind independently, but our competitiondata indicate that they do not.

Our finding that ncd and kinesin share a commonsite brings the problem of ncd/kinesin directionalityinto sharper focus. In a walking mechanism, thebound head acts in some way to target the unboundhead, restricting its choice of binding sites. In thekinesin case, the bound head directs the unboundhead towards the plus end of the MTs, and in the ncdcase, towards the minus end. It is unclear how thisis achieved, but the current results constitute furtherevidence for functional homology between thestrong binding states of ncd and kinesin, and are thusconsistent with our earlier suggestion that the sourceof directionality might be directionally biased weakbinding of the motor to the MT (Lockhart & Cross,1994).

Materials and Methods

Construction of plasmids for kinesin andncd expression

The construction of the plasmid for GST-MC5 hasbeen described (Chandra et al., 1993). ND327, containingresidues 327 to 700 of ncd was constructed by amplifyinga 660 bp fragment from the ncd clone pET/ncd (Chandraet al., 1993) using 5'-GAGACCTGCAAACATATGCTCT-TCCAGTCG-3' and 5'-GGTGATTTGGATCCAGAACCG-3'

as the forward and reverse primers, the forward primerintroducing an NdeI site onto the 5'-end of the amplifiedproduct. All PCR reactions were performed using theproof-reading thermostable DNA polymerase Vent (NewEngland Biolabs) according to the manufacturers instruc-tions. The amplified DNA was purified from an agarosegel, digested using NdeI/BamHI and cloned intoNdeI/BamHI-cut pET/ncd.

KD340 and KD401 were constructed from a full-length rat kinesin heavy chain clone and contain residues1 to 340 and 1 to 401, respectively, from this clone.For KD340 a 1020 bp fragment was amplified using 5'-CCGCTCTACATATGGCGGACCCAGCCGAATGCAGC-3'and 5'-TTTCTGGATCCACTATTCTGCTGTTAGTTCC-3'as the forward and reverse primers. The forward primerintroduced an NdeI site on the 5'-end of the amplifiedproduct and silently removed a BamHI site. The reverseprimer introduced a BamHI site and a stop codon onto the3'-end of the amplified DNA. The forward primer forKD401 was the same as that used for KD340 and the reverseprimer sequence was 5'-CCACAGGATCCTAGTTGTCTAT-GATGGG-3'. This primer introduced a BamHI site and astop codon onto the 3'-end of the amplified DNA. Theamplified DNA was purified from an agarose gel, cut usingNdeI/BamHI and cloned into NdeI/BamHI-cut pET17b(AMS Biotechnology Ltd).

Expression and purification of recombinant proteins

The overexpression and purification of GST-MC5 hasbeen described (Lockhart & Cross, 1994). The expressionof the remaining constructs was performed in BL21(DE3)cells. Overnight cultures of bacteria containing theappropriate construct were diluted 1 in 40 into 2 × TYmedium supplemented with ampicillin (50 mg/ml) andgrown, with shaking, at 37°C until the absorbanceat 600 nm was 1. The cell cultures were then shaken at22°C for 30 minutes before induction with isopropylb-D-thiogalactopyranoside (final concentration 0.4 mM)and after a further 16 hours shaking at 22°C the cells wereharvested by centrifugation. The cell pellets were thenfrozen in liquid nitrogen and stored at −80°C.

KD340, KD401 and ND327 were purified as follows. Thebacterial cell pellets were resuspended (1 g/3 ml) in bufferA (20 mM Pipes (pH 6.9). 1 mM DTT, 2 mM MgCl2, 1 mMEDTA and 1 mM phenylmethylsulphonyl fluoride) andincubated on ice with lysozyme (0.1 mg/ml; Sigma) for

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20 minutes. The cell lysate was supplemented with furtherMgCl2 to 10 mM and deoxyribonuclease I (40 mg/ml;Sigma) and incubated for a further 20 minutes on ice. Thesupernatant was clarified by centrifugation (27,000 g,40 minutes, 4°C) and the cell pellet discarded.

All chromatography steps were performed at 4°C. Thesupernatants were immediately loaded onto a columnpacked with 10 ml of Mono SP resin (Pharmacia)equilibrated in buffer A. After extensive washing of thecolumn with buffer A, ND327 was eluted using buffer Asupplemented with 200 mM NaCl. Column fractions wererapidly analysed for purity using SDS-PAGE and the peakfractions pooled. Typical yields of ND327 from 100 g ofcells were 6 ml at 10 mg/ml.

KD340 and KD401 were eluted from the Mono SP resinusing buffer A supplemented with 100 mM NaCl. Columnfractions were rapidly analysed using SDS-PAGE and thosecontaining the protein pooled and applied directly to a 1 mlMono Q column (Pharmacia) equilibrated in buffer A.KD340 and KD401 were eluted from the Mono Q columnusing buffer A supplemented with 300 mM NaCl and thepeak fractions pooled. Typical yields of KD340 and K401from 100 g of cells were 6 ml at 4 mg/ml.

Frozen protein stocks were prepared by supplementingthe purified protein solutions with 20% (v/v) glycerol,dividing into 50 ml aliquots, flash freezing in liquidnitrogen and storing at −80°C. When stored in this mannerthe proteins were stable for at least two months and oncethawed were used immediately.

Calculation of protein concentrations

Protein estimations were calculated spectrophotometri-cally based on extinction coefficients calculated from theprotein primary sequence, using values of 5690 M−1 cm−1

and 1280 M−1 cm−1 for the extinction coefficients oftryptophan and tyrosine at 280 nm, respectively (Gilbert &Johnson, 1993). As the proteins are all purified withstoichiometric bound ADP an extra extinction coefficientof 2500 M−1 cm−1 per ADP was added to the calculatedextinction coefficients (Gilbert & Johnson, 1993). Thecalculated extinction coefficients for KD340, KD401,ND327 and GST-MC5 were 15300, 27780, 29100 and71100 M−1 cm−1, respectively. All concentrations are ex-pressed per single chain.

Purification and polymerisation of tubulin

Tubulin was purified from porcine brain as described in(Lockhart & Cross, 1994). Tubulin (typically 5 mg/ml in50 mM Pipes (pH 6.9), 1 mM EGTA, 0.2 mM MgCl2,0.01 mM GTP and 20% glycerol) was polymerised by theaddition of MgCl2 and GTP to a final concentration of 2 and1 mM, respectively. The tubulin was incubated for 30minutes at 37°C, at which point Taxol (Sigma) was addedto a final concentration of 20 mM. The Taxol-stabilised MTswere pelleted by centrifugation (100,000 g, 20 minutes,25°C) in order to remove guanine nucleotides. Thesupernatant was carefully removed and the MT pelletcarefully resuspended in BRB 80 buffer (80 mM K-Pipes(pH 6.8), 1 mM MgCl2, 1 mM EGTA) containing20 mMTaxol.

mantATP turnovers

mantATP was synthesised as described (Hiratsuka,1983) and purified on a DEAE-cellulose column with alinear 0.1 M to 1 M triethylamine (pH 7.5) gradient.

mantATP turnovers by the purified proteins wereperformed as described (Lockhart & Cross, 1994).

MT activated ATPase rates

Steady-state MT activated ATPase rates were performedusing a pyruvate kinase/lactate dehydrogenase linkedassay as described (Lockhart & Cross, 1994). Values forVmax and Kact for the different proteins were obtained byleast-squares fitting MT activation data to rectangularhyperbolae using Kaleidagraph 2.1.3 (Synergy Software).The conditions used in the kinetic competition exper-iments are described in the relevant section of the paper.

MT pelleting assays

Pelleting assays were performed in 20 mM sodiumphosphate (pH 7.2), 1 mM DTT, 25 mM KCI, 2 mM MgCl2.The motor proteins (1, 2, 4, 5, 7.5 and 10 mM) were addedto Taxol-stabilised MTs (5.6 mM) and the volume adjustedto 40 ml. For KD340 only, the concentrations of motor usedin the assay were 0.7, 1.4, 2.8, 3.5, 5.25 and 7 mM. The finalNaCl concentration in the assays was 20 mM. The sampleswere gently mixed and centrifuged in a Beckman TL100ultracentrifuge at 100,000g for ten minutes at 20°C. Thesupernatants were carefully removed and the pelletsresuspended in 1 M NaCl. SDS-gel loading buffer wasadded to the samples and equal amounts of pellet andsupernatant were analysed by SDS-PAGE on a 5% to 20%(w/v) polycrylamide microslab gel (Laemmli, 1970). Afterelectrophoresis the gels were stained with Coomassiebrilliant blue R-250 (Sigma) to visualise the protein bandsfor analysis.

Grayscale CCD video images of the stained gels werecaptured into a Macintosh computer using a Scion AG-5framegrabber and the relative integrated intensities of theprotein bands in the supernatants and pellets determinedusing NIH Image 1.55 (Freeware). The amount of sampleloaded onto the gels was optimised to ensure a linearrelationship between the amount of protein loaded andthe integrated stain density. Binding parameters wereobtained by fitting rectangular hyperbolae to plots of theconcentration of motor in the pellet versus its concentrationin the supernatant using Kaleidagraph 2.1.3.

MT pelleting assays: competition assays usingKD401 and GST-MC5 + /– AMP-PNP

The buffer conditions used in the competition assayswere as described above except that the final volume of theassays was 80 ml and the assays were performed +/−1 mMAMP-PNP (Sigma). Increasing amounts of one motor (0.5,1, 2, 2.5, 3.75 and 5 mM) were incubated with MTs (2.5 mM)and to this a fixed amount (5 mM of the other motor added.The samples were gently mixed and centrifuged in aBeckman TL100 ultracentrifuge at 100,000g for ten minutesat 20°C. The centrifuged samples were then treated andanalysed as described above in order to determine therelative amounts of KD401 and GST-MC5 in the MT pellets.

Estimation of Stokes radius

Stokes radii (Rs) were determined using a Superose12 gel-filtration column (Pharmacia) equilibrated in20 mM Pipes (pH 6.9), 1 mM DTT, 2 mM MgCl2, 200 mMNaCl. The column was calibrated with proteins ofknown Rs: bovine pancreas ribonuclease A (1.64 nm),bovine pancreas chymotrypsinogen (2.09 nm), hen egg

JMB—MS 596

Interaction of Kinesin and ncd with Microtubules 771

ovalbumin (3.05 nm), bovine serum albumin (3.55 nm)and yeast alcohol dehydrogenase (4.6 nm).

Estimation of sedimentation coefficients

Sedimentation coefficients (s20, w) were determined byrate zonal centrifugation on a 15% to 35% (v/v) glycerolgradient in 20 mM Pipes (pH 6.9), 1 mM DTT, 2 mMMgCl2, 200 mM NaCl. The gradients were centrifuged at50,000 revs/min in a TST60.4 rotor (Sorvall) for 21 hours.Bovine serum albumin (4.6 S), rabbit muscle creatinekinase (5.3 S), rabbit muscle aldolase (7.3 S) and bovineliver catalase (11.3 S) were run as standards in a separatecentrifuge tube. The molecular mass of KD401, KD340 andND327 was calculated according to Freifelder (1982).

Acknowledgements

We thank Dr Sharyn Endow for the kind gift of thencd clone and the GST-MC5 clone, Dr Anne Sperry andDr Scott Brady for the kind gift of the rat heavy chainclone, and Dr Danny McKillop for helpful discussionsthroughout this work.

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Edited by J. Karn

(Received 23 January 1995; accepted 4 April 1995)