Nucleotide-Dependent Interaction of the N-Terminal Domainof MukB with Microtubules

Andrew Lockhart1 and John Kendrick-Jones

Structural Studies Division, MRC-Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom

Received June 10, 1998, and in revised form October 29, 1998

The MukB protein from Escherichia coli has adomain structure that is reminiscent of the eukary-otic motor proteins kinesin and myosin: N-terminalglobular domains, a region of coiled-coil, and aspecialised C-terminal domain. Sequence alignmentof the N-terminal domain of MukB with the kinesinmotor domain indicated an D22% sequence identity.These observations raised the possibility that MukBmight be a prokaryotic motor protein and, due tothe sequence homology shared with kinesin, mightbind to microtubules (Mts). We found that a con-struct encoding the first 342 residues of MukB(Muk342) binds specifically to Mts and shares anumber of properties with the motor domain ofkinesin. Visualisation of the Muk342 decorated Mtcomplexes using negative stain electron microscopyindicated that the Muk342 smoothly decorates theoutside of Mts. Biochemical data demonstrate thatMuk342 decorates Mts with a binding stoichiometryof one Muk342 monomer per tubulin monomer. Thesefindings strongly suggest that MukB has a role inforce generation and that it is a prokaryotic homo-logue of kinesin and myosin. r 1998 Academic Press

Key Words: chromosome partition; FtsZ; kinesin;motor; MukB; tubulin.

INTRODUCTION

The crystal structures of the motor proteins kine-sin (Kull et al., 1996) and myosin (Rayment et al.,1993) revealed that they shared a surprising numberof secondary structural elements. One explanation isthat they evolved from a common ancestor, but asyet, there is no clear evidence that such a motorexists in prokaryotes (Sakowicz and Goldstein, 1996).However, one possible candidate is MukB, which is a

large multidomain protein (Mr 5 177 kDa) involvedin the process of chromosome partition in Esch-erichia coli (Niki et al., 1991). This process results inthe accurate positioning of the replicated nucleoidsto the one-quarter and three-quarter cell lengths ofthe bacterium before cytokinesis occurs. The gene forMukB was identified from a genetic screen of parti-tion-deficient strains as being defective in the posi-tioning of replicated chromosomes (Niki et al., 1991).

Despite being biochemically uncharacterised, ithas been suggested that the protein is a bacteriallinear motor protein (Rothfield, 1994; Levin andGrossman, 1998). This suggestion is based mainly onthe predicted domain structure of MukB bearing aresemblance to both kinesin and myosin. MukBforms homodimers and its twin N-terminal domainsare separated from its much larger C-terminal do-mains by a region of predicted coiled-coil (Niki et al.,1991, 1992). In addition, the N-terminal domain of,340 residues is around the same size as that of thekinesin motor domain and contains predicted nucleo-tide-binding motifs (Niki et al., 1991). The largerC-terminal globular domain consists of around 870residues and because it contains sequence elementsthat may form zinc finger motifs, it has been postu-lated to be involved in interactions with other pro-teins and/or DNA (Niki et al., 1991). Although a pointmutation in this domain abolishes the DNA-bindingactivity of the protein (Saleh et al., 1996), it is stillunclear what the sequence specificity of MukB is forDNA.

Since a previous sequence comparison (Niki et al.,1991) indicated that the N-terminal domain of MukBhad a significant level of homology to the Mt-bindingprotein dynamin, we aligned the sequence of thisdomain with those of the motor domains of twomembers of the kinesin superfamily, kinesin andNcd (Fig. 1). The alignments indicated a strongsequence homology (34%) between the proteins witha sequence identity of 22%. We also noted that MukBshared a high degree of conservation around the

1 To whom correspondence should be addressed at presentaddress: Imutran Ltd. (A Novartis Pharma AG Co.), P.O. Box 399,Cambridge CB2 2YP, UK. Fax: 144 1223 847467. E-mail:andrew.lockhart@pharma.Novartis.com.

JOURNAL OF STRUCTURAL BIOLOGY 124, 303–310 (1998)ARTICLE NO. SB984056

303 1047-8477/98 $25.00Copyright r 1998 by Academic Press

All rights of reproduction in any form reserved.

nucleotide-binding sequences (labelled N-1 to N-4 inFig. 1) of kinesin (Kull et al., 1996) and Ncd (Sablin etal., 1996). The overall level of sequence identity iswithin the same range as that displayed between themotor domains of distantly related kinesin-like pro-teins, such as Smy1, Nod, Ncd, and Kar3, which arearound 25% identical (Lillee and Brown, 1992).

These sequence comparisons raised two intriguingpossibilities. First, that MukB may be a prokaryotichomologue of kinesin and myosin and that, second, itmight bind to Mts. In addition, the demonstration ofa specific interaction between MukB and Mts wouldprovide strong circumstantial evidence that the invivo binding partner of MukB is the prokaryotictubulin homologue FtsZ (Erickson and Stoffler, 1996).

The crystal structures of both FtsZ (Lowe andAmos, 1998) and tubulin (Nogales et al., 1998) haverecently been solved and indicate that both proteinsdisplay a strong degree of structural homology. Fur-thermore, a number of studies have demonstratedthat tubulin and FtsZ also share functional homolo-gies in that they can assemble into a range offilamentous polymer structures both in vivo and invitro (reviewed in Erickson, 1995). However, com-pared with the large amount of biochemical dataavailable on the polymerisation of tubulin, and thepresence of a range of Mt stabilising drugs, fewsimilar quantitative studies have been performedwith FtsZ and no compounds are yet available tostabilise FtsZ polymers in the absence of GTP. Thesefactors, at present, limit the use of FtsZ in quantita-tive biochemical assays. We therefore used a previ-ously characterised recombinantly expressed N-ter-

minal domain of MukB (Muk342; Lockhart andKendrick-Jones, 1998) to see whether we could de-tect, and characterise, the binding of this protein tomicrotubules.

MATERIALS AND METHODS

Construction of pETMuk342 and pETMukR250A. The con-struction of pETMuk342 is described in Lockhart and Kendrick-Jones (1998) and the plasmid contains a polyhistidine tag(RGSH6) on its C-terminus. MukR250A was made by digestingpETMuk342 with HindIII and RsrII to release an ,155-bpfragment. This fragment was gel-purified and then amplifiedusing polymerase chain reaction with a oligonucleotide primercorresponding to the 58 end of the fragment and a mutagenicprimer encoding the 38 end of the fragment and containing thearginine-to-alanine mutation. The amplified DNA was digestedwith HindIII/RsrII and re-ligated into gel-purified HindIII/RsrII-cut pETMuk342 to form the plasmid pETMukR250A. Sequencingconfirmed the presence of the mutation and the protein,MukR250A, was expressed and purified in the same manner asMuk342 (described below).

Expression and purification of Muk342 and MukR250A.Muk342 and MukR250A were expressed and purified as describedin Lockhart and Kendrick-Jones (1998). The concentration of thepurified protein was determined at 280 nm using an extinctioncoefficient of 18 200 M21 cm21 and is expressed per Muk342monomer.

Cloning and expression of GST-FtsZ. GST-FtsZ was clonedand expressed as described in Lockhart and Kendrick-Jones(1998). The concentration of the purified protein was determinedat 280 nm using an extinction coefficient of 44 800 M21 cm21 andis expressed per GST-FtsZ monomer.

Microtubule pelleting assays. Tubulin was purified and poly-merised as described (Lockhart et al., 1995). All assays wereperformed in 40-µl volumes and were spun in a TLA100.1 rotorusing a Beckman TLX ultracentrifuge at 50 000 rpm for 15 min at20°C. After the spin, supernatants were removed and added to 20µl of SDS-gel loading buffer and the Mt pellets were resuspended

FIG. 1. Sequence alignments of the N-terminal 342 residues of MukB (Muk342) with the motor domains of human kinesin (KHC_Hu)and Drosophila nonclaret disjunctional (Ncd_Dr). The alignments were generated using the ClustalW package (Thompson et al., 1994).Residues that are identical are coloured blue and those that are homologous are coloured green. Six residues that are involved in theinteraction of kinesin with microtubules (Woehlke et al., 1997) and that are conserved in the MukB sequence are marked with a redasterisk. The arginine residues coloured red are R250 (Muk342), R278 (KHC_Hu), and R623 (Ncd_Dr) (see text for discussion). Thesequences underlined and labelled N-1 to N-4 are those residues identified from the Ncd (Sablin et al., 1996) and kinesin (Kull et al., 1996)crystal structures as being involved in nucleotide binding.

304 LOCKHART AND KENDRICK-JONES

in 60 µl of SDS-gel loading buffer. Equal volumes of pellet andsupernatant were analysed by SDS–PAGE; after electrophoresisthe gels were stained with Coomassie brilliant blue R-250 tovisualise the protein bands and then destained thoroughly beforeanalysis. In order to determine the relative amounts of protein inthe supernatant and pellet fractions the gels were scanned using aMolecular Dynamics Model 300A densitometer and quantitatedusing the associated ImageQuant software. Measured dissocia-tion constants and binding stoichiometries were obtained byfitting the data to rectangular hyperbolae using Kaleidograph 3.0(Synergy Software). Values were corrected for the small amountsof Muk342 (typically ,5%) that were pelleted in the absence ofMts. Assays using a fixed concentration of Muk342 (2.5 µM) wereperformed in 50 mM potassium phosphate, pH 7.4, 2 mM DTT, 5mM MgOAC, 0.1 mg/ml BSA, and 20 µM Taxol supplemented witheither 2 mM AMPPNP or 2 mM Mg-ADP. Taxol-stabilised Mts(Lockhart et al., 1995) were added from 1.25 to 20 µM (allconcentrations are expressed per tubulin monomer). The finalconcentration of NaCl in the assays was 30 mM.

Pelleting assays using a fixed concentration of Mts (4 µM) wereperformed in 50 mM potassium phosphate, pH 6.5, 2 mM DTT, 5mM MgOAC, 20 µM Taxol, 0.1 mg/ml BSA, and 2 mM AMPPNP.Muk342 was added from 0.5 to 8 µM. Experiments with MukR250Awere performed in an identical buffer and the concentration ofprotein used was 0.45 to 7.2 µM. The final NaCl concentration inthese assays was 56 mM.

Competition assays between Muk342 and Ncd. The construc-tion, purification, and characterisation of Ncd327 have alreadybeen described (Lockhart et al., 1995). The protein spans residues327 to 700 of Ncd, is monomeric, and has a polypeptide molecularmass of 42.7 kDa. The buffer for these assays was 50 mMpotassium phosphate, pH 6.5, 2 mM DTT, 5 mM MgOAC, 20 µMTaxol, and 2 mM AMPPNP. Assays were performed using (i) afixed concentration of Muk342 (4 µM) and 8 and 16 µM of Ncd327and (ii) a fixed concentration of Ncd327 (8 µM) and 4 and 6 µMMuk342. In both cases 4 µM Mts was added and the final NaClconcentrations were 40 mM.

Negative stain electron microscopy of Muk342 decorated Mts.Mts (0.1 µM) were decorated with Muk342 (0.4 µM) in 50 mMpotassium phosphate, pH 6.5, 2 mM DTT, 5 mM MgOAC, 20 µMTaxol, 0.1 mg/ml BSA, and 2 mM AMPPNP in a 20-µl volume for 5min at 20°C. Two microliters of this reaction mixture was thencarefully spread over a carbon-coated grid, left for 2 min, blotted,and then stained with 1% uranyl acetate solution. Grids wereobserved using a Philips transmission electron microscope (ModelEM208).

FtsZ pelleting assays. Assays using a fixed concentration ofMuk342 (2.0 µM) titrated against increasing concentrations ofGST-FtsZ (0.87 to 21 µM) were performed in 50 mM potassiumphosphate, pH 6.5, 2 mM DTT, 5 mM MgOAc, 50 mM KCl, and 2.5mM GTP using 40-µl volumes and were immediately spun afterbeing mixed at 50 000 rpm for 15 min at 20°C. Equal volumes ofpellet and supernatant were analysed by SDS–PAGE and afterelectrophoresis the gels were stained with Coomassie brilliantblue R-250 to allow quantitation of the amounts of Muk342 andGST-FtsZ in the supernatants and pellets.

RESULTS

Expression and Characterisation of Muk342

In this study we used a previously characterisedrecombinantly expressed protein encoding the N-ter-minal 342 residues of MukB (Muk342). The proteinwas expressed with a C-terminal polyhistidine tag tofacilitate its rapid purification from bacterial ly-

sates. The purified protein is monomeric and is ableto hydrolyse both ATP and GTP slowly in the pres-ence of Mg21 ions (Lockhart and Kendrick-Jones,1998).

Muk342 Interacts Specifically with Microtubules

We measured the binding of the Muk342 to Mtsusing pelleting assays as these have been used in anumber of previous studies to quantitate the bindingof kinesin superfamily members to Mts (Lockhart etal., 1995; Lockhart and Cross, 1996). In the pelletingassays as controls for nonspecific binding and toaccount for protein trapping in the polymer weincluded bovine serum albumin (BSA) and alsopolymerised F-actin (Fig. 2). In the absence of eitherMts or F-actin less than 5% of Muk342 pellets,whereas the addition of Mts (10 µM) to the assaysresults in over 40% of the Muk342 pelleting and lessthan 5% of the BSA pelleting. When the assay wasperformed in the presence of polymerised actin (10µM) greater than 90% of the Muk342 remains in thesupernatant, demonstrating no binding with actinand that the interaction of Muk342 with Mts istherefore specific.

Nucleotide-Dependent Interaction between Muk342and Microtubules

The binding of Muk342 to Mts was measured bytitrating a fixed concentration of the protein againstincreasing concentrations of Mts. The presence ofnucleotide-binding motifs and the Mg-NTPase activ-ity of Muk342 encouraged us to perform these bind-ing assays under two different nucleotide conditions,i.e., in the presence of either 2 mM adenosine58-imidotriphosphate (AMPPNP, a nonhydrolysableATP analogue) or 2 mM Mg-ADP (Fig. 3A). Theseassays first of all indicated that the interaction ofMuk342 with Mts was saturable and confirmed thespecificity of the interaction, with the maximal levelsof saturation estimated to be 0.7 and 0.82, in the

FIG. 2. Greyscale image of a Coomassie-stained SDS gelshowing the pelleting of Muk342 (2.5 µM, Mr 5 39.9 kDa) with (i)no added polymer, (ii) Mts (10 µM, Mr 5 50 kDa), and (iii)polymerised actin (10 µM, Mr 5 42 kDa). Assays were performedin 50 mM potassium phosphate, pH 7.4, 2 mM DTT, 5 mMMgOAC, 0.1 mg/ml BSA (Mr 5 66 kDa), and 20 µM Taxol. S,supernatant; P, pellet.

305INTERACTION OF MukB WITH MICROTUBULES

presence of AMPPNP and Mg-ADP, respectively. Inaddition, the assays indicated that the binding wasnucleotide dependent, with AMPPNP producing thestrongest interaction (Kd 5 3.4 µM) and ADP theweakest interaction (Kd 5 9.7 µM). The assays werealso carried out in the presence of no added nucleo-tide and produced an intermediate affinity value of5.4 µM (data not shown).

The Binding of Muk342 to Mts Saturates at OneMuk342 Monomer per Tubulin Monomer

We found that the affinity of MukB for Mts wasboth ionic strength- and pH-dependent and gener-ally decreased as either factor increased. Pelletingassays using a fixed concentration of Mts and increas-ing amounts of Muk342 (Fig. 3B) were performed ata lower pH, 6.3–6.5 instead of 7.4, and this allowedthe use of lower concentrations of Muk342 withwhich to saturate the Mts. We found that the bestconditions for these assays was in 50 mM potassiumphosphate, pH 6.3 to 6.5, with between 30 and 50mM NaCl, which in the presence of 2 mM AMPPNPgave a Kd 5 0.4 µM. A binding stoichiometry of 1Muk342 monomer per tubulin monomer was alsodetermined from these assays, which is twice thebinding stoichiometry found for monomeric kinesinmotor domains (Huang and Hackney, 1994; Lockhartet al., 1995).

Visualisation of the Muk342.Mt Complexesby Electron Microscopy

Mts were decorated with Muk342, in the presenceof AMPPNP, and then negatively stained for trans-mission electron microscopy. The resulting micro-graphs (Fig. 4) demonstrated that Muk342 smoothlydecorated the outside of Mts. The absence of largeMuk342 aggregates on the surface of the decoratedMts is consistent with MukB interacting specificallywith the polymerised tubulin. Furthermore, opticaldiffraction patterns of the Muk342 decorated Mtsdemonstrated no 8-nm layer line in either decoratedor undecorated Mts and is consistent with Muk342decorating each tubulin subunit (data not shown).This is different than the characteristic appearanceof an 8-nm layer line found with kinesin-decoratedMts, which is due to periodic binding of the motordomain to every other tubulin subunit (Song andMandelkow, 1993).

Muk342 Competes for Mt Binding Siteswith the Motor Domain of Ncd

The results of the assays described above areconsistent with the presence of two Muk342-bindingsites per tubulin heterodimer and suggested that theprotein may share one of its binding sites with

FIG. 3. (A) Greyscale images of Coomassie-stained SDS gelsshowing the pelleting of a fixed concentration of Muk342 (2.5 µM)with increasing concentrations of Mts (1.25 to 20 µM). Bindingisotherms are shown for assays performed in the presence of 2 mMAMPPNP (.) and 2 mM Mg-ADP (m). These gave Kd values of 3.4and 9.7 µM, respectively. (B) Greyscale image of a Coomassie-stained SDS gel showing the pelleting of a fixed concentration ofMts (4 µM) and increasing amounts of Muk342 (0.5 to 8 µM).Binding isotherms are shown at the bottom for Muk342 (.) andthe mutant protein MukR250A (see text for details, m). Thesegave Kd values of 0.4 and 3.3 µM, respectively.

306 LOCKHART AND KENDRICK-JONES

kinesin. Binding assays were used to investigatewhether Muk342 and the motor domain of kinesinhomologue Ncd (Lockhart et al., 1995) (Ncd327)could competitively displace each other from Mts.Previous studies demonstrated that the Ncd motordomain is monomeric and binds to an identical siteon the Mt as the kinesin motor domain (Lockhart etal., 1995; Hirose et al., 1997). The assays wereperformed in 2 mM AMPPNP using conditions se-lected to maximise the binding of Muk342 andNcd327.

Figure 5A illustrates the effect of adding a fixedconcentration of Muk342 (4 µM) to Mts (4 µM) andthen adding to this mixture either 8 or 16 µMNcd327. The displacement of Muk342 from the Mtpellet by the Ncd327 can be seen by the increase inthe amount of Muk342 in the supernatant lanes ofthis gel. Figure 5B shows the reverse experiment inwhich Ncd327 (8 µM) is mixed with Mts and Muk342(4 and 6 µM) and then added to the mixture. In thiscase the displacement of Ncd327 from the Mt pelletcan be observed by its decrease in the Mt pelletlanes.

Quantitation of Fig. 5Ashows that the total amountof protein binding in the Mt pellet remains constant

at ,4 µM (Fig. 5C) and is consistent with the totalamount of Mt-binding sites available for both Muk342and Ncd327. In the absence of Ncd327, around 90%of the binding sites are occupied by Muk342, support-ing our earlier finding that Muk342 binds to twosites per tubulin heterodimer. As Ncd327 binds withonly half the stoichiometry of Muk342, it should beable to displace only half the Muk342 bound to theMts. Consistent with this expectation, the additionof 16 µM Ncd327 results in the displacement ofaround 30% of the Muk342 from the Mts and theamount of Muk342 in the Mt pellet has plateaued atslightly over 2 µM, which is around half its startingbinding stoichiometry. In the reverse experimentMuk342 should be able to displace almost all of theNcd327 from the Mts and again this is supported bythe almost linear decrease in the amount of Ncd327in the Mt pellet (Fig. 5D). These results providefurther corroborative data that Muk342 decorateseach tubulin monomer and that it shares one of thesesites with kinesin superfamily members.

Mutation of an Arginine (R250) Decreasesthe Affinity of MukB for Mts 10-fold

An alanine-scanning mutagenesis study of thekinesin motor domain recently identified a numberof residues involved in its interaction with microtu-bules (Woehlke et al., 1997). Aligning the sequencesof kinesin and Muk342 indicated that a number ofthese residues were conserved (Fig. 1B). In particu-lar, arginine 250 (R250) in Muk342 was homologousto arginine 278 (R278) in the human kinesin motordomain (Fig. 1B). Of all the residues changed in theabove study, mutation of R278 to alanine producedthe most dramatic decrease in the affinity of kinesinfor Mts (Woehlke et al., 1997). We therefore mutatedR250 to alanine and found that this produced analmost identical effect on the binding of Muk342 toMts (Fig. 3B). The mutant protein, MukR250A, hadaround a 10-fold weaker affinity (Kd 5 3.3 µM) forMts than the wild-type Muk342 (Kd 5 0.4 µM).

DISCUSSION

Sequence Homology of the MukB N-TerminalDomain with the Kinesin Motor Domain

Sequence alignment of Muk342 with the motordomains of both kinesin and Ncd demonstrated asignificant homology (,34%) between the proteins.It is clear from this alignment (Fig. 1) that althoughsome of the kinesin superfamily nucleotide-bindingmotifs are well conserved (such as N-4), others areeither weakly conserved (N-1 and N-2) or very poorlyconserved (N-3). Other characteristic kinesin super-family motifs, such as the HIPYR sequence (corre-

FIG. 4. Visualisation of Muk342 decorated Mts by negativestain electron microscopy. (A) Undecorated Mts. (B) Muk342decorated Mts.

307INTERACTION OF MukB WITH MICROTUBULES

sponding to residues 274 to 278 in kinesin motordomain), are also weakly conserved in Muk342(LEAIR, residues 245 to 250). However, as we demon-strated biochemically with the MukR250A mutant,this arginine residue is clearly playing a similar rolein both proteins, which reinforces our initial observa-tion that the two proteins may share the sameprotein fold.

Functional Homology of the MukB N-TerminalDomain with the Kinesin Motor Domain

Although the sequence alignment data were sug-gestive that MukB and kinesin were homologues,the biochemical data unambiguously demonstratethat Muk342 shares a number of functional proper-ties with the kinesin motor domain. Muk342 bindswith high affinity, and in a nucleotide-dependentmanner, to Mts. Two striking conclusions can bedrawn from these results. First, the finding that theinteraction between Muk342 and Mts is nucleotide-dependent; this observation is the signature forforce-generating motor proteins. These proteins areable to generate force and movement by undergoingcyclic changes in affinity for their polymer tracks asthey pass through the different nucleotide statesassociated with ATP hydrolysis (Amos and Cross,1997). Second, the pattern of nucleotide-dependentMt affinity is similar to that found for kinesinsuperfamily members where for example theAMPPNP and no nucleotide states are stronglybinding and the ADP state is weakly binding. Indeedthis AMPPNP-induced tight binding of kinesin to

Mts was the basis of its initial discovery (Lasek andBrady, 1985) and is characteristic of all the superfam-ily members thus far characterised.

A direct comparison of the dissociation constantsobtained for Muk342 with those recently reported forkinesin superfamily members (Amos and Cross,1997) is complicated by the use of different assayconditions, although the magnitude of the nucleotide-dependent changes reported here are clearly not asgreat as those for dimeric kinesin constructs, whichcan undergo up to a 1000-fold changes in affinity formicrotubules. However, when kinesin is expressed ina monomeric form the nucleotide-dependent changesin microtubule affinity can be as low as 20-fold(Rosenfeld et al., 1996) and whilst still larger thanthat we report for Muk342 it clearly demonstratesthat nucleotide-dependent changes are very depen-dent on the exact nature of the construct used.

We also found that Muk342 bound to Mts with astoichiometry of one Muk342 monomer per tubulinmonomer. This result differs from that of kinesinsuperfamily members, which bind with half thisstoichiometry to Mts, i.e., one motor domain pertubulin heterodimer (Huang and Hackney, 1994;Lockhart et al., 1995). Crosslinking studies suggestthat both kinesin and Ncd contact both tubulinmonomers (Walker, 1995) and three-dimensionalreconstructions of kinesin decorated Mts indicatethat the kinesin motor domain binds across thejunction between the a and b tubulin monomers(reviewed in Amos and Hirose, 1997). There is evi-dence that monomeric kinesin domains will, when

FIG. 5. Greyscale images of Coomassie-stained SDS gels showing the competition for Mt-binding sites using (A) a fixed concentrationof Muk342 (4 µM) and increasing concentrations of Ncd327 (8 and 16 µM, Mr 5 42.3 kDa) and (B) a fixed concentration of Ncd327 (8 µM)and increasing concentrations of Muk342 (4 and 6 µM). (C and D) Quantitation of the amounts of Muk342 (d), Ncd327 (j), and the totalprotein in the Mt pellets (m) from the images in (A) and (B), respectively.

308 LOCKHART AND KENDRICK-JONES

high concentrations of motor relative to Mts areused, decorate Mts with higher than expected stoichi-ometries (Huang and Hackney, 1994) and this mayrepresent its weak interaction with a second bindingsite.

MukB and Motility Assays

The ability of Muk342 to translocate Mts in an invitro motility assay was also investigated using aconstruct with glutathione S-transferase fused to itsN-terminus (GST-Muk342). The fusion protein, al-though able to attach Mts to the coverslip surface,proved to be nonmotile under these conditions. Thereare a number of possible reasons for this. First, thelack of motility may reflect steric limitations im-posed by the geometry of the motility assays. Thiseffect has been observed with kinesin superfamilymembers where a progressive shortening of the stalkregion results in inhibition of motility (Stewart et al.,1993). Attempts to express longer MukB constructsencoding part of the tail region have thus far beenunsuccessful due to the insolubility of such con-structs. Second, microtubules are clearly not thecorrect substrate for MukB to walk along and al-though Muk342 can bind with high affinity to them,it may not be able to undergo the conformationalchanges required for motility. This latter view is alsosupported by the inability of Mts to activate theATPase activity of Muk342 and the relatively mod-est changes in nucleotide-induced binding affinity.

Evidence That MukB Binds to FtsZ

The data strongly argue that there is a significantdegree of functional homology between the N-ter-minal domain of MukB and the kinesin motor do-main. In addition, combined with sequence homol-ogy between these proteins, the results furthersuggest that MukB may represent an evolutionaryhomologue of the eukaryotic motor proteins. Giventhe sequence and structural similarities betweentubulin and FtsZ, these results also suggested thatMukB may interact with the prokaryotic tubulinhomologue FtsZ. Do we have any direct evidence tosupport this hypothesis?

To address this question we have recombinantlyexpressed FtsZ as a glutathione S-transferase fusionprotein (GST-FtsZ; Lockhart and Kendrick-Jones,1998). Purified GST-FtsZ behaves in a manner iden-tical to nonfusion FtsZ with approximately 50–60%of the protein forming a high-speed pellet in thepresence of 2 mM GTP (Fig. 6, lane ii; Mukherjee andLutkenhaus, 1998). Addition of Muk342 to the pellet-ing assay results in around 80–90% of the proteincosedimenting with the GST-FtsZ pellet, clearly dem-onstrating that MukB has a high affinity for FtsZ.Interestingly, in contrast to Muk342’s interaction

with microtubules, pelleting assays performed withthe motor domain of the kinesin homologue Ncd327indicated that the protein did not bind significantlyto polymerised GST-FtsZ (Fig. 6, lanes iii and iv). Adetailed biochemical characterisation of the interac-tion between Muk342 and FtsZ is reported in Lock-hart and Kendrick-Jones (1998).

Alanine scanning mutagenesis (Woehlke et al.,1997) and protease mapping (Alonso et al., 1998) ofthe binding interface between kinesin and Mts indi-cate that multiple sites are involved in this interac-tion and that it is strongly electrostatic in nature(Woehlke et al., 1997). Our results suggest that sinceMuk342 can bind with high affinity to either poly-mer, they may share some common patches of,perhaps, similar charge on their surfaces. This viewis supported by our finding that the interactionbetween Muk342 and Mts is both pH- and ionicstrength-dependent.

In conclusion, we believe this work provides thefirst compelling evidence of a functional homologybetween the kinesin motor domain and the N-ter-minal domain of MukB, which is highly suggestivethat the two proteins are operating in an analogusmanner and, in addition, provides further evidenceof the functional homology between FtsZ and tubu-lin.

We are grateful to Professor Sota Hiraga for providing thepAX814 MukB clone, Dr. Isabelle Crevel for providing the tubulin,and Juan Fan for technical assistance with the electron micro-scope. We also thank Drs. Linda Amos and Rob Cross for helpfuladvice and discussion. This work was supported by a MRCResearch Fellowship to A.L.

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