The ATPase domain of HscC (DnaK homolog) is essential forinterfering c

70 activity in E. coli

Mohammad Arifuzzaman a, Taku Oshima b;c, Hirotada Mori b;c;�

a Department of Cell Biology, NARA Institute of Science and Technology, Ikoma 630-0101, Japanb Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma 630-0101, Japan

c CREST, JST (Japan Science and Technology), Osaka, Japan

Received 24 September 2003; received in revised form 14 November 2003; accepted 14 November 2003

First published online 2 December 2003

Abstract

HscC, a DnaK homolog in Escherichia coli, consists of adenosine triphosphatase (ATPase), substrate-binding and C-terminal domains.Overexpression of HscC markedly inhibits growth of host cell and reduces the c

70-dependent promoter activity presumably by forming acomplex with c

70. To identify the region(s) of HscC responsible for growth inhibition and complex formation with c70, domain swapping

experiments were carried out between DnaK and HscC. Thus the chimeric proteins carrying the ATPase domain of HscC and substrate-binding domains of either HscC or DnaK were found to inhibit the growth of the cell, reduce the c

70-dependent promoter activity andform a complex with c

70. These results indicate that the ATPase domain of HscC rather than the substrate-binding domain is importantfor determining its functional specificity.2 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: HscC; DnaK homolog; c70 ; Adenosine triphosphatase domain

1. Introduction

Members of the Hsp70 family are highly conserved mo-lecular chaperones that play important roles in dealingwith cellular proteins that are unfolded or damaged duringsynthesis or exposure to environmental stress [1,2]. Chap-erone proteins that belong to the Hsp70 family have aden-osine triphosphatase (ATPase) activity and exhibit typicalstructural features, such as ATPase, substrate-binding andC-terminal domains [3]. Crystal structure of the ATPasedomain of bovine Hsc70 reveals a bi-lobed domain with adeep cleft in which the adenosine triphosphate (ATP) mol-ecule is bound [4]. The substrate-binding domain is neces-sary and su⁄cient for substrate binding; however, normalregulation of the substrate-binding activity does not occurin the absence of the ATPase domain [5^8]. Communica-tion between ATPase and substrate-binding domains of

Hsp70 is fundamental to its function because adenine nu-cleotides regulate the interaction with substrate proteins[9].

In Escherichia coli three Hsp70 proteins, DnaK, HscA(Hsc66) and HscC (Hsc62) [10,11] and six Hsp40 proteins,DnaJ, CbpA, DjlA, HscB (Hsc20), YbeV and YbeS havebeen identi¢ed [11]. Three Hsp40 proteins, DnaJ, CbpAand DjlA, have been shown to serve as co-chaperones forDnaK [12^14], whereas HscB was reported to act similarlyfor HscA [15]. DnaJ interacts with DnaK at either theATPase domain and/or the substrate-binding domainand these interactions have been shown to be importantfor physiological functions of DnaK [16,17]. DnaJ accel-erates the ATPase activity of DnaK several folds [18]whereas CbpA can compensate for biochemical and phys-iological function of DnaJ [13,19,20]. Similarly, HscB in-teracts with HscA and enhances the ATPase activity ofHscA in vitro [15].

The HscC protein with a molecular mass of 62 kDa isthe smallest among members of the Hsp70 family in E. coli[21]. The ATPase and C-terminal domains of HscC aresmaller compared to those of DnaK [11]. Nevertheless,HscC displays signi¢cant ATPase activity and binds togelatin in the same manner as DnaK, consistent with its

0378-1097 / 03 / $22.00 2 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.doi :10.1016/S0378-1097(03)00863-2

* Corresponding author. Tel. : +81 (743) 72 5660;Fax: +81 (743) 72 5669.

E-mail address: hmori@gtc.aist-nara.ac.jp (H. Mori).

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www.fems-microbiology.org

entry as a member of the Hsp70 family [21]. The ATPaseactivity of HscC is enhanced by YbeV [22].

We recently reported that HscC physically interacts withc

70 responsible for transcription of most of the genes dur-ing exponential growth and negatively modulates its activ-ity [23]. As might have been expected, the cloned hscCgene on multicopy plasmid did not complement the tem-perature sensitivity of vdnaK52 mutant. Instead, overex-pression of HscC caused severe inhibition of cell growth[23]. In this study, we carried out domain swapping experi-ments to identify the region(s) of HscC responsible forgrowth inhibition and for physical interaction with c

70

leading to the inhibition of transcription of numerousgenes.

2. Materials and methods

2.1. Bacterial strains and plasmid

E. coli MC4100 (F3 araD139 v(argF-lac)U19 rpsL150relI £bB53O1 deoC1 ptsF25 rbsR) and MARF4 (MC4100/V pF13-Pbla : :lacZ) strains were used in this study.pTrc99A is a derivative of pBR322 [23].

2.2. Media, enzymes and chemicals

Cells were grown in Luria^Bertani (LB) broth. Whennecessary, di¡erent concentrations (w/v) of IPTG (isopro-pylthio-L-D-galactoside, 0.05, 0.1, 0.3 and 0.5 mM) wereadded. Ampicillin and chloramphenicol (Wako, Japan)were used at 50 Wg ml31. Restriction enzymes, ligationkit, PCR kit (polymerase chain reaction kit), T4 polynu-cleotide kinase, alkaline phosphatase (calf intestine) andT4 DNA polymerase were supplied by Takara ShuzoCo., Kyoto, Japan. All chemicals used were supplied byWako, Japan, except for sodium chloride (Nacalai, Ja-pan), yeast extract, and tryptone peptone (Difco Labora-tories).

2.3. L-Galactosidase assay

The bla promoter activity was monitored by assayingL-galactosidase expressed from bla promoter. Cells weregrown at 37‡C in LB medium to OD600W0.3. 0.3 mMIPTG was added, and samples were taken after 1 h incu-bation. L-Galactosidase activity was determined accordingto Miller [24].

2.4. Puri¢cation of His-tagged proteins

MC4100 cells producing histidine-tagged versions ofDnaK, HscC, putative ATPase and substrate-binding do-main-carrying chimera molecules were grown at 37‡C in200 ml of LB cm31 medium to OD600 0.3. IPTG (0.3 mM)was added to induce proteins, and samples were taken

after 2 h. Cells were collected by centrifugation (4000Ug,10 min at 4‡C) and resuspended in 20 ml of cold TEGbu¡er (20 mM Tris^HCl (pH 7.5), 100 mM NaCl, 0.1 mMethylenediamine tetraacetic acid (EDTA), 20% glycerol).All subsequent manipulations were done at 4‡C. Crudeextracts were obtained by sonication (6U20 s, level 5,Astrason ultrasonic processor) and centrifugation(11 900Ug, 30 min) and were loaded onto a 1 ml nickel(Ni2þ) column (prepared according to manufacturer’s in-structions (Probond Resin, Invitrogene) and equilibratedwith bu¡er TEG). A⁄nity chromatography of extractswas performed at 4‡C. Loaded columns were ¢rst washedwith 20 ml of bu¡er TEG, eluted with a 0^1000 mMimidazole linear gradient, and fractions of 0.5 ml werecollected. Homogeneity of proteins (about 90%) was con-¢rmed by sodium dodecyl sulfate^polyacrylamide gel elec-trophoresis (SDS^PAGE).

2.5. SDS^PAGE and Western blotting

Protein samples were mixed with 4Usample bu¡er (400mM dithiothreitol (DTT), 40% glycerol, 8% SDS, 0.04%bromophenol blue, 200 mM Tris^HCl (pH 6.8)), boiledfor a few minutes and subjected to electrophoresis in12.5% SDS^polyacrylamide gels. Proteins on the gelswere analyzed by Western blotting on polyvinylidene di-£uoride membranes (PVDF; Immobilon, Millipore). Themembranes were ¢rst incubated with antibodies againstc

70, or RGS-His (antibody that recognizes the arginine^glycine^serine^histidine epitope found on proteins en-coded by 6UHis fragment of pCA24N vectors derivedfrom pQE9, Qiagen) and then with HRP (horseradish per-oxidase)-conjugated anti-rabbit IgG antibody (Cappel) oranti-mouse IgG (for RGS-His (Dako)) according to thestandard procedures. Immunoreactive bands were visual-ized by using the enhanced chemiluminescence (ECL) de-tection kit (Amersham) according to the manufacturer’sinstructions.

3. Results

3.1. Construction of chimeric hscC/dnaK genes

To perform domain swapping of ATPase, substrate-binding and C-terminal domains of HscC and DnaK, Aat-II and PmaCI restriction sites were introduced into inter-domain regions separating the ATPase and substrate-bind-ing domains, and those separating the substrate-bindingand the C-terminal domains, respectively. Introductionof the restriction sites did not a¡ect the overall functionof chaperones signi¢cantly (data not shown). Besides theamino acid changes caused by creating the restriction sitesin hscC (pIRZ20) did not change the toxicity pro¢les incomparison to the wild-type HscC (Fig. 2). The set ofstrains carrying chimeric genes shown in Fig. 1 were

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then constructed by inserting into pTrc99A vector at thecloning site downstream of an IPTG-inducible Ptrc pro-moter and transforming into MC4100 cells. All chimericgenes carried by a series of pIRZ plasmids (pIRZ10^pIRZ13 and pIRZ19^pIRZ26) could be expressed uponaddition of IPTG and the products were obtained mostlyin soluble form as visualized by SDS^PAGE followed bystaining with Coomassie brilliant blue (data not shown).

3.2. Chimeras carrying the ATPase domain of HscC showtoxicity

It was reported that overexpression of HscC shows se-vere toxicity [23]. Therefore, to identify domains respon-sible for this e¡ect, we constructed a series of pIRZ plas-

mids that produce chimeric HscC and DnaK proteins.MC4100 wild-type strain was transformed with plasmidsand plated on LB agar plates containing IPTG. Afterovernight incubation at 37‡C growth inhibition was ob-served by the overexpression of HscC with or without itsC-terminal domain or the chimeras carrying the ATPasedomain of HscC (Fig. 2; pIRZ12, pIRZ20, pIRZ21,pIRZ22 and pIRZ23). Subsequently, no growth inhibitionwas observed by the overexpression of putative ATPasedomain of HscC, DnaK with or without its C-terminaldomain or chimeras carrying the ATPase domain ofDnaK (Fig. 2; pIRZ13, pIRZ19, pIRZ10, pIRZ11,pIRZ24, pIRZ25 and pIRZ26). Furthermore, the overex-pression of putative HscC containing substrate-bindingand C-terminal domains did not show any growth inhibi-tion (data not shown). This result indicates that the ATP-ase domain of HscC plays a critical role for the speci¢cgrowth inhibition e¡ect.

3.3. Chimeras carrying the ATPase domain of HscCspeci¢cally inhibit the c

70-dependent bla promoteractivity

It was previously reported that overexpression of HscCspeci¢cally inhibits the c

70-dependent promoter activity[23]. To identify the domain(s) that are responsible forthis inhibition, we investigated the e¡ect of the above setof chimeric proteins on functioning of the c

70-dependent

Fig. 1. Construction and structure of chimeric dnaK and hscC genes onthe multicopy pIRZ plasmids. The domain structures of the DnaK(open) or HscC (hatched) proteins and the positions of domain bound-aries are schematically presented. Amino acid changes caused by the in-troduction of restriction sites are indicated. Two di¡erent restrictionsites were introduced by sequence-speci¢c mutagenesis. An AatII sitewas introduced into the boundary separating the ATPase and substrate-binding domains (385/351 amino acid positions for DnaK or HscC, re-spectively) leading to I351V (isoleucine to valine) changes for HscC,whereas a PmaCI site was created within the interdomain region be-tween the substrate-binding and C-terminal domains (513/452 aminoacid positions for DnaK or HscC, respectively) leading to E452H (gluta-mic acid to histidine) changes for HscC. The set of chimeric or trun-cated genes shown here were constructed by using these modi¢ed proto-type genes. Letters on the right side of each construct give the name ofthe plasmid.

Fig. 2. Overproductions of chimeras carrying the ATPase domain ofHscC inhibit cell growth. MC4100 cells containing each of the HscC/DnaK chimeras were treated with di¡erent concentrations of IPTG asindicated and tested for growth at 37‡C. Growth was assayed by deter-mining the ability of the cells to form colonies on LB agar plates.+, normal growth, and 3, no growth.

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bla promoter in a strain carrying VPbla-lacZ by measuringL-galactosidase activity. The results showed that overex-pression of HscC with or without its C-terminal domain orthe chimeras carrying the ATPase domain of HscC inhib-its the c

70-dependent bla promoter activity appreciably(Fig. 3; pIRZ12, pIRZ20, pIRZ21, pIRZ22 andpIRZ23). As shown for the e¡ect on cell growth, the C-terminal domain is not required for this e¡ect. In contrast,overexpression of putative ATPase domain of HscC,DnaK with or without its C-terminal domain or chimerascarrying the ATPase domain of DnaK combined withsubstrate-binding domain of HscC or DnaK failed to in-hibit bla promoter activity (Fig. 3; pIRZ13, pIRZ19,pIRZ10, pIRZ11, pIRZ24, pIRZ25 and pIRZ26). It thusseems evident that the ATPase domain rather than thesubstrate-binding domain of HscC is primarily responsiblefor speci¢c inhibition of cell growth and of c70-dependentpromoter activity.

3.4. c70 is co-puri¢ed with the chimeric Hsp70 proteins

containing the ATPase domain of HscC

Previous observations suggested that overexpression ofHscC physically interacts with c

70 and this interaction iscritical for cell growth [23]. Therefore, to identify the do-main(s) of HscC that interacts speci¢cally with c

70, weinvestigated the interaction between chimeric HscC/DnaK proteins and c

70 in vivo by employing a co-puri¢-

cation strategy. We constructed plasmids expressing His-tagged chimeric proteins that were functionally active asjudged by the inhibitory e¡ects on cell growth (data notshown). The proteins were induced by addition of IPTGto MC4100 cells containing each plasmid and puri¢edfrom crude cell extracts by Ni2þ column chromatography,followed by imidazole elution. Upon immunoblottinganalysis with antibody against c70 and His-tag, all chimer-ic proteins containing the ATPase domain of HscC, aswell as intact HscC, were co-eluted with c

70 (Fig. 4;pIRZ28, pIRZ29, pIRZ30, pIRZ31 and pIRZ16). In con-trast, no signi¢cant amount of c

70 was co-eluted with theATPase domain of HscC by itself (pIRZ17) or with thechimeric proteins containing the ATPase domain of DnaK(Fig. 4; pIRZ27, pIRZ32, pIRZ33, pIRZ34, pIRZ14 andpIRZ15). In addition, the putative HscC without ATPasedomain of HscC could not interact with c

70 (data notshown). These observations strongly suggest that the ATP-ase domain of HscC is primarily responsible for the spe-ci¢c interaction with c

70 which presumably reduces itsactivity and for the eventual inhibition of host cell growth.

4. Discussion

We observed that chimeric proteins that consist of theATPase domain of HscC and substrate-binding domain ofeither HscC or DnaK inhibit cell growth presumably by

Fig. 3. Overproductions of chimeric proteins carrying the ATPase domain of HscC inhibit the c70-dependent bla promoter activity. Cultures of

MARF4 (MC4100/V pF13-Pbla : :lacZ) carrying the indicated plasmids were grown at 37‡C to OD600 0.3; aliquots were treated with or without IPTG(0.3 mM) for 1 h, and samples were assayed for L-galactosidase activity. Enzyme activities (average of three individual experiments) are presented inMiller units. Black bars and open bars represent the samples treated with or without IPTG, respectively. Lower panel shows the domain features ofeach chimeric gene: C or K indicates HscC or DnaK, respectively.

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inhibiting transcription from c70-dependent promoters

(Figs. 2 and 3). In agreement with these results, all thechimeric proteins carrying the ATPase domain of HscCcombined with other domains of either HscC or DnaKwere found to form complexes with c

70 (Fig. 4). Theseobservations strongly suggested that the ATPase domainis important for speci¢c binding of HscC with c

70 thatwould reduce its activity in initiating transcription fromc

70-dependent promoters.Similar domain-exchange experiments between two

Hsp70 proteins in yeast SsbI and SsaI were reported, thechimeric proteins carrying the ATPase domain of SsbI(but not SsaI) and two other domains of SsbI or SsaIcomplement the cold sensitive phenotype of vssbI mutant[25]. The ATPase domain is therefore thought to have acrucial role in determining speci¢city of Hsp70 moleculesin their characteristic response to environmental changes.The present results support the idea that the ATPase do-main of HscC plays a central role in determining the func-tional speci¢city of HscC. The ATPase domain by itself,however, exhibits no inhibitory activities characteristic ofHscC, because overexpression of ATPase domain of HscCalone failed to show any inhibition of growth or c

70 ac-tivity (Figs. 2 and 3). A major question that remains to beresolved is how the chimera Hsp70 molecules carrying theATPase domain of HscC inhibit transcription from c

70-dependent promoter and exhibit cellular toxicity. One pos-sible explanation is that the two functional domains (ATP-ase and substrate-binding domains) of HscC play impor-tant role for the physiological function; the ATPasedomain is crucial for the substrate recognition through

physical interaction with the substrate (c70) and the sub-strate-binding domain is important for substrate stabiliza-tion. We could not also ignore the other possibility thatsubstrate (c70) initially interacts with a co-chaperone,which might be important for HscC activity, then co-chap-erone^substrate complex interacts with the ATPase do-main of HscC; a subsequent conformational change ofHscC could allow the substrate bound to co-chaperoneto be transferred to the substrate-binding domain ofHscC. It has been hypothesized that the functional specif-icity of Hsp70 is determined by physical interaction be-tween one or more Hsp70 domains and Hsp40 proteins[17,25]. The DnaJ protein, a major Hsp40 homolog inE. coli, interacts with DnaK through the ATPase andsubstrate-binding domains and this interaction is crucialfor physiological function of DnaK [17]. Moreover, anoth-er Hsp40 homolog in E. coli, HscB, interacts with HscAand enhances the ATPase activity of HscA [15], suggestingthat co-chaperones play a crucial role for physiologicalfunction of Hsp70 proteins. HscC might also require aco-chaperone for its function that interacts with the ATP-ase domain. Recently, Yoshimune et al. observed thatYbeV, encoding a DnaJ homolog, is closely linked tohscC in E. coli, and stimulates ATPase activity of HscC,thus implicating YbeV to be the co-chaperone for HscC[22].

In this analysis, the ATPase domain was found to be thekey factor for determination of the functional speci¢city ofHscC and substrate-binding domains might assist its phys-iological function of HscC probably through stabilizationof binding to the target proteins. This approach should

Fig. 4. c70 co-puri¢ed with the chimeric proteins containing the ATPase domain of HscC. The domain structures of the DnaK (open) or HscC

(hatched) proteins are schematically presented. Cultures of MC4100 containing di¡erent histidine-tagged plasmids were grown to OD600 0.3 and inducedby 0.3 mM IPTG for 2 h. Proteins were puri¢ed by Ni2þ a⁄nity chromatography as described in Section 2. Elution fraction numbers are indicated atthe top. The eluted fractions were used for Western blotting analysis with anti-c70 and anti-RGS-His antibody. His-tagged indicates the puri¢ed chimer-ic proteins and c

70 indicates the co-puri¢ed c70 protein.

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provide further insights into the mechanism of determina-tion of functional speci¢city among the members of Hsp70families.

Acknowledgements

This work was supported by a Grant-in-Aid for Scien-ti¢c Research on Priority Areas from the Ministry of Ed-ucation, Culture, Sports, Science and Technology of Ja-pan, a grant from CREST, JST (Japan Science andTechnology) and in part from NEDO (New Energy andIndustrial Technology Development Organization). Wethank Takashi Yura for manuscript preparation.

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