Escherichia coli DnaK and rat Hsc70 are members of the highly conserved 70-kDa heat shock protein [Hsp70] family that showstrong sequence and structure similarities and comparable functional properties in terms of interactions with peptides and unfolded proteins and cooperation with cochaperones . We show here that,while the DnaK protein is, as expected, able to complement anE . coli dnaK mutant strain for growth at high temperatures and phage propagation, Hsc70 protein is not . However, an Hsc70in which the peptide-binding domain has been replaced by thatof DnaK is able to complement this strain for both phenotypes,suggesting that the peptide-binding domain of DnaK is essentialto fulfill the specific functions of this protein necessaryfor growth at high temperatures and for phage replication.The implications of these findings on the functional specificitiesof the Hsp70s and the role of protein-protein interactions inthe DnaK chaperone system are discussed.

 
The heat shock proteins of 70 kDa [Hsp70s] are among the most conserved proteins in nature and are found in most prokaryoticcells and in most compartments of all eukaryotic cells [1, 22].They are known to protect cells against damage by high temperaturesand to assist protein folding and assembly by ATP-dependentcycles of substrate binding and release . They cooperate in thesefunctions with various cofactors, such as the ubiquitous membersof the DnaJ chaperone and GrpE families [6, 11, 14].

Escherichia coli DnaK and rat Hsc70 are two prominent members of this family that have been extensively studied . While bacterial DnaK is a bona fide heat shock protein, for it is strongly inducible by heat shock [1] and is able to efficiently protect cells athigh temperatures, eukaryotic Hsc70 is not and is in fact a constitutive protein expressed at normal temperatures [10, 16]that plays little or no role in heat stress protection . DnaKis involved in negative regulation of the heat shock response,in host and bacteriophage replication, in the prevention of protein denaturation and aggregation during stress, and in the refolding of heat-denatured proteins [18], while Hsc70 interactswith a wide range of specific and well-folded cellular proteinsand possesses specialized functions, such as clathrin uncoatingfrom coated vesicles [8] . Moreover, these proteins differ intheir abilities to interact with a defined set of cochaperones.For instance, while DnaK and Hsc70 chaperones are both slowATPases that have similar hydrophobic peptide-binding specificities,they cooperate with different cochaperones to accomplish theirfunctional cycles of substrate binding and release through nucleotidehydrolysis and exchange . E . coli DnaK uses the ATPase-activatingfactor DnaJ and the nucleotide exchange factor GrpE [13, 26,31], whereas Hsc70 does not bind to GrpE, although it stillinteracts with Hsp40, a DnaJ homolog, and uses Hip and Bag-1,a set of cochaperones with no counterpart in E . coli [15, 29, 32] . In fact, it was proposed that the interaction of GrpE withDnaK, but not Hsc70, is at the basis of the diversification and functional specificity of Hsp70 chaperone systems [4].

Nevertheless, these two relatives have very similar three-dimensional structures, as indicated by the X-ray and nuclear magnetic resonance structures available [12, 13, 21, 23, 34], and are both madeof three domains: an N-terminal ATPase domain, a peptide-bindingdomain composed essentially of a ß sandwich with a shallow peptide-binding pocket followed by an -helical segmentsupposed to form a lid controlling the accessibility to the peptide-binding pocket, and a C-terminal -helical domain [7,9, 12, 23] [Fig . 1].

 
Thus, in spite of a high sequence and structure similarity,these proteins appear to have different functional properties.To gain insight into the structural origin of these differences,a series of chimeric proteins, made by swapping respective domainshaving similar structures but different functions, have beengenerated and analyzed in vivo for the complementation of twoE . coli phenotypes, growth at high temperatures and propagationof phage . The results of this in vivo study are discussed withrespect to the available in vitro structural and functionalinformation for these two proteins.

 
Plasmids, strains, and media. The various chimeric proteins used in this work have been constructedwith the pDnaK and pUHE21-2Fd12 plasmids, a kind gift from BerndBukau [University of Heidelberg, Heidelberg, Germany] . All strainsand plasmids are listed in Table 1.

 
Ultracompetent cells from E . coli strain XL2-Blue were used for the various constructions and were from Stratagene . TheE . coli strain used for complementation studies, BB2393 [C600 dnaK103[Am] thr::Tn10], is from Bernd Bukau.

Luria-Bertani [LB] medium was used for bacterial growth . Tryptone, yeast extract, and agar were obtained from Difco Laboratories,while ampicillin and kanamycin were from Sigma.

Construction of Hsc70/DnaK chimeric proteins. To obtain a plasmid coding for rat Hsc70, the hsc70 coding sequenceof pFB7 [2] was inserted between the BamHI and HindIII restrictionsites of pUHE21-2fd12 . This was performed after modifying theinternal HindIII site of the hsc70 coding sequence, with theQuikChange kit [Stratagene], and introducing the 5' and 3' restrictionsites by PCR . The resulting plasmid was used to transform XL2-Blueultracompetent cells . Single colonies were picked for overnightculture at 30°C, and the plasmids were purified by the MidiPrepskit [Bio 101].

For the construction of chimeras, restriction sites were introduced in the coding sequence of dnaK and hsc70 by site-directed mutagenesisusing the QuikChange kit . Since the restriction sites had tobe unique sites and identical in both plasmids in order to perform the domain swap, only the possibilities that resulted in minimal changes in the amino acid sequence have been retained . Thus, and based on structural alignment of the two proteins [34], AflII sites were introduced into the coding sequences for the interdomain region separating the ATPase domain [N] and the peptide-binding domain [P] [positions 386 to 387 in DnaK and389 to 390 in Hsc70], and SpeI sites were introduced into thecoding sequences for the loop separating the peptide-bindingdomain [P] and the C-terminal domain [C], between helix 2 andhelix 3 [positions 557 to 558 in DnaK and 563 to 564 in Hsc70][Fig. 1] . The creation of AflII and SpeI sites in DnaK coding sequence led to the replacement of valine 386 by a leucine and the insertion of a valine in position 558, whereas, in Hsc70,valine 389 was replaced by a leucine, glutamine 390 was replacedby a lysine, isoleucine 563 was replaced by a leucine, and asparagine564 was replaced by a valine . Complementation properties withthese plasmids were indistinguishable from those with the parental, unmodified plasmids.

After gel electrophoresis in 2% agarose, products of digestion were purified with the Geneclean kit provided by Bio 101, andthe desired restriction fragments were mixed in order to obtaina given chimera . The DNA sequence corresponding to all chimericproteins was verified by automatic sequencing [MWG-Biotec, Ebersberg,Germany].

High-temperature growth studies. To ensure a strong repression of the lac promoter under thecontrol of which DnaK, Hsc70, and their chimeras are expressed,strain BB2393 was transformed with the pDMI.1 plasmid encodingthe LacI repressor . The resulting strain was then transformedby the various constructions . Transformant cells were platedon LB media containing ampicillin [100 µg/ml] and kanamycin[25 µ/ml].

For each construction, a single colony was picked and inoculated into 2 ml of LB medium containing ampicillin and kanamycin foran overnight culture at 30°C . Aliquots of 10 µl ofthis sample and successive 10-fold dilutions of it were spottedon an LB agar plate containing ampicillin and kanamycin withor without IPTG [isopropyl-ß-D-thiogalactopyranoside;100 µM] . For each construction, test plates were incubatedat 30 and 43°C for 24 h . After the test, to control theresults, each plasmid was purified and used to transform againcompetent BB2393 cells carrying the pDMI.1 plasmid . Each testwas performed three times.

phage growth studies. Tests measuring the levels of phage resistance or sensitivityof the different E . coli strains were performed after growthovernight at 30°C in kanamycin- and ampicillin-containingLB medium supplemented with 10 mM MgSO₄ and 0.2% maltose, withor without IPTG [100 µM] . The cells were then spread with0.8% top agar on agar plates containing the same components.Serial dilutions of a vir phage stock [5 x 10⁹ PFU/ml] were spotted on the top agar, and plates were incubated overnightat 30°C, resulting in lysis or no lysis of each bacterialstrain.

SDS-PAGE, immunoblots, and quantification. Exponentially growing 30°C cultures of MC4100, BB1553, BB2393,and BB2393 carrying the different constructions and the pDMI.1plasmid were induced by using 100 µM IPTG for 5 h to allowexpression of wild-type or chimeric proteins . Two millilitersof each culture was subjected to sonication and then centrifugation.To partially purify the wild-type and chimeric Hsp70s from theextracts, 300 µl of the soluble protein fraction was incubatedfor 5 min with 100 µl of ATP agarose beads in buffer A[20 mM Tris-HCl [pH 7.5], 3 mM MgCl₂, 1 mM ß-mercaptoethanol,1 mM EDTA] containing 20 mM KCl . After three washes with bufferB [buffer A containing 250 mM KCl], Hsp70s were released fromthe beads with 100 µl of buffer E [buffer A containing 20 mM KCl and 3 mM ATP] . A degree of purification of about 80% could be achieved by this procedure . Cell extracts as well as partially purified proteins were subjected to sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis [SDS-12% PAGE], stained with Coomassie blue or transferred to nitrocellulosepaper [Hybond-C; Amersham], and then immunoblotted with anti-DnaK polyclonal rabbit antibodies [provided by Bernd Bukau] . Detectionwas performed with the ECL detection system [Amersham] as describedby the manufacturer.

To determine the cellular levels of relevant proteins, 2 mlof each exponentially growing culture at 30°C, induced with100 µM IPTG for 5 h, was subjected to sonication and thencentrifugation . The pellets were resuspended in 1 ml of bufferA, and protein concentration was determined by Lowry assay.Loading of the SDS-12% PAGE gel for each sample was adjustedbased on the protein concentration data . To obtain a linearrange of detection for immunoblot quantification, increasingamounts of purified DnaK ranging from 0 to 20 ng were treatedin the same manner . The contents of the gels were then transferredto nitrocellulose membranes [Hybond-C; Amersham] and immunoblottedwith rabbit anti-DnaK polyclonal antibodies [DAKO], followedby incubation with I¹²⁵-protein A . Detection was performed witha PhosphorImager, and quantification was obtained with ImageQuantsoftware.

 
Rat Hsc70 is unable to complement an E . coli DnaK-deficient strain for growth at high temperatures and phage propagation. The BB2393 strain used for the complementation studies reportedhere carries an amber mutation on the dnaK gene, dnaK103[Am]and is devoid of a functional DnaK protein [19] [Table 1] . Theabsence of the DnaK protein was verified on immunoblots of cellextracts with polyclonal anti-DnaK antibodies . As shown in Fig.2, whereas DnaK is present in cell extracts of the wild-typestrain [Fig . 2A, lane 2], it is absent in those of the BB2393strain, just as it is absent in those of BB1553 [dnaK52], astrain in which the dnaK gene has been deleted [Fig. 2A, lanes3 and 4] . The same results were observed after partial purificationof DnaK from these strains [Fig. 2B] . The BB2393 dnaK103[Am]strain was chosen in this study over the BB1553 [dnaK52] deletionstrain since it has about normal levels of functional DnaJ cochaperoneby contrast to the deletion strain, in which the essential DnaJcochaperone level is reduced by more than 95% [19, 28] . Normalamounts of DnaJ have been shown to be of great importance forstudies of the complementation of E . coli DnaK defects by Bacillussubtilis DnaK [20].

 
As shown in Fig . 3B, line 1, the BB2393 dnaK103[Am] strain doesnot grow at 43°C, although it grows normally at 30°C, and is unable to support the growth of phage, by contrast tothe MC4100 wild-type strain, which grows normally at 30 and43°C and which supports the growth of phage [not shown].As expected, the IPTG-induced expression of the wild-type DnaKprotein [NPC [Fig . 1]] in this strain complemented these twophenotypes [Fig . 3B, line 2] . However, expression of rat Hsc70[N'P'C', where N', P', and C' are the domains of Hsc70 thatcorrespond to DnaK N, P, and C, respectively] was not able todo so, and neither thermoresistance at 43°C nor growth of phage was observed [Fig . 3B, line 3] even though the protein was present [Fig . 3C, line 3] at an intracellular level comparableto that of DnaK [Fig . 3D, lines 2 and 3] . Thus, there seemsto be no correlation between the protein expression level andcomplementation properties . Note, however, that DnaK and Hsc70are overexpressed in these strains at levels about 20 timesthose for the wild-type strain, which has about 5 ng of DnaK/µg of total soluble proteins.

 
Based on this result, it was therefore of interest to determinethe structural elements of DnaK required to ensure growth athigh temperatures and propagation of phage.

Rationale for the design of the Hsc70-DnaK chimeric proteins by domain swapping. The rationale for the design Hsc70-DnaK chimeric proteins wasthat of whole-domain exchange between Hsc70 and DnaK, takinginto account the modular structure of these proteins . Indeed, the fact that the three domains composing the Hsp70s can be expressed separately in and purified from E . coli or obtained by limited proteolysis indicates that these domains behave astrue independent folding and structural units . Moreover, thestructure of the three isolated domains has been establishedby X-ray crystallography and nuclear magnetic resonance, andtheir associated functional properties have been studied [7, 9, 12, 23] . Thus, the respective domains of the different membersof the Hsp70 family can be swapped with confidence since thestructural integrity and overall stability of the parent proteinsshould be maintained in the resulting chimeric proteins.

Therefore, the eight possible combinations among the three respective domains, N', P', and C' of Hsc70 and N, P, and C of DnaK, were constructed and analyzed for their ability to complement the temperature sensitivity phenotype of the BB2393 dnaK103 strain and growth of phage . Two splice junctions corresponding tothe domain boundaries defined by structural and functional studieswere introduced in solvent-accessible connecting loops [Fig.1]: a first junction point at residue 386 between the N andP domains of DnaK, corresponding to residue 389 in Hsc70, anda second junction point at residue 557 between the P and C domains[at the end of helix 2, which forms the putative lid], whichcorresponds to residue 563 of Hsc70 . The introduction of thesejunction points entailed some substitutions and insertions inthe protein sequences [see Material and Methods] . Nevertheless,even though these modifications, located at solvent-exposedloops connecting the domains, were not expected to change thefunctional properties of the proteins, it was verified thatthe complementation properties of DnaK and Hsc70 were not affectedby these changes and were indistinguishable from those of thewild-type proteins reported in Fig . 3 [results not shown].

The peptide-binding domain of DnaK is essential for growth of E . coli cells at high temperatures and for phage replication. As shown in Fig . 4, cells bearing the NP'C' chimera, havingthe N-terminal domain of DnaK and the peptide-binding and C-terminaldomains of Hsc70, do not grow at 43°C and do not support phage growth [Fig . 4B, line 2], even though the protein is expressed at levels comparable to those of other chimeras [Fig. 4C and D, line 2] . This indicates that the presence of the N-terminaldomain of DnaK in the chimera is not sufficient to restore growth.However, its counterpart, chimera N'PC, having the peptide andC-terminal domains of DnaK and the N-terminal domain of Hsc70, is able to restore growth [Fig . 4B, line 1], indicating thatthe presence of the peptide-binding and C-terminal domains of DnaK in the hybrid protein is necessary for complementationof both phenotypes . This is due to the sole presence of the peptide-binding domain of DnaK in the chimeric protein, sincea strain carrying Hsc70 in which only the peptide-binding domainis replaced by that of DnaK [N'PC'] is able to grow at 43°Cand to support phage growth [Fig . 4B, line 4] . As shown inFig. 4B and D, the difference in complementation properties between the various chimeras is not related to differences in intracellular amounts of the relevant protein, since all chimerasare expressed at comparable levels, but rather reflects intrinsic functional differences . Thus, whether the N-terminal and C-terminal domains come from Dnak or Hsc70 [N or N' and C or C', respectively] in the hybrid protein, only the peptide-binding domain of DnaK[P] appears to be the determinant for complementation of thednaK strain for growth at high temperatures and for propagationof phage [compare lines 1, 4, and 5 of Fig . 4B].

 
From these studies, it appears that rat Hsc70, which has morethan 50% sequence identity in the N-terminal domain and peptide-binding domain with E . coli DnaK [3, 34], is unable to ensure growthof the BB2393 dnaK103[Am] strain at high temperatures or tosupport the growth of phage . Nevertheless, an Hsc70 in whichthe peptide-binding domain is replaced by that of DnaK [chimeraN'PC'] can restore these two phenotypes, indicating that theP domain of DnaK is the determining factor for growth at hightemperatures and phage propagation . Most importantly, the Pdomain seems also to have a species specificity since an E.coli DnaK in which only the P domain is replaced by that ofrat Hsc70 [chimera NP'C] is inefficient and unable to ensurethermoresistance and phage growth . These findings, which suggestthat functional specificity is related to peptide binding specificity,are in contrast with those reported for Saccharomyces cerevisiaeHsp70 Ssa-Ssb chimeric proteins [17] . However, the phenotypesanalyzed in such studies, cold sensitivity and hygromycin Bsensitivity, are distinct from thermoresistance and phage growth,addressed in this work, . Moreover, the chimeras used by Jameset al . [17] were made using Ssa, a yeast "classical" Hsp70 thatis functionally related to DnaK and the Hsc70 family, and Ssb,an "unconventional" Hsp70 that has divergent functional properties[24].

Functional specificity of the peptide-binding domain of DnaKfor growth at high temperatures and phage multiplication shoulddepend on the peptide-binding site itself and/or on the dynamicsof the helical lid . In this respect, the peptide-binding domainof DnaK [P] and that of Hsc70 [P'] are both composed of tworegions [Fig . 1]: a ß sandwich subdomain, holdingthe peptide-binding site, and an -helical region, which formsa lid controlling the accessibility to the peptide-binding pocket[9, 23, 34] . As far as the substrate-binding site is concerned, it is exceptionally well conserved in Hsp70s in general andin DnaK and Hsc70 in particular, and most residues involvedin peptide binding are identical in both proteins . Moreover,substrate specificities in vitro for Hsc70 and DnaK are comparable;both proteins bind short peptides of 5 to 7 residues, mostlyhydrophobic [12a, 32, 33, 34], and Hsc70 can substitute forDnaK in protein renaturation in vitro [35] . Thus, it is unlikelythat functional specificity of the peptide-binding domain ofDnaK is due exclusively to the peptide-binding site, unlessthe latter has a more stringent peptide binding specificityin vivo than in vitro . However, functional specificity couldbe related to the helical region forming the lid over the bindingsite, which regulates access to it by a latch-like mechanism[20a, 34] . Indeed, even though P and P' are very similar inthe substrate-binding site, there is a strong sequence variationbetween them in the helical region that forms the lid . In fact,it has been proposed that changes in amino acid composition[25] and orientation [21] of this latch in Hsc70 relative toDnaK are the determinant of DnaK chaperone activity [19] . Hence,dynamics in the latch opening and closing may be involved indiscriminating substrates in vivo and ultimately in conferringa specific target protein-binding capacity to P but not to P'.Finally, the ability of P, but not of P', to complement mayalso be due to specific interactions in vivo with the cochaperonesDnaJ and GrpE, or yet-unknown interactions with critical componentsof the cell machinery

It is well established that thermoresistance and phage propagationin E . coli are based on the ability of the DnaK-DnaJ-GrpE chaperonesystem to prevent heat-induced damage and to interact with thephage replication protein complex [20, 27] . DnaJ is known tobind to the N-terminal ATPase domain of DnaK and other Hsp70s,including Hsc70 . However, GrpE is known to bind to DnaK butnot to Hsc70 since the latter lacks the primary binding sitesin the N-terminal ATPase domain [4] . Thus, all the proteinsstudied here that are able to complement can in principle bindto DnaJ through their N-terminal domains be it N' of Hsc70 orN of DnaK . However, only proteins having the ATPase domain ofDnaK [N] bind GrpE . In spite of this, two chimeras having theATPase domain of Hsc70 [N'PC and N'PC'] are still able to complement,even though they may not be able to bind GrpE . It is then possiblethat these chimeras do not need GrpE binding to activate nucleotideexchange, since nucleotide exchange is already fast, and thatthey do not have to be stimulated, as has been shown for Hsc70 [15] . Alternatively, GrpE may interact with the P domains ofthese chimeras, as has been proposed on the basis of crystallographicand mutagenesis data that additional GrpE binding sites in theC-terminal domain of DnaK do exist [13] . This is corroboratedby the fact that even the chimeras in which the N-terminal domainof DnaK is present and where an interaction with GrpE is expectedto be effective can complement the loss of DnaK only if thepeptide-binding domain of DnaK is present.

Altogether, complementation results presented here indicatethat the peptide-binding domain of DnaK is essential for theprotection of E . coli cells at high temperatures and for phagegrowth.

 
We thank Bernd Bukau and Axel Mogk for the gift of plasmidsand strains.

 
* Corresponding author . Mailing address: University P . & M . Curie, CNRS, 96 Bd . Raspail, 75006 Paris, France . Phone: 33 1 53 63 40 90 . Fax: 33 1 42 22 13 98 . E-mail: ladjimi@ccr.jussieu.fr .

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