Biochemical properties of the Escherichia coli dnaK heat shock protein and its mutant derivatives

The dnaK protein of Escherichia coli has been shown to possess both autophosphorylating and 5'-nucleotidase activities. The dnaK protein has been shown to bind avidly to ATP, but hydrolyzing it slowly. In vitro autophosphorylation occurs at a threonine residue when either ATP or GTP are used as phosphate donors. The extent of autophosphorylation is low; only a few percent of the molecules are phosphorylated. This activity is stimulated at least tenfold in the presence of Ca2+ ions with either ATP or GTP as the donor. The autophosphorylating activity of the mutant dnaK756 protein in the presence or absence of Ca2+ is reduced compared to that of the wild type.     

Functional defects of the DnaK756 mutant chaperone of Escherichia coli indicate distinct roles for amino- and carboxyl-terminal residues in substrate and co-chaperone interaction and interdomain communication

The first discovery of an Hsp70 chaperone gene was the isolation of an Escherichia coli mutant, dnaK756, which rendered the cells resistant to lytic infection with bacteriophage lambda. The DnaK756 mutant protein has since been used to establish many of the cellular roles and biochemical properties of DnaK. DnaK756 has three glycine-to-aspartate substitutions at residues 32, 455, and 468, which were reported to result in defects in intrinsic and GrpE-stimulated ATPase activities, substrate binding, stability of the substrate-binding domain, interdomain communication, and, consequently, defects in chaperone activity. To dissect the effects of the different amino acid substitutions in DnaK756, we analyzed two DnaK variants carrying only the amino-terminal (residue 32) or the two carboxyl-terminal (residues 455 and 468) substitutions. The amino-terminal substitution interfered with the GrpE-stimulated ATPase activity. The carboxyl-terminal mutations (i) affected stability and function of the substrate-binding domain, (ii) caused a 10-fold elevated ATP hydrolysis rate, but (iii) did not severely affect domain coupling. Surprisingly, DnaK chaperone activity was more severely compromised by the amino-terminal than by the carboxyl-terminal amino acid substitutions both in vivo and in vitro. In the in vitro refolding of denatured firefly luciferase, the defect of the DnaK variant carrying the amino-terminal substitution results from its inability to release, upon GrpE-mediated nucleotide exchange, bound luciferase in a folding competent state. Our results indicate that the DnaK-DnaJ-GrpE chaperone system can tolerate suboptimal substrate binding, whereas the tight kinetic control of substrate dissociation by GrpE is essential.     

Purification and properties of the Escherichia coli dnaK replication protein

The Escherichia coli dnaK+ gene was cloned into the "runaway" plasmid vector pMOB45 resulting in a large overproduction of the dnaK protein. The dnaK protein was purified by following its ability to complement the replication of single-stranded M13 bacteriophage DNA in a reaction system dependent on the presence of the lambda O and P DNA replication proteins. The DNA replication activity of the dnaK protein is also essential for lambda dv DNA replication in vitro, since antibodies against it were shown to inhibit the reaction. Purified dnaK protein preparations possess a weak ATPase activity and an autophosphorylating activity which copurify with its DNA replication activity throughout all purification steps. The dnaK protein is an acidic largely monomeric protein of Mr = 72,000 and 78,400 under denaturing and native conditions, respectively. The amino acid composition and N-terminal amino acid sequence match those predicted from the DNA sequence of the dnaK gene (Bardwell, J.C.A., and Craig, E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 848-852).     

The heat-shock DnaK protein is required for plasmid R1 replication and it is dispensable for plasmid ColE1 replication.

Plasmid R1 replication in vitro is inactive in extracts prepared from a dnaK756 strain but is restored to normal levels upon addition of purified DnaK protein. Replication of R1 in extracts of a dnaKwt strain can be specifically inhibited with polyclonal antibodies against DnaK. RepA-dependent replication of R1 in dnaK756 extracts supplemented with DnaKwt protein at maximum concentration is partially inhibited by rifampicin and it is severely inhibited at sub-optimal concentrations of DnaK protein. The copy number of a run-away R1 vector is reduced in a dnaK756 background at 30 degrees C and at 42 degrees C the amplification of the run-away R1 vector is prevented. However a runaway R1 vector containing dnaK gene allows the amplification of the plasmid at high temperature. These data indicate that DnaK is required for both in vitro and in vivo replication of plasmid R1 and show a partial compensation for the low level of DnaK by RNA polymerase. In contrast ColE1 replication is not affected by DnaK as indicated by the fact that ColE1 replicates with the same efficiency in extracts from dnaKwt and dnaK756 strains.     

The dnaK protein modulates the heat-shock response of Escherichia coli

E. coli bacteria respond to a sudden upward shift in temperature by transiently overproducing a small subset of their proteins, one of which is the product of the dnaK gene. Mutations in dnaK have been previously shown to affect both DNA and RNA synthesis in E. coli. Bacteria carrying the dnaK756 mutation fail to turn off the heat-shock response at 43 degrees C. Instead, they continue to synthesize the heat-shock proteins in large amounts and underproduce other proteins. Both reversion and P1 transduction analyses have shown that the failure to turn off the heat-shock response is the result of the dnaK756 mutation. In addition, bacteria that overproduce the dnaK protein at all temperatures undergo a drastically reduced heat-shock response at high temperature. We conclude that the dnaK protein is an inhibitor of the heat-shock response in E. coli.     

The DnaK chaperone is necessary for alpha-complementation of beta-galactosidase in Escherichia coli

We show here the involvement of the molecular chaperone DnaK from Escherichia coli in the in vivo alpha-complementation of the beta-galactosidase. In the dnaK756(Ts) mutant, alpha-complementation occurs when the organisms are grown at 30 degrees C but not at 37 or 40 degrees C, although these temperatures are permissive for bacterial growth. Plasmid-driven expression of wild-type dnaK restores the alpha-complementation in the mutant but also stimulates it in a dnaK(+) strain. In a mutant which contains a disrupted dnaK gene (DeltadnaK52::Cm(r)), alpha-complementation is also impaired, even at 30 degrees C. This observation provides an easy and original phenotype to detect subtle functional changes in a protein such as the DnaK756 chaperone, within the physiologically relevant temperature.     

The in vivo and in vitro characterization of DnaK from Agrobacterium tumefaciens RUOR

Molecular chaperones of the heat shock protein 70 family (Hsp70; also called DnaK in prokaryotes) play an important role in the folding and functioning of cellular protein machinery. The dnaK gene from the plant pathogen Agrobacterium tumefaciens RUOR was amplified using the polymerase chain reaction and the DnaK protein (Agt DnaK) was over-produced as a His-tagged protein in Escherichia coli. The Agt DnaK amino acid sequence was 96% identical to the A. tumefaciens C58 DnaK sequence and 65% identical to the E. coli DnaK sequence. Agt DnaK was shown to be able to functionally replace E. coli DnaK in vivo using complementation assays with an E. coli dnaK756 mutant strain and a dnaK52 deletion strain. Over-production and purification of Agt DnaK was successful, and allowed for further characterization of the protein. Kinetic analysis of the basal ATPase activity of purified Agt DnaK revealed a Vmax of 1.3 nmol phosphate released per minute per milligram DnaK, and a Km of 62 microM ATP. Thus, this is the first study to provide both in vivo and in vitro evidence that Agt DnaK has the properties of a molecular chaperone of the Hsp70 family.     

Involvement of the chaperonin dnaK in the rapid degradation of a mutant protein in Escherichia coli.

The ability of Escherichia coli rapidly to degrade abnormal proteins is inhibited by mutations affecting any of several heat shock proteins (hsps). We therefore tested whether a short-lived mutant protein might become associated with hsps as part of its degradation. At 30 degrees C, the non-secreted mutant form of alkaline phosphatase, phoA61, is relatively stable, and very little phoA61 is found associated with the hsp dnaK. However, raising the temperature to 37 degrees C or 41 degrees C stimulated the degradation of this protein, and up to 30% of cellular phoA61 became associated with dnaK, as shown by immunoprecipitation and Western blot analysis. Also found in complexes with phoA61 were the hsps, protease La and grpE (but no groEL, or groES). The rapid degradation of phoA61 at 37 degrees C and 41 degrees C is in part by protease La, since it decreased by 50% in lon mutants. This process also requires dnaK, since deletion of this gene prevented phoA61 degradation almost completely (unless a wild-type dnaK gene was introduced). In contrast, the missense mutation, dnaK756, enhanced phoA61 degradation. The dnaK756 protein also was associated with phoA61, but this complex, unlike that containing wild-type dnaK could not be dissociated by ATP addition. Furthermore, in a grpE mutant, the degradation of phoA61 and the amount associated with dnaK increased, while in a dnaJ mutant, phoA61 degradation and its association with dnaK decreased. Thus, complex formation with dnaK appears essential for phoA61 degradation by protease La and some other cell proteases, and a failure of the dnaK to dissociate normally may accelerate proteolytic attack.(ABSTRACT TRUNCATED AT 250 WORDS)     

<Emphasis Type="Italic">Plasmodium falciparum</Emphasis> heat shock protein 70 is able to suppress the thermosensitivity of an <Emphasis Type="Italic">Escherichia coli</Emphasis> DnaK mutant strain

Heat shock protein 70 (Hsp70) and heat shock protein 40 (Hsp40) are molecular chaperones that ensure that the proteins of the cell are properly folded and functional under both normal and stressful conditions. The malaria parasite Plasmodium falciparum is known to overproduce a heat shock protein 70 (PfHsp70) in response to thermal stress; however, the in vivo function of this protein still needs to be explored. Using in vivo complementation assays, we found that PfHsp70 was able to suppress the thermosensitivity of an Escherichia coli dnaK756 strain, but not that of the corresponding deletion strain (dnaK52) or dnaK103 strain, which produces a truncated DnaK. Constructs were generated that encoded the ATPase domain of PfHsp70 fused to the substrate-binding domain (SBD) of E. coli DnaK (referred to as PfK), and the ATPase domain of E. coli DnaK coupled to the SBD of PfHsp70 (KPf). PfK was unable to suppress the thermosensitivity of any of the E. coli strains. In contrast, KPf was able to suppress the thermosensitivity in the E. coli dnaK756 strain. We also identified two key amino acid residues (V401 and Q402) in the linker region between the ATPase domain and SBD that are essential for the in vivo function of PfHsp70. This is the first example of an Hsp70 from a eukaryotic parasite that can suppress thermosensitivity in a prokaryotic system. In addition, our results also suggest that interdomain communication is critical for the function of the PfHsp70 and PfHsp70-DnaK chimeras. We discuss the implications of these data for the mechanism of action of the Hsp70-Hsp40 chaperone machinery.     

Escherichia coli dnaK null mutants are inviable at high temperature.

DnaK, a major Escherichia coli heat shock protein, is homologous to major heat shock proteins (Hsp70s) of Drosophila melanogaster and humans. Null mutations of the dnaK gene, both insertions and a deletion, were constructed in vitro and substituted for dnaK+ in the E. coli genome by homologous recombination in a recB recC sbcB strain. Cells carrying these dnaK null mutations grew slowly at low temperatures (30 and 37 degrees C) and could not form colonies at a high temperature (42 degrees C); furthermore, they also formed long filaments at 42 degrees C. The shift of the mutants to a high temperature evidently resulted in a loss of cell viability rather than simply an inhibition of growth since cells that had been incubated at 42 degrees C for 2 h were no longer capable of forming colonies at 30 degrees C. The introduction of a plasmid carrying the dnaK+ gene into these mutants restored normal cell growth and cell division at 42 degrees C. These null mutants showed a high basal level of synthesis of heat shock proteins except for DnaK, which was completely absent. In addition, the synthesis of heat shock proteins after induction in these dnaK null mutants was prolonged compared with that in a dnaK+ strain. The well-characterized dnaK756 mutation causes similar phenotypes, suggesting that they are caused by a loss rather than an alteration of DnaK function. The filamentation observed when dnaK mutations were incubated at a high temperature was not suppressed by sulA or sulB mutations, which suppress SOS-induced filamentation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Escherichia coli DnaK and GrpE heat shock proteins interact both in vivo and in vitro.

Previous studies have demonstrated that the Escherichia coli dnaK and grpE genes code for heat shock proteins. Both the Dnak and GrpE proteins are necessary for bacteriophage lambda DNA replication and for E. coli growth at all temperatures. Through a series of genetic and biochemical experiments, we have shown that these heat shock proteins functionally interact both in vivo and in vitro. The genetic evidence is based on the isolation of mutations in the dnaK gene, such as dnaK9 and dnaK90, which suppress the Tr- phenotype of bacteria carrying the grpE280 mutation. Coimmunoprecipitation of DnaK+ and GrpE+ proteins from cell lysates with anti-DnaK antibodies demonstrated their interaction in vitro. In addition, the DnaK756 and GrpE280 mutant proteins did not coimmunoprecipitate efficiently with the GrpE+ and DnaK+ proteins, respectively, suggesting that interaction between the DnaK and GrpE proteins is necessary for E. coli growth, at least at temperatures above 43 degrees C. Using this assay, we found that one of the dnaK suppressor mutations, dnaK9, reinstated a protein-protein interaction between the suppressor DnaK9 and GrpE280 proteins.     

Roles of the Escherichia coli Small Heat Shock Proteins IbpA and IbpB in Thermal Stress Management: Comparison with ClpA, ClpB, and HtpG In Vivo

We have constructed an Escherichia coli strain lacking the small heat shock proteins IbpA and IbpB and compared its growth and viability at high temperatures to those of isogenic cells containing null mutations in the clpA, clpB, or htpG gene. All mutants exhibited growth defects at 46°C, but not at lower temperatures. However, the clpA, htpG, and ibp null mutations did not reduce cell viability at 50°C. When cultures were allowed to recover from transient exposure to 50°C, all mutations except Δibp led to suboptimal growth as the recovery temperature was raised. Deletion of the heat shock genes clpB and htpG resulted in growth defects at 42°C when combined with the dnaK756 or groES30 alleles, while the Δibp mutation had a detrimental effect only on the growth of dnaK756 mutants. Neither the overexpression of these heat shock proteins nor that of ClpA could restore the growth of dnaK756 or groES30 cells at high temperatures. Whereas increased levels of host protein aggregation were observed in dnaK756 and groES30 mutants at 46°C compared to wild-type cells, none of the null mutations had a similar effect. These results show that the highly conserved E. coli small heat shock proteins are dispensable and that their deletion results in only modest effects on growth and viability at high temperatures. Our data also suggest that ClpB, HtpG, and IbpA and -B cooperate with the major E. coli chaperone systems in vivo.     

Site-directed mutagenesis alters DnaK-dependent folding process

The overproduction of d-aminoacylase (A6-d-ANase) of Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) is accompanied by aggregation of the overproduced protein, and its soluble expression is facilitated by the coexpression of DnaK-DnaJ-GrpE (DnaKJE). When the A6-d-ANase gene was expressed in the Escherichia coli dnaK mutant dnaK756, little activity was observed in the soluble fraction, and it was restored by the coexpression of DnaKJE or the substitution of the R354 residue of A6-d-ANase for lysine. These results suggest that the guanidino group of the R354 residue of A6-d-ANase disturbs its proper folding in the absence of DnaK and the disturbance is eliminated by binding of DnaK to the R354 residue in the presence of DnaK. This is the first report that the DnaK-dependent folding process of the enzyme is altered by site-directed mutagenesis.     

Deletion analysis of the C-terminal region of a molecular chaperone DnaK from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Bacillus licheniformis DnaK (BlDnaK) is predicted to consist of a 45-kDa N-terminal ATPase domain and a 25-kDa C-terminal substrate-binding domain. In this study, the full-length BlDnaK and its T86W and three C-terminally truncated mutants were constructed to evaluate the role of up to C-terminal 255 amino acids of the protein. The steady-state ATPase activity for BlDnaK, T86W, T86W/ΔC120, T86W/ΔC249, and T86W/ΔC255 was 65.68, 53.21, 116.04, 321.38, and 90.59 nmol Pi/min per mg, respectively. In vivo, BldnaK, T86W and T86W/ΔC120 genes allowed an E. coli dnaK756-ts mutant to grow at 44°C. Except for T86W/ΔC255, simultaneous addition of B. licheniformis DnaJ and GrpE, and NR-peptide synergistically stimulated the ATPase activity of BlDnaK, T86W, T86W/ΔC120, and T86W/ΔC249 by 16.9-, 13.9-, 33.9-, 9.9-fold, respectively. Measurement of intrinsic tryptophan fluorescence revealed significant alterations of microenvironment of aromatic amino acids in the C-terminally truncated mutants. The temperature-dependent signal in the far-UV region for T86W was consistent with that of BlDnaK, but the C-terminally truncated mutant proteins showed a higher sensitivity toward temperature-induced denaturation. These results suggest that C-terminal truncations alter the ATPase activity and thermal stability of BlDnaK and induce the conformation change of the ATPase domain. Wan-Chi Liang and Min-Guan Lin contributed equally to this work.     

Phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase by the gene products of dnaK and dnaJ in Escherichia coli K-12 cells

In Escherichia coli K-12, the heat shock protein DnaK and DnaJ participate in phosphorylation of both glutaminyl-tRNA synthetase and threonyl-tRNA synthetase since when cellular proteins extracted from the dnaK7(Ts), dnaK756(Ts) and dnaJ259(Ts) mutant cells labeled with 32Pi at 42 degrees C were analyzed by two-dimensional gel electrophoresis, no phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase was observed while phosphorylation of both aminoacyl-tRNA synthetases was detected in the samples extracted from wild-type cells.     

Escherichia coli Hsp31 functions as a holding chaperone that cooperates with the DnaK-DnaJ-GrpE system in the management of protein misfolding under severe stress conditions

Escherichia coli Hsp31 is a homodimeric protein that exhibits chaperone activity in vitro and is a representative member of a recently recognized family of heat shock proteins (Hsps). To gain insights on Hsp31 cellular function, we deleted the hchA gene from the MC4100 chromosome and combined the resulting null allele with lesions in other cytoplasmic chaperones. Although the hchA mutant only exhibited growth defects when cultivated at 48 degrees C, loss of Hsp31 had a strong deleterious effect on the ability of cells to survive and recover from transient exposure to 50 degrees C, and led to the enhanced aggregation of a subset of host proteins at this temperature. The absence of Hsp31 did not significantly affect the ability of the ClpB-DnaK-DnaJ-GrpE system to clear thermally aggregated proteins at 30 degrees C suggesting that Hsp31 does not possess disaggregase activity. Although it had no effect on the growth of groES30, Delta clpB or Delta ibpAB cells at high temperatures, the hchA deletion aggravated the temperature sensitive phenotype of dnaK756 and grpE280 mutants and led to increased aggregation in stressed dnaK756 cells. On the basis of biochemical, structural and genetic data, we propose that Hsp31 acts as a modified holding chaperone that captures early unfolding intermediates under prolonged conditions of severe stress and releases them when cells return to physiological conditions. This additional line of defence would complement the roles of DnaK-DnaJ-GrpE, ClpB and IbpB in the management of thermally induced cellular protein misfolding.     

Characterization of the dnaK multigene family in the Cyanobacterium Synechococcus sp. strain PCC7942

The cyanobacterium Synechococcus sp. strain PCC7942 has three dnaK homologues (dnaK1, dnaK2, and dnaK3), and a gene disruption experiment was carried out for each dnaK gene by inserting an antibiotic resistance marker. Our findings revealed that DnaK1 was not essential for normal growth, whereas DnaK2 and DnaK3 were essential. We also examined the effect of heat shock on the levels of these three DnaK and GroEL proteins and found a varied response to heat shock, with levels depending on each protein. The DnaK2 and GroEL proteins exhibited a typical heat shock response, that is, their synthesis increased upon temperature upshift. In contrast, the synthesis of DnaK1 and DnaK3 did not respond to heat shock; in fact, the level of DnaK1 protein decreased. We also analyzed the effect of overproduction of each DnaK protein in Escherichia coli cells using an inducible expression system. Overproduction of DnaK1 or DnaK2 resulted in defects in cell septation and formation of cell filaments. On the other hand, overproduction of DnaK3 did not result in filamentous cells; rather a swollen and twisted cell morphology was observed. When expressed in an E. coli dnaK756 mutant, dnaK2 could suppress the growth deficiency at the nonpermissive temperature, while dnaK1 and dnaK3 could not suppress this phenotype. On the contrary, overproduction of DnaK1 or DnaK3 resulted in growth inhibition at the permissive temperature. These results suggest that different types of Hsp70 in the same cellular compartment have specific functions in the cell.     

The chaperone function of DnaK requires the coupling of ATPase activity with substrate binding through residue E171.

Central to the chaperone function of Hsp70 stress proteins including Escherichia coli DnaK is the ability of Hsp70 to bind unfolded protein substrates in an ATP-dependent manner. Mg2+/ATP dissociates bound substrates and, furthermore, substrate binding stimulates the ATPase of Hsp70. This coupling is proposed to require a glutamate residue, E175 of bovine Hsc70, that is entirely conserved within the Hsp70 family, as it contacts bound Mg2+/ATP and is part of a hinge required for a postulated ATP-dependent opening/closing movement of the nucleotide binding cleft which then triggers substrate release. We analyzed the effects of dnaK mutations which alter the corresponding glutamate-171 of DnaK to alanine, leucine or lysine. In vivo, the mutated dnaK alleles failed to complement the delta dnaK52 mutation and were dominant negative in dnaK+ cells. In vitro, all three mutant DnaK proteins were inactive in known DnaK-dependent reactions, including refolding of denatured luciferase and initiation of lambda DNA replication. The mutant proteins retained ATPase activity, as well as the capacity to bind peptide substrates. The intrinsic ATPase activities of the mutant proteins, however, did exhibit increased Km and Vmax values. More importantly, these mutant proteins showed no stimulation of ATPase activity by substrates and no substrate dissociation by Mg2+/ATP. Thus, glutamate-171 is required for coupling of ATPase activity with substrate binding, and this coupling is essential for the chaperone function of DnaK.     

Identification of a gene, closely linked to dnaK, which is required for high-temperature growth of Escherichia coli.

We have constructed four deletion derivatives of the cloned dnaK gene. Plasmid pDD1, in which the last 10 amino acids of the DnaK protein have been replaced by three different amino acids derived from the pBR322 vector, was as effective as plasmid pKP31, from which it was derived, in restoring the ability of a dnaK null mutant, Escherichia coli BB1553, to plate lambda phage and to grow at high temperatures. The other three mutations, involving much larger deletions of the dnaK gene, did not restore the ability to plate lambda phage or the ability to grow at high temperatures. Plasmid pKUC2, which contains the whole dnaK gene and its promoters, was capable of restoring the ability of E. coli BB1553 to plate lambda phage but, surprisingly, it did not restore the ability to grow at high temperatures, even though it was shown that the DnaK protein was efficiently expressed in these cultures. By transposon mutagenesis and sub-cloning, we have shown the presence of a second gene in plasmid pKP31 which is required for high-temperature growth of E. coli BB1553. This gene, which we call htg A, is presumably also defective in the dnaK null mutant E. coli BB1553. We have also demonstrated that the inability of E. coli K756 to grow above 43.5 degrees C is complemented by sub-clones which contain the htg A gene, but not by plasmid pKUC2.     

A novel dnaK operon from Porphyromonas gingivalis

The nucleotide sequence of the dnaK operon cloned from Porphyromonas gingivalis revealed that the operon does not contain homologues of either dnaJ or grpE. However, there were two genes which encode small heat shock proteins immediately downstream from the dnaK and they were transcribed together with dnaK as one unit. The ATPase activity of the P. gingivalis DnaK was synergistically stimulated up to 40-fold in the simultaneous presence of Escherichia coli DnaJ and GrpE. These results suggest that the DnaK homologue of P. gingivalis, with its unique genetic structure and evolutionary features, works as a member of the DnaK chaperone system.