Real time kinetics of the DnaK/DnaJ/GrpE molecular chaperone machine action

Applying stopped-flow fluorescence spectroscopy for measuring conformational changes of the DnaK molecular chaperone (bacterial Hsp70 homologue) and its binding to target peptide, we found that after ATP hydrolysis, DnaK is converted to the DnaK*(ADP) conformation, which possesses limited affinity for peptide substrates and the GrpE cochaperone but efficiently binds the DnaJ chaperone. In the presence of DnaJ (bacterial Hsp40 homologue), the DnaK*(ADP) form is converted back to the DnaK conformation, and the resulting DnaJ-DnaK(ADP) complex binds to peptide substrates more tightly. Formation of the DnaJ(substrate-DnaK(ADP)) complex is a rate-limiting reaction. The presence of GrpE and ATP hydrolysis promotes the fast release of the peptide substrate from the chaperone complex and converts DnaK to the DnaK*(ADP) conformation. We conclude that in the presence of DnaJ and GrpE, the binding-release cycle of DnaK is stoichiometrically coupled to the adenosine triphosphatase activity of DnaK.     

The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system DnaK, DnaJ, and GrpE

Molecular chaperones of the Hsp70 class bind unfolded polypeptide chains and are thought to be involved in the cellular folding pathway of many proteins. DnaK, the Hsp70 protein of Escherichia coli, is regulated by the chaperone protein DnaJ and the cofactor GrpE. To gain a biologically relevant understanding of the mechanism of Hsp70 action, we have analyzed a model reaction in which DnaK, DnaJ, and GrpE mediate the folding of denatured firefly luciferase. The binding and release of substrate protein for folding involves the following ATP hydrolysis-dependent cycle: (i) unfolded luciferase binds initially to DnaJ; (ii) upon interaction with luciferase-DnaJ, DnaK hydrolyzes its bound ATP, resulting in the formation of a stable luciferase-DnaK-DnaJ complex; (iii) GrpE releases ADP from DnaK; and (iv) ATP binding to DnaK triggers the release of substrate protein, thus completing the reaction cycle. A single cycle of binding and release leads to folding of only a fraction of luciferase molecules. Several rounds of ATP-dependent interaction with DnaK and DnaJ are required for fully efficient folding.     

Control of the DnaK chaperone cycle by substoichiometric concentrations of the co-chaperones DnaJ and GrpE

The polypeptide binding and release cycle of the molecular chaperone DnaK (Hsp70) of Escherichia coli is regulated by the two co-chaperones DnaJ and GrpE. Here, we show that the DnaJ-triggered conversion of DnaK.ATP (T state) to DnaK.ADP.Pi (R state), as monitored by intrinsic protein fluorescence, is monophasic and occurs simultaneously with ATP hydrolysis. This is in contrast with the T-->R conversion in the absence of DnaJ which is biphasic, the first phase occurring simultaneously with the hydrolysis of ATP (Theyssen, H., Schuster, H.-P., Packschies, L., Bukau, B., and Reinstein, J. (1996) J. Mol. Biol. 263, 657-670). Apparently, DnaJ not only stimulates ATP hydrolysis but also couples it with conformational changes of DnaK. In the absence of GrpE, DnaJ forms a tight ternary complex with peptide.DnaK.ADP.Pi (Kd = 0.14 microM). However, by monitoring complex formation between DnaK (1 microM) and a fluorophore-labeled peptide in the presence of ATP (1 mM), DnaJ (1 microM), and varying concentrations of the ADP/ATP exchange factor GrpE (0.1-3 microM), substoichiometric concentrations of GrpE were found to shift the equilibrium from the slowly binding and releasing, high-affinity R state of DnaK completely to the fast binding and releasing, low-affinity T state and thus to prevent the formation of a long lived ternary DnaJ. substrate.DnaK.ADP.Pi complex. Under in vivo conditions with an estimated chaperone ratio of DnaK:DnaJ:GrpE = 10:1:3, both DnaJ and GrpE appear to control the chaperone cycle by transient interactions with DnaK.     

Purification and biochemical properties of Saccharomyces cerevisiae's Mge1p, the mitochondrial cochaperone of Ssc1p

Previous biochemical and genetic studies have demonstrated the universal conservation of the DnaK (Hsp70) chaperone machine. Its three members, DnaK, DnaJ, and GrpE, in Escherichia coli work synergistically to promote protein protection, disaggregation, and import into the various organelles. In the mitochondria of Saccharomyces cerevisiae the three corresponding members are designated as Ssc1p, Mdj1p, and Mge1p, respectively. The MGE1 gene was previously cloned by us and others, and its product has been shown to be absolutely essential for protein transport into mitochondria and hence cell viability. To better understand its biological role, we have proceeded to overexpress and purify the mature Mge1p in E. coli through the construction of the appropriate vector clone. Mge1p has been shown to functionally substitute for its E. coli GrpE counterpart in a variety of its biological functions, including suppression of the bacterial temperature-sensitive phenotype of the grpE280 mutation, formation of a stable complex with DnaK, stimulation of DnaK's ATPase activity, and the refolding of denatured luciferase by the DnaK/DnaJ chaperone proteins. Thus, the function of the GrpE homologues appears to be highly conserved across the biological kingdoms.     

Chaperone protein GrpE and the GroEL/GroES complex promote the correct folding of tobacco mosaic virus coat protein for ribonucleocapsid assembly in vivo

Several prokaryotic chaperone proteins were shown to promote the correct folding and in vivo assembly of tobacco mosaic virus coat protein (TMV CP) using a chimaeric RNA packaging system in control or chaperone-deficient mutant strains of Escherichia coli. Mutations in groEL or dnaK reduced the amount of both total and soluble TMV CP, and the yield of assembled TMV-like particles, several-fold. Thus both GroEL and DnaK have significant direct or indirect effects on the overall expression, stability, folding and assembly of TMV CP in vivo. In contrast, while cells carrying a mutation in grpE expressed TMV CP to a higher overall level than control E. coli, the amounts of both soluble CP and assembled TMV-like particles were below control levels, suggesting a negative effect of GrpE on overall CP accumulation, but positive role(s) in CP folding and assembly. Curiously, cells with mutations in groES and, to a lesser extent, dnaJ expressed total, soluble and assembled forms of TMV CP significantly above control values, suggesting some form of negative control by these chaperone proteins. To avoid pleiotropic effects or artefacts in chaperone-null mutants, selected chaperone proteins were also over-expressed in control E. coli cells. Overproduction of GroEL or GroES alone had little effect. However, co-overexpression of GroEL and GroES resulted in a two-fold increase in soluble TMV CP and a four-fold rise in assembled TMV-like (pseudovirus) particles in vivo. Moreover, TMV CP was shown to interact directly with GroEL in vivo. Together, these results suggest that GrpE and the GroEL/GroES chaperone complex promote the correct folding and assembly of TMV CP into ribonucleocapsids in vivo.     

The small heat-shock protein IbpB from Escherichia coli stabilizes stress-denatured proteins for subsequent refolding by a multichaperone network

The role of small heat-shock proteins in Escherichia coli is still enigmatic. We show here that the small heat-shock protein IbpB is a molecular chaperone that assists the refolding of denatured proteins in the presence of other chaperones. IbpB oligomers bind and stabilize heat-denatured malate dehydrogenase (MDH) and urea-denatured lactate dehydrogenase and thus prevent the irreversible aggregation of these proteins during stress. While IbpB-stabilized proteins alone do not refold spontaneously, they are specifically delivered to the DnaK/DnaJ/GrpE (KJE) chaperone system where they refold in a strict ATPase-dependent manner. Although GroEL/GroES (LS) chaperonins do not interact directly with IbpB-released proteins, LS accelerate the rate of KJE-mediated refolding of IbpB-released MDH, and to a lesser extent lactate dehydrogenase, by rapidly processing KJE-released early intermediates. Kinetic and gel-filtration analysis showed that denatured MDH preferentially transfers from IbpB to KJE, then from KJE to LS, and then forms a active enzyme. IbpB thus stabilizes aggregation-prone folding intermediates during stress and, as an integral part of a cooperative multichaperone network, is involved in the active refolding of stress-denatured proteins.     

Analysis of three DnaK mutant proteins suggests that progression through the ATPase cycle requires conformational changes

DnaK, the bacterial homolog of the eukaryotic hsp70 proteins, is an ATP-dependent chaperone whose basal ATPase is stimulated by synthetic peptides and its cohort heat shock proteins, DnaJ and GrpE. We have used three mutant DnaK proteins, E171K, D201N, and A174T (corresponding to Glu175, Asp206, and Ala179, respectively, in bovine heat stable cognate 70) to probe the ATPase cycle. All of the mutant proteins exhibit some alteration in basal ATP hydrolysis. However, they all exhibit more severe defects in the regulated activities. D201N and E171K are completely defective in all regulated activities of the protein and also in making the conformational change exhibited by the wt protein upon binding ATP. We suggest that the inability of D201N and E171K to achieve the ATP activated conformation prevents both stimulation by all effectors and the ATP-mediated release of GrpE. In contrast, the defect of A174T is much more specific. It exhibits normal binding and release of GrpE and normal stimulation of ATPase activity by DnaJ. However, it is defective in the synergistic activation of its ATPase by DnaJ and GrpE. We suggest that this mutant protein is specifically defective in a DnaJ/GrpE mediated conformational change in DnaK necessary for the synergistic action of DnaJ+GrpE.     

The ins and outs of a molecular chaperone machine

Genetic and biochemical work has highlighted the biological importance of the GroEL/GroES (Hsp60/Hsp10; cpn60/cpn10) chaperone machine in protein folding. GroEL's donut-shaped structure has attracted the attention of structural biologists because of its elegance as well as the secrets (substrates) it can hide. The recent determination of the GroES and GroEL/GroES structures provides a glimpse of their plasticity, revealing dramatic conformational changes that point to an elaborate mechanism, coupling ATP hydrolysis to substrate release by GroEL.     

Temperature-controlled activity of DnaK-DnaJ-GrpE chaperones: protein-folding arrest and recovery during and after heat shock depends on the substrate protein and the GrpE concentration

Heat-shock proteins DnaK, DnaJ, and GrpE (KJE) from Escherichia coli constitute a three-component chaperone system that prevents aggregation of denatured proteins and assists the refolding of proteins in an ATP-dependent manner. We found that the rate of KJE-mediated refolding of heat- and chemically denatured proteins is decreased at high temperatures. The efficiency and reversibility of protein-folding arrest during and after heat shock depended on the stability of the complex between KJE and the denatured proteins. Whereas a thermostable protein was released and partially refolded during heat shock, a thermolabile protein remained bound to the chaperone. The apparent affinity of GrpE and DnaJ for DnaK was decreased at high temperatures, thereby decreasing futile consumption of ATP during folding arrest. The coupling of ATP hydrolysis and protein folding was restored after the stress. This strongly indicates that KJE chaperones are heat-regulated heat-shock proteins which can specifically arrest the folding of aggregation-prone proteins during stress and preferentially resume refolding under conditions that allow individual proteins to reach and maintain a stable native conformation.     

One founder/one gene hypothesis in a new expanding population: Saguenay (Quebec, Canada)

Here we report a method of immobilising the chaperonins GroEL and GroES to a glass matrix. The immobilised chaperone system has been used to successfully refold target proteins denatured by guanidine hydrochloride and produce substantially higher levels of active protein than occur on dilution into aqueous solution alone. The chaperone system has been shown to refold proteins from each of the three categories of GroEL substrate. The refolding of the enzyme glycerol dehydrogenase from Bacillus stearothermophilus shows a two-fold increase in activity in the presence of immobilised GroEL compared to that in free solution. The lactate dehydrogenase from B. stearothermophilus also shows a two-fold higher yield of activity in the presence of the immobilised GroEL and ATP. The presence of immobilised GroEL in the absence of ATP arrests the refolding of LDH. The enzyme citrate synthetase from porcine heart demonstrates a three-fold increase in activity when refolded in the presence of immobilised GroEL, ATP and free GroES. Similar results are obtained in the presence of free GroEL, immobilised GroES and ATP. The matrix-bound chaperone can be removed from the refolding mixture by centrifugation, producing a reusable system that can be easily isolated and purified from the refolded substrate.     

Structure-function analyses of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli

The DnaK, DnaJ, and GrpE proteins of Escherichia coli have been universally conserved across the biological kingdoms and work together to constitute a highly efficient molecular chaperone machine. We have examined the extent of functional conservation of Saccharomyces cerevisiae Ssc1p, Mdj1p, and Mge1p by analyzing their ability to substitute for their corresponding E. coli homologs in vivo. We found that the expression of yeast Mge1p, the GrpE homolog, allowed for the deletion of the otherwise essential grpE gene of E. coli, albeit only up to 40 degrees C. The inability of Mge1p to substitute for GrpE at very high temperatures is consistent with our previous finding that it specifically failed to stimulate DnaK's ATPase at such extreme conditions. In contrast to Mge1p, overexpression of Mdj1p, the DnaJ homolog, was lethal in E. coli. This toxicity was specifically relieved by mutations which affected the putative zinc binding region of Mdj1p. Overexpression of a truncated version of Mdj1p, containing the J- and Gly/Phe-rich domains, partially substituted for DnaJ function at high temperature. A chimeric protein, consisting of the J domain of Mdj1p coupled to the rest of DnaJ, acted as a super-DnaJ protein, functioning even more efficiently than wild-type DnaJ. In contrast to the results with Mge1p and Mdj1p, both the expression and function of Ssc1p, the DnaK homolog, were severely compromised in E. coli. We were unable to demonstrate any functional complementation by Ssc1p, even when coexpressed with its Mdj1p cochaperone in E. coli.     

Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaJ

Chaperones of the Hsp70 family bind to unfolded or partially folded polypeptides to facilitate many cellular processes. ATP hydrolysis and substrate binding, the two key molecular activities of this chaperone, are modulated by the cochaperone DnaJ. By using both genetic and biochemical approaches, we provide evidence that DnaJ binds to at least two sites on the Escherichia coli Hsp70 family member DnaK: under the ATPase domain in a cleft between its two subdomains and at or near the pocket of substrate binding. The lower cleft of the ATPase domain is defined as a binding pocket for the J-domain because (i) a DnaK mutation located in this cleft (R167H) is an allele-specific suppressor of the binding defect of the DnaJ mutation, D35N and (ii) alanine substitution of two residues close to R167 in the crystal structure, N170A and T173A, significantly decrease DnaJ binding. A second binding determinant is likely to be in the substrate-binding domain because some DnaK mutations in the vicinity of the substrate-binding pocket are defective in either the affinity (G400D, G539D) or rate (D526N) of both peptide and DnaJ binding to DnaK. Binding of DnaJ may propagate conformational changes to the nearby ATPase catalytic center and substrate-binding sites as well as facilitate communication between these two domains to alter the molecular properties of Hsp70.     

The 77-kDa echinoderm microtubule-associated protein (EMAP) shares epitopes with the mammalian brain MAPs, MAP-2 and tau

Heat shock proteins not only can protect host cells against heat stress, they can also enable freeze tolerance as well. With respect to this unexpected feature, we are able to show that, at least in Escherichia coli, the heat shock proteins DnaK/DnaJ and GroEL play a very significant role. We found that the recovery rate of E. coli cultures that had been stored at -80 degreesC in the absence of any cryoprotectant was related to the abundance of these heat shock proteins accumulated before the freeze treatment. Before freezing, the DnaK in the bacterial cells was induced to accumulate to a level comparable to that produced in response to heat shock. After the freezing treatment, the recovery rate of the induced culture was very similar to that of the heat-shocked culture. Over production of GroEL was also protective but less effective. While freezing inevitably leads to protein denaturation, we propose that advance synthesis of DnaK/DnaJ and GroEL can accordingly prevent irreversible denaturation by chaperoning the unfolded polypeptides during freezing.     

Mutations in the DnaK chaperone affecting interaction with the DnaJ cochaperone

Hsp70 chaperones assist protein folding by ATP-controlled cycles of substrate binding and release. ATP hydrolysis is the rate-limiting step of the ATPase cycle that causes locking in of substrates into the substrate-binding cavity of Hsp70. This key step is strongly stimulated by DnaJ cochaperones. We show for the Escherichia coli Hsp70 homolog, DnaK, that stimulation by DnaJ requires the linked ATPase and substrate-binding domains of DnaK. Functional interaction with DnaJ is affected by mutations in an exposed channel located in the ATPase domain of DnaK. It is proposed that binding to this channel, possibly involving the J-domain, allows DnaJ to couple substrate binding with ATP hydrolysis by DnaK. Evolutionary conservation of the channel and the J-domain suggests conservation of the mechanism of action of DnaJ proteins.     

Role of the J-domain in the cooperation of Hsp40 with Hsp70

The Escherichia coli Hsp40 DnaJ and Hsp70 DnaK cooperate in the binding of proteins at intermediate stages of folding, assembly, and translocation across membranes. Binding of protein substrates to the DnaK C-terminal domain is controlled by ATP binding and hydrolysis in the N-terminal ATPase domain. The interaction of DnaJ with DnaK is mediated at least in part by the highly conserved N-terminal J-domain of DnaJ that includes residues 2-75. Heteronuclear NMR experiments with uniformly 15N-enriched DnaJ2-75 indicate that the chemical environment of residues located in helix II and the flanking loops is perturbed on interaction with DnaK or a truncated DnaK molecule, DnaK2-388. NMR signals corresponding to these residues broaden and exhibit changes in chemical shifts in the presence of DnaK(MgADP). Addition of MgATP largely reversed the broadening, indicating that NMR signals of DnaJ2-75 respond to ATP-dependent changes in DnaK. The J-domain interaction is localized to the ATPase domain of DnaK and is likely to be dominated by electrostatic interactions. The results suggest that the J-domain tethers DnaK to DnaJ-bound substrates, which DnaK then binds with its C-terminal peptide-binding domain.     

Mechanism of chaperonin action: GroES binding and release can drive GroEL-mediated protein folding in the absence of ATP hydrolysis

As a basic principle, assisted protein folding by GroEL has been proposed to involve the disruption of misfolded protein structures through ATP hydrolysis and interaction with the cofactor GroES. Here, we describe chaperonin subreactions that prompt a re-examination of this view. We find that GroEL-bound substrate polypeptide can induce GroES cycling on and off GroEL in the presence of ADP. This mechanism promotes efficient folding of the model protein rhodanese, although at a slower rate than in the presence of ATP. Folding occurs when GroES displaces the bound protein into the sequestered volume of the GroEL cavity. Resulting native protein leaves GroEL upon GroES release. A single-ring variant of GroEL is also fully functional in supporting this reaction cycle. We conclude that neither the energy of ATP hydrolysis nor the allosteric coupling of the two GroEL rings is directly required for GroEL/GroES-mediated protein folding. The minimal mechanism of the reaction is the binding and release of GroES to a polypeptide-containing ring of GroEL, thereby closing and opening the GroEL folding cage. The role of ATP hydrolysis is mainly to induce conformational changes in GroEL that result in GroES cycling at a physiologically relevant rate.     

A novel factor required for the assembly of the DnaK and DnaJ chaperones of Thermus thermophilus

We previously reported the isolation of T.DnaK.DnaJ chaperone complex from Thermus thermophilus. Here, we show that a novel factor is necessary for the assembly of T.DnaK and T.DnaJ into the complex. A dnaK gene cluster of T. thermophilus contained five genes, dnaK-grpE-dnaJ-orf4-clpB. Interestingly, T.DnaJ lacks the whole "cysteine-rich region" that has been postulated to be necessary to bind unfolded proteins. The orf4 gene encodes a novel 78-amino acid protein. Curiously, T.DnaK and T.DnaJ expressed in Escherichia coli did not form the complex. Careful reexamination of the T.DnaK.DnaJ complex revealed the presence of a small protein in the complex, which turned out to be a product of orf4. As expected, expression of three genes, dnaK-dnaJ-orf4, resulted in production of a T.DnaK.DnaJ complex in E. coli that was indistinguishable from the authentic complex in its ability to interact with nucleotide and denatured protein. The product of orf4 was also required for in vitro reconstitution of the complex and named T.DafA (T.DnaK.DnaJ assembly factor A). The complex comprises three copies each of T.DnaK, T.DnaJ, and T.DafA. Even though a definite homolog of T.DafA has not been found in the data base, this finding raises a possibility that interaction between DnaK and DnaJ chaperones in other organisms is also mediated by a small protein yet unnoticed.     

Characterization of interactions between the anti-apoptotic protein BAG-1 and Hsc70 molecular chaperones

The anti-cell death protein BAG-1 binds to 70-kDa heat shock proteins (Hsp70/Hsc70) and modulates their chaperone activity. Among other facilitory roles, BAG-1 may serve as a nucleotide exchange factor for Hsp70/Hsc70 family proteins and thus represents the first example of a eukaryotic homologue of the bacterial co-chaperone GrpE. In this study, the interactions between BAG-1 and Hsc70 are characterized and compared with the analogous GrpE-DnaK bacterial system. In contrast to GrpE, which binds DnaK as a dimer, BAG-1 binds to Hsc70 as a monomer with a 1:1 stoichiometry. Dynamic light scattering, sedimentation equilibrium, and circular dichroism measurements provided evidence that BAG-1 exists as an elongated, highly helical monomer in solution. Isothermal titration microcalorimetry was used to determine the complex stoichiometry and an equilibrium dissociation constant, KD, of 100 nM. Kinetic analysis using surface plasmon resonance yielded a KD consistent with the calorimetrically determined value. Molecular modeling permitted a comparison of structural features between the functionally homologous BAG-1 and GrpE proteins. These data were used to propose a mechanism for BAG-1 in the regulation of Hsp70/Hsc70 chaperone activity.     

Mammalian cytosolic DnaJ homologues affect the hsp70 chaperone-substrate reaction cycle, but do not interact directly with nascent or newly synthesized proteins

Members of the hsp70 family of molecular chaperones interact with and stabilize nascent polypeptides during synthesis and/or translocation into organelles. The bacterial hsp70 homologue DnaK requires the DnaJ cofactor for its reaction cycle with polypeptide substrates. DnaJ stimulates the ATPase activity of the DnaK chaperone and thereby is thought to regulate the affinity of DnaK for its protein target. Herein we have analyzed some of the biochemical properties of two mammalian cytosolic DnaJ homologues, the hdj-1 and hdj-2 proteins. We were particularly interested in examining the proposal that DnaJ homologues are the first molecular chaperones to interact directly with nascent polypeptides. Nascent/newly synthesized proteins, nascent polypeptides released from the ribosome by puromycin, or polypeptides misfolded as a result of incorporation of an amino acid analogue were not found in complexes with either of the two HeLa cell DnaJ homologues. We still were unable to demonstrate any interactions between hdj-1p and nascent/newly synthesized proteins even after chemical cross-linking. We did find that hdj-1p, like bacterial DnaJ, stimulated the ATPase activity of hsp70. Stable complex formation between hsp70 and an unfolded polypeptide substrate in vitro was found to be reduced in the presence of hdj-1p and ATP. Thus, while hdj-1p likely does function as a cofactor for the hsp70 chaperone, having effects on hsp70's ATPase activity and conformation/oligomeric structure and the stability of hsp70-substrate complexes, it was not observed to interact directly with nascent/newly synthesized proteins. Rather, hdj-1p likely serves a regulatory role, governing the reaction cycle of hsp70 with polypeptide substrates.     

Chaperone-mediated reduction of RepA dimerization is associated with RepA conformational change

RepA, the initiator protein of plasmid P1, binds to multiple sites (iterons) in the origin. The binding normally requires participation of chaperones, DnaJ, DnaK and GrpE. When purified, RepA appears dimeric and is inactive in iteron binding. On reaction with chaperones, a species active in iteron binding is formed and found to be monomeric. To test whether the chaperones can reduce dimerization, RepA was used to replace the dimerization domain of the lambda repressor. The hybrid protein repressed the lambda operator efficiently, indicating that RepA can dimerize in vivo. A further increase in repressor activity was seen in dnaJ mutant cells. These results are consistent with a chaperone-mediated reduction of RepA dimerization. We also found that RepA mutants defective in dimerization still depend on DnaJ for iteron binding. Conversely, RepA mutants that no longer require chaperones for iteron binding remain dimerization proficient. These results indicate that the chaperone dependence of RepA activity is not solely owing to RepA dimerization. Our results are most simply explained by a chaperone-mediated conformational change in RepA protomer that activates iteron binding. This conformational change also results in reduced RepA dimerization.