DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage.

Members of the conserved Hsp70 chaperone family are assumed to constitute a main cellular system for the prevention and the amelioration of stress-induced protein damage, though little direct evidence exists for this function. We investigated the roles of the DnaK (Hsp70), DnaJ and GrpE chaperones of Escherichia coli in prevention and repair of thermally induced protein damage using firefly luciferase as a test substrate. In vivo, luciferase was rapidly inactivated at 42 degrees C, but was efficiently reactivated to 50% of its initial activity during subsequent incubation at 30 degrees C. DnaK, DnaJ and GrpE did not prevent luciferase inactivation, but were essential for its reactivation. In vitro, reactivation of heat-inactivated luciferase to 80% of its initial activity required the combined activity of DnaK, DnaJ and GrpE as well as ATP, but not GroEL and GroES. DnaJ associated with denatured luciferase, targeted DnaK to the substrate and co-operated with DnaK to prevent luciferase aggregation at 42 degrees C, an activity that was required for subsequent reactivation. The protein repair function of DnaK, GrpE and, in particular, DnaJ is likely to be part of the role of these proteins in regulation of the heat shock response.     

Transient interactions of a slow-folding protein with the Hsp70 chaperone machinery

Most known proteins have at least one local Hsp70 chaperone binding site. Does this mean that all proteins interact with Hsp70 as they fold? This study makes an initial step to address the above question by examining the interaction of the E.coli Hsp70 chaperone (known as DnaK) and its co-chaperones DnaJ and GrpE with a slow-folding E.coli substrate, RNase H^D. Importantly, this protein is a nonobligatory client, and it is able to fold in vitro even in the absence of chaperones. We employ stopped-flow mixing, chromatography, and activity assays to analyze the kinetic perturbations induced by DnaK/DnaJ/GrpE (K/J/E) on the folding of RNase H^D. We find that K/J/E slows down RNase H^D''s apparent folding, consistent with the presence of transient chaperone-substrate interactions. However, kinetic retardation is moderate for this slow-folding client and it is expected to be even smaller for faster-folding substrates. Given that the interaction of folding-competent substrates such as RNase H^D with the K/J/E chaperones is relatively short-lived, it does not significantly interfere with the timely production of folded biologically active substrate. The above mode of action is important because it preserves K/J/E bioavailability, enabling this chaperone system to act primarily by assisting the folding of other misfolded and (or) aggregation-prone cellular proteins that are unable to fold independently. When refolding is carried out in the presence of K/J and absence of the nucleotide exchange factor GrpE, some of the substrate population becomes trapped as a chaperone-bound partially unfolded state.     

Effective cotranslational folding of firefly luciferase without chaperones of the Hsp70 family

Molecular chaperones of the Hsp70 family (bacterial DnaK, DnaJ, and GrpE) were shown to be strictly required for refolding of firefly luciferase from a denatured state and thus for effective restoration of its activity. At the same time the luciferase was found to be synthesized in an Escherichia coli cell-free translation system in a highly active state in the extract with no chaperone activity. The addition of the chaperones to the extract during translation did not raise the activity of the enzyme. The abrupt arrest of translation by the addition of a translational inhibitor led to immediate cessation of the enzyme activity accumulation, indicating the cotranslational character of luciferase folding. The results presented suggest that the chaperones of the Hsp70 family are not required for effective cotranslational folding of firefly luciferase.     

Conserved,Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

The 70-kDa heat shock proteins (Hsp70s) function as molecular chaperones through the allosteric coupling of their nucleotide- and substrate-binding domains, the structures of which are highly conserved. In contrast, the roles of the poorly structured, variable length C-terminal regions present on Hsp70s remain unclear. In many eukaryotic Hsp70s, the extreme C-terminal EEVD tetrapeptide sequence associates with co-chaperones via binding to tetratricopeptide repeat domains. It is not known whether this is the only function for this region in eukaryotic Hsp70s and what roles this region performs in Hsp70s that do not form complexes with tetratricopeptide repeat domains. We compared C-terminal sequences of 730 Hsp70 family members and identified a novel conservation pattern in a diverse subset of 165 bacterial and organellar Hsp70s. Mutation of conserved C-terminal sequence in DnaK, the predominant Hsp70 in Escherichia coli, results in significant impairment of its protein refolding activity in vitro without affecting interdomain allostery, interaction with co-chaperones DnaJ and GrpE, or the binding of a peptide substrate, defying classical explanations for the chaperoning mechanism of Hsp70. Moreover, mutation of specific conserved sites within the DnaK C terminus reduces the capacity of the cell to withstand stresses on protein folding caused by elevated temperature or the absence of other chaperones. These features of the C-terminal region support a model in which it acts as a disordered tether linked to a conserved, weak substrate-binding motif and that this enhances chaperone function by transiently interacting with folding clients.     

Beyond Transcription—New Mechanisms for the Regulation of Molecular Chaperones

Molecular chaperones are an essential part of the universal heat shock response that allows organisms to survive stress conditions that cause intracellular protein unfolding. During the past few years, two new mechanisms have been found to control the activity of several chaperones under stress conditions—the regulation of chaperone activity by the redox state and by the temperature of the environment. Hsp33, for example, is redox-regulated. Hsp33 is specifically activated by disulfide bond formation during oxidative stress, where it becomes a highly efficient chaperone holdase that binds tightly to unfolding proteins. Certain small heat shock proteins, such as Hsp26 and Hsp16.9, on the other hand, are temperature regulated. Exposure to heat shock temperatures causes these oligomeric proteins to disassemble, thereby changing them into highly efficient chaperones. The ATP-dependent chaperone folding system DnaK/DnaJ/GrpE also appears to be temperature regulated, switching from a folding to a holding mode during heat stress. Both of these novel post-translational regulatory strategies appear to have one ultimate goal: to significantly increase the substrate binding affinity of the affected chaperones under exactly those stress conditions that require their highest chaperone activity. This ensures that protein folding intermediates remain bound to the chaperones under stress conditions and are released only after the cells return to non-stress conditions.     

Beyond transcription--new mechanisms for the regulation of molecular chaperones

Molecular chaperones are an essential part of the universal heat shock response that allows organisms to survive stress conditions that cause intracellular protein unfolding. During the past few years, two new mechanisms have been found to control the activity of several chaperones under stress conditions-the regulation of chaperone activity by the redox state and by the temperature of the environment. Hsp33, for example, is redox-regulated. Hsp33 is specifically activated by disulfide bond formation during oxidative stress, where it becomes a highly efficient chaperone holdase that binds tightly to unfolding proteins. Certain small heat shock proteins, such as Hsp26 and Hsp16.9, on the other hand, are temperature regulated. Exposure to heat shock temperatures causes these oligomeric proteins to disassemble, thereby changing them into highly efficient chaperones. The ATP-dependent chaperone folding system DnaK/DnaJ/GrpE also appears to be temperature regulated, switching from a folding to a holding mode during heat stress. Both of these novel post-translational regulatory strategies appear to have one ultimate goal: to significantly increase the substrate binding affinity of the affected chaperones under exactly those stress conditions that require their highest chaperone activity. This ensures that protein folding intermediates remain bound to the chaperones under stress conditions and are released only after the cells return to non-stress conditions.     

Structure-function analysis of HscC,the Escherichia coli member of a novel subfamily of specialized Hsp70 chaperones

Hsp70 chaperones assist protein folding processes through nucleotide-controlled cycles of substrate binding and release. In our effort to understand the structure-function relationship within the Hsp70 family of proteins, we characterized the Escherichia coli member of a novel Hsp70 subfamily, HscC, and identified considerable differences to the well studied E. coli homologue, DnaK, which together suggest that HscC is a specialized chaperone. The basal ATPase cycle of HscC had k(cat) and K(m) values that were 8- and 10,000-fold higher than for DnaK. The HscC ATPase was not affected by the nucleotide exchange factor of DnaK GrpE and stimulated 8-fold by DjlC, a DnaJ protein with a putative transmembrane domain, but not by other DnaJ proteins tested. Substrate binding dynamics and substrate specificity differed significantly between HscC and DnaK. These differences are explicable by distinct structural variations. HscC does not have general chaperone activity because it did not assist refolding of a denatured model substrate. In vivo, HscC failed to complement temperature sensitivity of DeltadnaK cells. Deletion of hscC caused a slow growth phenotype that was suppressed after several generations. Triple knock-outs of all E. coli genes encoding Hsp70 proteins (DeltadnaK DeltahscA DeltahscC) were viable, indicating that Hsp70 proteins are not strictly essential for viability. An extensive search for DeltahscC phenotypes revealed a hypersensitivity to Cd(2+) ions and UV irradiation, suggesting roles of HscC in the cellular response to these stress treatments. Together our data show that the Hsp70 structure exhibits an astonishing degree of adaptive variations to accommodate requirements of a specialized function.     

A small heat shock protein cooperates with heat shock protein 70 systems to reactivate a heat-denatured protein

Small heat shock proteins (sHsps) are a diverse group of heat-induced proteins that are conserved in prokaryotes and eukaryotes and are especially abundant in plants. Recent in vitro data indicate that sHsps act as molecular chaperones to prevent thermal aggregation of proteins by binding non-native intermediates, which can then be refolded in an ATP-dependent fashion by other chaperones. We used heat-denatured firefly luciferase (Luc) bound to pea (Pisum sativum) Hsp18.1 as a model to define the minimum chaperone system required for refolding of a sHsp-bound substrate. Heat-denatured Luc bound to Hsp18.1 was effectively refolded either with Hsc/Hsp70 from diverse eukaryotes plus the DnaJ homologs Hdj1 and Ydj1 (maximum = 97% Luc reactivation with k(ob) = 1.0 x 10(-2)/min), or with prokaryotic Escherichia coli DnaK plus DnaJ and GrpE (100% Luc reactivation, k(ob) = 11.3 x 10(-2)/min). Furthermore, we show that Hsp18.1 is more effective in preventing Luc thermal aggregation than the Hsc70 or DnaK systems, and that Hsp18.1 enhances the yields of refolded Luc even when other chaperones are present during heat inactivation. These findings integrate the aggregation-preventive activity of sHsps with the protein-folding activity of the Hsp70 system and define an in vitro system for further investigation of the mechanism of sHsp action.     

Successive and synergistic action of the Hsp70 and Hsp100 chaperones in protein disaggregation

Proteins belonging to the B-subtype of the Hsp100/Clp chaperone family execute a crucial role in cellular thermotolerance. They cooperate with the Hsp70 chaperones in reactivation of thermally aggregated protein substrates. We investigated the initial events of the disaggregation reaction in real time using denatured, aggregated green fluorescent protein (GFP) as a substrate. Bacterial Hsp70 (DnaK), its co-chaperones (DnaJ and GrpE), and Hsp100 (ClpB) were incubated with aggregated GFP, and the increase in GFP fluorescence was monitored. Incubation of aggregated GFP with DnaK/DnaJ/GrpE but not with ClpB resulted in the rapid initiation of the disaggregation reaction. Under the same conditions a complex between DnaK, DnaJ, and GFP, but not ClpB, was formed as demonstrated by sedimentation analysis and light scattering experiments. Chaperone-dependent disaggregation of chemically denatured aggregated luciferase showed that, similar to GFP disaggregation, incubation with Hsp70 results in the rapid start of the reactivation reaction. For both aggregated GFP and luciferase, incubation with Hsp70 chaperones changes the initial rate but not the overall efficiency or rate of the refolding reaction. Our results clearly demonstrate that the interaction of DnaK and its co-chaperones with aggregated substrate is the rate-limiting reaction at the initial steps of disaggregation.     

Protein folding rates and thermodynamic stability are key determinants for interaction with the Hsp70 chaperone system

The Hsp70 family of molecular chaperones participates in vital cellular processes including the heat shock response and protein homeostasis. E. coli''s Hsp70, known as DnaK, works in concert with the DnaJ and GrpE co-chaperones (K/J/E chaperone system), and mediates cotranslational and post-translational protein folding in the cytoplasm. While the role of the K/J/E chaperones is well understood in the presence of large substrates unable to fold independently, it is not known if and how K/J/E modulates the folding of smaller proteins able to fold even in the absence of chaperones. Here, we combine experiments and computation to evaluate the significance of kinetic partitioning as a model to describe the interplay between protein folding and binding to the K/J/E chaperone system. First, we target three nonobligatory substrates, that is, proteins that do not require chaperones to fold. The experimentally observed chaperone association of these client proteins during folding is entirely consistent with predictions from kinetic partitioning. Next, we develop and validate a computational model (CHAMP70) that assumes kinetic partitioning of substrates between folding and interaction with K/J/E. CHAMP70 quantitatively predicts the experimentally measured interaction of RNase H^D as it refolds in the presence of various chaperones. CHAMP70 shows that substrates are posed to interact with K/J/E only if they are slow-folding proteins with a folding rate constant k_f <50 s⁻¹, and/or thermodynamically unstable proteins with a folding free energy ΔG⁰_UN ≥−2 kcal mol⁻¹. Hence, the K/J/E system is tuned to use specific protein folding rates and thermodynamic stabilities as substrate selection criteria.     

Functional analysis of CbpA, a DnaJ homolog and nucleoid-associated DNA-binding protein

DnaK/Hsp70 proteins are universally conserved ATP-dependent molecular chaperones that help proteins adopt and maintain their native conformations. DnaJ/Hsp40 and GrpE are co-chaperones that assist DnaK. CbpA is an Escherichia coli DnaJ homolog. It acts as a multicopy suppressor for dnaJ mutations and functions in vitro in combination with DnaK and GrpE in protein remodeling reactions. CbpA binds nonspecifically to DNA with preference for curved DNA and is a nucleoid-associated protein. The DNA binding and co-chaperone activities of CbpA are modulated by CbpM, a small protein that binds specifically to CbpA. To identify the regions of CbpA involved in the interaction of CbpA with CbpM and those involved in DNA binding, we constructed and characterized deletion and substitution mutants of CbpA. We discovered that CbpA interacted with CbpM through its N-terminal J-domain. We found that the region C-terminal to the J-domain was required for DNA binding. Moreover, we found that the CbpM interaction, DNA binding, and co-chaperone activities were separable; some mutants were proficient in some functions and defective in others.     

In vivo and in vitro interaction of DnaK and a chloroplast transit peptide

Chloroplast transit peptides have been proposed to function as substrates for Hsp70 molecular chaperones. Many models of chloroplast protein import depict Hsp70s as the translocation motors that drive protein import into the organelle, but to our knowledge, no direct evidence has demonstrated that transit peptides function either in vivo or in vitro as substrates for the chaperone. In this report, we demonstrate that DnaK binds SStp (the full-length transit peptide for the precursor to the small subunit of Rubisco) in vivo when fused to either glutathione-S-transferase (GST) or to an His6-S-peptide tag (His-S) via an ATP-dependent mechanism. Three independent biophysical and biochemical assays confirm the ability of DnaK and SStp to interact in vitro. The cochaperones, DnaJ and GrpE, were also associated with the DnaK/SStp complex. Therefore, both GST-SStp and His-S-SStp can be used as affinity-tagged substrates to study prokaryotic chaperone/transit peptide interactions as well as to provide a novel functional probe to study the dynamics of DnaK/DnaJ/GrpE interactions in vivo. The combination of these results provides the first experimental support for a transit peptide-dependent interaction between a chloroplast precursor and Hsp70. These results are discussed in light of a general mechanism for protein translocation into chloroplasts and mitochondria.     

Identification of a redox-regulated chaperone network

We have identified and reconstituted a multicomponent redox-chaperone network that appears to be designed to protect proteins against stress-induced unfolding and to refold proteins when conditions return to normal. The central player is Hsp33, a redox-regulated molecular chaperone. Hsp33, which is activated by disulfide bond formation and subsequent dimerization, works as an efficient chaperone holdase that binds to unfolding protein intermediates and maintains them in a folding competent conformation. Reduction of Hsp33 is catalyzed by the glutaredoxin and thioredoxin systems in vivo, and leads to the formation of highly active, reduced Hsp33 dimers. Reduction of Hsp33 is necessary but not sufficient for substrate protein release. Substrate dissociation from Hsp33 is linked to the presence of the DnaK/DnaJ/GrpE foldase system, which alone, or in concert with the GroEL/GroES system, then supports the refolding of the substrate proteins. Upon substrate release, reduced Hsp33 dimers dissociate into inactive monomers. This regulated substrate transfer ultimately links substrate release and Hsp33 inactivation to the presence of available DnaK/DnaJ/GrpE, and, therefore, to the return of cells to non-stress conditions.     

Effect of molecular chaperones on the soluble expression of alginate lyase inE. coli

When the alginate lyase gene (aly) fromPseudoalteromonas elyakovii was expressed inE. coli, most of the gene product was organized as aggregated insoluble particles known as inclusion bodies. To examine the effects of chaperones on soluble and nonaggregated form of alginate lyase inE. coli, we constructed plasmids designed to permit the coexpression ofaly and the DnaK/DnaJ/GrpE or GroEL/ES chaperones. The results indicate that coexpression ofaly with the Dnak/DnaJ/GrpE chaperone together had a marked effect on the yield alginate lyase as a soluble and active form of the enzyme. It is speculated this result occurs through facilitation of the correct folding of the protein. The optimal concentration ofl-arabinose required for the induction of the DnaK/DnaJ/GrpE chaperone was found to be 0.05 mg/mL. An analysis of the protein bands on SDS-PAGE gel indicated that at least 37% of total alginate lyase was produced in the soluble fraction when the DnaK/DnaJ/GrpE chaperone was coexpressed.     

Molecular characterization of a plant mitochondrial chaperone GrpE

Escherichia coli DnaK (Hsp70) cooperates with DnaJ and GrpE in its essential role as a molecular chaperone. Function of mitochondrial Hsp70 (mHsp70) in protein folding and organellar import in eukaryotes is critically dependent on GrpE. We cloned two genes from tobacco (Nicotiana tabacum) BY2 cells based on peptide sequences from a purified protein. The predicted amino acid sequences of both clones resembled that of GrpE from E. coli and its homologues from eukaryotes, and a cDNA clone from Arabidopsis thaliana. One gene (Type 1) encoded a deduced protein that was identical to the purified protein while the other (Type 2) encoded a deduced protein that has 80% sequence identity to Type 1. Both tobacco and Arabidopsis thaliana GrpE homologues bound to DnaK and ATP inhibited this binding. The tobacco GrpE homologue contained a typical N-terminal mitochondrial target presequence of 64 residues and the presequence directed the green fluorescent protein to tobacco mitochondria. The tobacco GrpE homologue also associated with mHsp70 when reintroduced into BY2 protoplasts, and this association was disrupted by ATP. A three-dimensional structure for the tobacco GrpE homologue was modeled based on the X-ray structure of E. coli GrpE complexed with DnaK. The modeled structure has the same overall structure as E. coli GrpE. We propose that the tobacco GrpE homologue interacts with mHsp70 in a manner analogous to E. coli GrpE with DnaK and designate it as tobacco mitochondrial GrpE (NtmGrpE).     

Analysis of sequence-specific binding of RNA to Hsp70 and its various homologs indicates the involvement of N- and C-terminal interactions.

Members of the 70-kDa family of molecular chaperones assist in a number of molecular interactions that are essential under both normal and stress conditions. These functions require ATP and co-chaperone molecules and are associated with a cyclic transition of intramolecular conformational changes. As a new putative function, we have previously shown that mammalian Hsp/Hsc70 as well as a distant relative, Hsp110, selectively bind certain RNA sequences via their N-terminal ATP-binding domain. To investigate this phenomenon in more detail, here we examined RNA-binding affinity and specificity of various deletion mutants of human Hsp70. We demonstrate, that, although the N-terminal ATPase domain alone is sufficient for RNA binding, its binding affinity is considerably reduced when compared to that of the full-length protein. Additionally, we provide evidence that binding of RNA to a membrane-immobilized protein partner results in complete loss of RNA sequence specificity. Using various Hsp70 homologs, we show distinct RNA-binding properties of these proteins judged by sequence specificity, ribopolymer sensitivity, and northwestern analysis. Finally, we present data disclosing that RNA binding by DnaK, the Escherichia coli homolog, is influenced by the activity of its co-chaperones, DnaJ and GrpE. We conclude that the RNA-binding capability of this class of molecular chaperones is a conserved feature and it is strongly influenced by the structural and conformational properties. Furthermore, the notion that RNA binding of some Hsp70 family members is influenced by co-chaperones suggests an RNA-binding cycle resembling the protein-binding property of the chaperones.     

Cooperative action of Escherichia coli ClpB protein and DnaK chaperone in the activation of a replication initiation protein

The Escherichia coli molecular chaperone protein ClpB is a member of the highly conserved Hsp100/Clp protein family. Previous studies have shown that the ClpB protein is needed for bacterial thermotolerance. Purified ClpB protein has been shown to reactivate chemically and heat-denatured proteins. In this work we demonstrate that the combined action of ClpB and the DnaK, DnaJ, and GrpE chaperones leads to the activation of DNA replication of the broad-host-range plasmid RK2. In contrast, ClpB is not needed for the activation of the oriC-dependent replication of E. coli. Using purified protein components we show that the ClpB/DnaK/DnaJ/GrpE synergistic action activates the plasmid RK2 replication initiation protein TrfA by converting inactive dimers to an active monomer form. In contrast, Hsp78/Ssc1/Mdj1/Mge1, the corresponding protein system from yeast mitochondria, cannot activate the TrfA replication protein. Our results demonstrate for the first time that the ClpB/DnaK/DnaJ/GrpE system is involved in protein monomerization and in the activation of a DNA replication factor.     

Functional similarities and differences of an archaeal Hsp70(DnaK) stress protein compared with its homologue from the bacterium Escherichia coli

Archaea are prokaryotes but some of their chaperoning systems resemble those of eukaryotes. Also, not all archaea possess the stress protein Hsp70(DnaK), in contrast with bacteria and eukaryotes, which possess it without any known exception. Further, the primary structure of the archaeal DnaK resembles more the bacterial than the eukaryotic homologues. The work reported here addresses two questions: Is the archaeal Hsp70 protein a chaperone, like its homologues in the other two phylogenetic domains? And, if so, is the chaperoning mechanism of bacterial or eukaryotic type? The data have shown that the DnaK protein of the archaeon Methanosarcina mazei functions efficiently as a chaperone in luciferase renaturation in vitro, and that it requires DnaJ, and the other bacterial-type chaperone, GrpE, to perform its function. The M. mazei DnaK chaperone activity was enhanced by interaction with the bacterial co-chaperone DnaJ, but not by the eukaryotic homologue HDJ-2. Both the bacterial GrpE and DnaJ stimulated the ATPase activity of the M. mazei DnaK. The M. mazei DnaK-dependent chaperoning pathway in vitro is similar to that of the bacterium Escherichia coli used for comparison. However, in vivo analyses indicate that there are also significant differences. The M. mazei dnaJ and grpE genes rescued E.coli mutants lacking these genes, but E.coli dnaK mutants were not complemented by the M. mazei dnaK gene. Thus, while the data from in vitro tests demonstrate functional similarities between the M. mazei and E.coli DnaK proteins, in vivo results indicate that, intracellularly, the chaperones from the two species differ.     

Partner proteins determine multiple functions of Hsp70

The 70 kDa heat shock proteins (Hsp70s) are ubiquitous molecular chaperones that are best known for their participation in protein folding. However, evidence is accumulating that Hsp70s perform several other cellular functions in cooperation with specific soluble or membrane-bound partner proteins. While the basic function of Hsp70s is explained by their ability to bind unfolded polypeptide segments, the partner proteins appear to customize them for specific roles such as involvement in protein traffic and folding, translocation of preproteins across membranes, and gene regulation.