Conserved Distal Loop Residues in the Hsp104 and ClpB Middle Domain Contact Nucleotide-binding Domain 2 and Enable Hsp70-dependent Protein Disaggregation

The homologous hexameric AAA⁺ proteins, Hsp104 from yeast and ClpB from bacteria, collaborate with Hsp70 to dissolve disordered protein aggregates but employ distinct mechanisms of intersubunit collaboration. How Hsp104 and ClpB coordinate polypeptide handover with Hsp70 is not understood. Here, we define conserved distal loop residues between middle domain (MD) helix 1 and 2 that are unexpectedly critical for Hsp104 and ClpB collaboration with Hsp70. Surprisingly, the Hsp104 and ClpB MD distal loop does not contact Hsp70 but makes intrasubunit contacts with nucleotide-binding domain 2 (NBD2). Thus, the MD does not invariably project out into solution as in one structural model of Hsp104 and ClpB hexamers. These intrasubunit contacts as well as those between MD helix 2 and NBD1 are different in Hsp104 and ClpB. NBD2-MD contacts dampen disaggregase activity and must separate for protein disaggregation. We demonstrate that ClpB requires DnaK more stringently than Hsp104 requires Hsp70 for protein disaggregation. Thus, we reveal key differences in how Hsp104 and ClpB coordinate polypeptide handover with Hsp70, which likely reflects differential tuning for yeast and bacterial proteostasis.     

Prokaryotic chaperones support yeast prions and thermotolerance and define disaggregation machinery interactions

Saccharomyces cerevisiae Hsp104 and Escherichia coli ClpB are Hsp100 family AAA+ chaperones that provide stress tolerance by cooperating with Hsp70 and Hsp40 to solubilize aggregated protein. Hsp104 also remodels amyloid in vitro and promotes propagation of amyloid prions in yeast, but ClpB does neither, leading to a view that Hsp104 evolved these activities. Although biochemical analyses identified disaggregation machinery components required for resolubilizing proteins, interactions among these components required for in vivo functions are not clearly defined. We express prokaryotic chaperones in yeast to address these issues and find ClpB supports both prion propagation and thermotolerance in yeast if it is modified to interact with yeast Hsp70 or if E. coli Hsp70 and its cognate nucleotide exchange factor (NEF) are present. Our findings show prion propagation and thermotolerance in yeast minimally require cooperation of species-specific Hsp100, Hsp70, and NEF with yeast Hsp40. The functions of this machinery in prion propagation were directed primarily by Hsp40 Sis1p, while thermotolerance relied mainly on Hsp40 Ydj1p. Our results define cooperative interactions among these components that are specific or interchangeable across life kingdoms and imply Hsp100 family disaggregases possess intrinsic amyloid remodeling activity.     

Hsp110 Is a Bona Fide Chaperone Using ATP to Unfold Stable Misfolded Polypeptides and Reciprocally Collaborate with Hsp70 to Solubilize Protein Aggregates

Structurally and sequence-wise, the Hsp110s belong to a subfamily of the Hsp70 chaperones. Like the classical Hsp70s, members of the Hsp110 subfamily can bind misfolding polypeptides and hydrolyze ATP. However, they apparently act as a mere subordinate nucleotide exchange factors, regulating the ability of Hsp70 to hydrolyze ATP and convert stable protein aggregates into native proteins. Using stably misfolded and aggregated polypeptides as substrates in optimized in vitro chaperone assays, we show that the human cytosolic Hsp110s (HSPH1 and HSPH2) are bona fide chaperones on their own that collaborate with Hsp40 (DNAJA1 and DNAJB1) to hydrolyze ATP and unfold and thus convert stable misfolded polypeptides into natively refolded proteins. Moreover, equimolar Hsp70 (HSPA1A) and Hsp110 (HSPH1) formed a powerful molecular machinery that optimally reactivated stable luciferase aggregates in an ATP- and DNAJA1-dependent manner, in a disaggregation mechanism whereby the two paralogous chaperones alternatively activate the release of bound unfolded polypeptide substrates from one another, leading to native protein refolding.     

The disaggregation activity of the mitochondrial ClpB homolog Hsp78 maintains Hsp70 function during heat stress

Molecular chaperones are important components of mitochondrial protein biogenesis and are required to maintain the organellar function under normal and stress conditions. We addressed the functional role of the Hsp100/ClpB homolog Hsp78 during aggregation reactions and its functional cooperation with the main mitochondrial Hsp70, Ssc1, in mitochondria of the yeast Saccharomyces cerevisiae. By establishing an aggregation/disaggregation assay in intact mitochondria we demonstrated that Hsp78 is indispensable for the resolubilization of protein aggregates generated by heat stress under in vivo conditions. The ATP-dependent disaggregation activity of Hsp78 was capable of reversing the preprotein import defect of a destabilized mutant form of Ssc1. This role in disaggregation of Ssc1 is unique for Hsp78, since the recently identified, Hsp70-specific chaperone Zim17 had no effect on the resolubilization reaction. We observed only a minor effect of the second mitochondrial Hsp100 family member Mcx1 on protein disaggregation. A "holding" activity of the mitochondrial Hsp70 system was a prerequisite for a successful resolubilization of aggregated proteins. We conclude that the protective role of Hsp78 in thermotolerance is mainly based on maintaining the molecular chaperone Ssc1 in a soluble and functional state.     

Unique peptide substrate binding properties of 110-kDa heat-shock protein (Hsp110) determine its distinct chaperone activity

The molecular chaperone 70-kDa heat-shock proteins (Hsp70s) play essential roles in maintaining protein homeostasis. Hsp110, an Hsp70 homolog, is highly efficient in preventing protein aggregation but lacks the hallmark folding activity seen in Hsp70s. To understand the mechanistic differences between these two chaperones, we first characterized the distinct peptide substrate binding properties of Hsp110s. In contrast to Hsp70s, Hsp110s prefer aromatic residues in their substrates, and the substrate binding and release exhibit remarkably fast kinetics. Sequence and structure comparison revealed significant differences in the two peptide-binding loops: the length and properties are switched. When we swapped these two loops in an Hsp70, the peptide binding properties of this mutant Hsp70 were converted to Hsp110-like, and more impressively, it functionally behaved like an Hsp110. Thus, the peptide substrate binding properties implemented in the peptide-binding loops may determine the chaperone activity differences between Hsp70s and Hsp110s.     

The elusive middle domain of Hsp104 and ClpB: location and function

Hsp104 in yeast and ClpB in bacteria are homologous, hexameric AAA+ proteins and Hsp100 chaperones, which function in the stress response as ring-translocases that drive protein disaggregation and reactivation. Both Hsp104 and ClpB contain a distinctive coiled-coil middle domain (MD) inserted in the first AAA+ domain, which distinguishes them from other AAA+ proteins and Hsp100 family members. Here, we focus on recent developments concerning the location and function of the MD in these hexameric molecular machines, which remains an outstanding question. While the atomic structure of the hexameric assembly of Hsp104 and ClpB remains uncertain, recent advances have illuminated that the MD is critical for the intrinsic disaggregase activity of the hexamer and mediates key functional interactions with the Hsp70 chaperone system (Hsp70 and Hsp40) that empower protein disaggregation.     

Modulation of heme/substrate binding cleft of neuronal nitric-oxide synthase (nNOS) regulates binding of Hsp90 and Hsp70 proteins and nNOS ubiquitination

Like other nitric-oxide synthase (NOS) enzymes, neuronal NOS (nNOS) turnover and activity are regulated by the Hsp90/Hsp70-based chaperone machinery, which regulates signaling proteins by modulating ligand binding clefts (Pratt, W. B., Morishima, Y., and Osawa, Y. (2008) J. Biol. Chem. 283, 22885-22889). We have previously shown that nNOS turnover is due to Hsp70/CHIP-dependent ubiquitination and proteasomal degradation. In this work, we use an intracellular cross-linking approach to study both chaperone binding and nNOS ubiquitination in intact HEK293 cells. Treatment of cells with N(G)-nitro-L-arginine, a slowly reversible competitive inhibitor that stabilizes nNOS, decreases both nNOS ubiquitination and binding of Hsp90, Hsp70, and CHIP. Treatment with the calcium ionophore A23187, which increases Ca(2+)-calmodulin binding to nNOS, increases nNOS ubiquitination and binding of Hsp90, Hsp70, and CHIP in a manner that is specific for changes in the heme/substrate binding cleft. Both Hsp90 and Hsp70 are bound to the expressed nNOS oxygenase domain, which contains the heme/substrate binding cleft, but not to the reductase domain, and binding is increased to an expressed fragment containing both the oxygenase domain and the calmodulin binding site. Overexpression of Hsp70 promotes nNOS ubiquitination and decreases nNOS protein, and overexpression of Hsp90 inhibits nNOS ubiquitination and increases nNOS protein, showing the opposing effects of the two chaperones as they participate in nNOS quality control in the cell. These observations support the notion that changes in the state of the heme/substrate binding cleft affect chaperone binding and thus nNOS ubiquitination.     

Mitochondrial Heat Shock Protein (Hsp) 70 and Hsp10 Cooperate in the Formation of Hsp60 Complexes

Mitochondrial Hsp70 (mtHsp70) mediates essential functions for mitochondrial biogenesis, like import and folding of proteins. In these processes, the chaperone cooperates with cochaperones, the presequence translocase, and other chaperone systems. The chaperonin Hsp60, together with its cofactor Hsp10, catalyzes folding of a subset of mtHsp70 client proteins. Hsp60 forms heptameric ring structures that provide a cavity for protein folding. How the Hsp60 rings are assembled is poorly understood. In a comprehensive interaction study, we found that mtHsp70 associates with Hsp60 and Hsp10. Surprisingly, mtHsp70 interacts with Hsp10 independently of Hsp60. The mtHsp70-Hsp10 complex binds to the unassembled Hsp60 precursor to promote its assembly into mature Hsp60 complexes. We conclude that coupling to Hsp10 recruits mtHsp70 to mediate the biogenesis of the heptameric Hsp60 rings.     

The Assembly and Intermolecular Properties of the Hsp70-Tomm34-Hsp90 Molecular Chaperone Complex

Maintenance of protein homeostasis by molecular chaperones Hsp70 and Hsp90 requires their spatial and functional coordination. The cooperation of Hsp70 and Hsp90 is influenced by their interaction with the network of co-chaperone proteins, some of which contain tetratricopeptide repeat (TPR) domains. Critical to these interactions are TPR domains that target co-chaperone binding to the EEVD-COOH motif that terminates Hsp70/Hsp90. Recently, the two-TPR domain-containing protein, Tomm34, was reported to bind both Hsp70 and Hsp90. Here we characterize the structural basis of Tomm34-Hsp70/Hsp90 interactions. Using multiple methods, including pull-down assays, fluorescence polarization, hydrogen/deuterium exchange, and site-directed mutagenesis, we defined the binding activities and specificities of Tomm34 TPR domains toward Hsp70 and Hsp90. We found that Tomm34 TPR1 domain specifically binds Hsp70. This interaction is partly mediated by a non-canonical TPR1 two-carboxylate clamp and is strengthened by so far unidentified additional intermolecular contacts. The two-carboxylate clamp of the isolated TPR2 domain has affinity for both chaperones, but as part of the full-length Tomm34 protein, the TPR2 domain binds specifically Hsp90. These binding properties of Tomm34 TPR domains thus enable simultaneous binding of Hsp70 and Hsp90. Importantly, we provide evidence for the existence of an Hsp70-Tomm34-Hsp90 tripartite complex. In addition, we defined the basic conformational demands of the Tomm34-Hsp90 interaction. These results suggest that Tomm34 represents a novel scaffolding co-chaperone of Hsp70 and Hsp90, which may facilitate Hsp70/Hsp90 cooperation during protein folding.     

Regulation of the Hsp104 Middle Domain Activity Is Critical for Yeast Prion Propagation

Molecular chaperones play a significant role in preventing protein misfolding and aggregation. Indeed, some protein conformational disorders have been linked to changes in the chaperone network. Curiously, in yeast, chaperones also play a role in promoting prion maintenance and propagation. While many amyloidogenic proteins are associated with disease in mammals, yeast prion proteins, and their ability to undergo conformational conversion into a prion state, are proposed to play a functional role in yeast biology. The chaperone Hsp104, a AAA+ ATPase, is essential for yeast prion propagation. Hsp104 fragments large prion aggregates to generate a population of smaller oligomers that can more readily convert soluble monomer and be transmitted to daughter cells. Here, we show that the middle (M) domain of Hsp104, and its mobility, plays an integral part in prion propagation. We generated and characterized mutations in the M-domain of Hsp104 that are predicted to stabilize either a repressed or de-repressed conformation of the M-domain (by analogy to ClpB in bacteria). We show that the predicted stabilization of the repressed conformation inhibits general chaperone activity. Mutation to the de-repressed conformation, however, has differential effects on ATP hydrolysis and disaggregation, suggesting that the M-domain is involved in coupling these two activities. Interestingly, we show that changes in the M-domain differentially affect the propagation of different variants of the [PSI+] and [RNQ+] prions, which indicates that some prion variants are more sensitive to changes in the M-domain mobility than others. Thus, we provide evidence that regulation of the M-domain of Hsp104 is critical for efficient prion propagation. This shows the importance of elucidating the function of the M-domain in order to understand the role of Hsp104 in the propagation of different prions and prion variants.     

Hsp33 Controls Elongation Factor-Tu Stability and Allows Escherichia coli Growth in the Absence of the Major DnaK and Trigger Factor Chaperones

Intracellular de novo protein folding is assisted by cellular networks of molecular chaperones. In Escherichia coli, cooperation between the chaperones trigger factor (TF) and DnaK is central to this process. Accordingly, the simultaneous deletion of both chaperone-encoding genes leads to severe growth and protein folding defects. Herein, we took advantage of such defective phenotypes to further elucidate the interactions of chaperone networks in vivo. We show that disruption of the TF/DnaK chaperone pathway is efficiently rescued by overexpression of the redox-regulated chaperone Hsp33. Consistent with this observation, the deletion of hslO, the Hsp33 structural gene, is no longer tolerated in the absence of the TF/DnaK pathway. However, in contrast with other chaperones like GroEL or SecB, suppression by Hsp33 was not attributed to its potential overlapping general chaperone function(s). Instead, we show that overexpressed Hsp33 specifically binds to elongation factor-Tu (EF-Tu) and targets it for degradation by the protease Lon. This synergistic action of Hsp33 and Lon was responsible for the rescue of bacterial growth in the absence of TF and DnaK, by presumably restoring the coupling between translation and the downstream folding capacity of the cell. In support of this hypothesis, we show that overexpression of the stress-responsive toxin HipA, which inhibits EF-Tu, also rescues bacterial growth and protein folding in the absence of TF and DnaK. The relevance for such a convergence of networks of chaperones and proteases acting directly on EF-Tu to modulate the intracellular rate of protein synthesis in response to protein aggregation is discussed.     

Hsp40s Specify Functions of Hsp104 and Hsp90 Protein Chaperone Machines

Hsp100 family chaperones of microorganisms and plants cooperate with the Hsp70/Hsp40/NEF system to resolubilize and reactivate stress-denatured proteins. In yeast this machinery also promotes propagation of prions by fragmenting prion polymers. We previously showed the bacterial Hsp100 machinery cooperates with the yeast Hsp40 Ydj1 to support yeast thermotolerance and with the yeast Hsp40 Sis1 to propagate [PSI⁺] prions. Here we find these Hsp40s similarly directed specific activities of the yeast Hsp104-based machinery. By assessing the ability of Ydj1-Sis1 hybrid proteins to complement Ydj1 and Sis1 functions we show their C-terminal substrate-binding domains determined distinctions in these and other cellular functions of Ydj1 and Sis1. We find propagation of [URE3] prions was acutely sensitive to alterations in Sis1 activity, while that of [PIN⁺] prions was less sensitive than [URE3], but more sensitive than [PSI⁺]. These findings support the ideas that overexpressing Ydj1 cures [URE3] by competing with Sis1 for interaction with the Hsp104-based disaggregation machine, and that different prions rely differently on activity of this machinery, which can explain the various ways they respond to alterations in chaperone function.     

Structural Analysis of the Ribosome-associated Complex (RAC) Reveals an Unusual Hsp70/Hsp40 Interaction

Yeast Zuotin and Ssz are members of the conserved Hsp40 and Hsp70 chaperone families, respectively, but compared with canonical homologs, they atypically form a stable heterodimer termed ribosome-associated complex (RAC). RAC acts as co-chaperone for another Hsp70 to assist de novo protein folding. In this study, we identified the molecular basis for the unusual Hsp70/Hsp40 pairing using amide hydrogen exchange (HX) coupled with mass spectrometry and mutational analysis. Association of Ssz with Zuotin strongly decreased the conformational dynamics mainly in the C-terminal domain of Ssz, whereas Zuotin acquired strong conformational stabilization in its N-terminal segment. Deletion of the highly flexible N terminus of Zuotin abolished stable association with Ssz in vitro and caused a phenotype resembling the loss of Ssz function in vivo. Thus, the C-terminal domain of Ssz, the N-terminal extension of Zuotin, and their mutual stabilization are the major structural determinants for RAC assembly. We furthermore found dynamic changes in the J-domain of Zuotin upon complex formation that might be crucial for RAC co-chaperone function. Taken together, we present a novel mechanism for converting Zuotin and Ssz chaperones into a functionally active dimer.     

Metazoan Hsp70 machines use Hsp110 to power protein disaggregation

Accumulation of aggregation‐prone misfolded proteins disrupts normal cellular function and promotes ageing and disease. Bacteria, fungi and plants counteract this by solubilizing and refolding aggregated proteins via a powerful cytosolic ATP‐dependent bichaperone system, comprising the AAA+ disaggregase Hsp100 and the Hsp70‐Hsp40 system. Metazoa, however, lack Hsp100 disaggregases. We show that instead the Hsp110 member of the Hsp70 superfamily remodels the human Hsp70‐Hsp40 system to efficiently disaggregate and refold aggregates of heat and chemically denatured proteins in vitro and in cell extracts. This Hsp110 effect relies on nucleotide exchange, not on ATPase activity, implying ATP‐driven chaperoning is not required. Knock‐down of nematode Caenorhabditis elegans Hsp110, but not an unrelated nucleotide exchange factor, compromises dissolution of heat‐induced protein aggregates and severely shortens lifespan after heat shock. We conclude that in metazoa, Hsp70‐Hsp40 powered by Hsp110 nucleotide exchange represents the crucial disaggregation machinery that reestablishes protein homeostasis to counteract protein unfolding stress.     

The Molecular Mechanism of Hsp100 Chaperone Inhibition by the Prion Curing Agent Guanidinium Chloride

The Hsp100 chaperones ClpB and Hsp104 utilize the energy from ATP hydrolysis to reactivate aggregated proteins in concert with the DnaK/Hsp70 chaperone system, thereby playing an important role in protein quality control. They belong to the family of AAA+ proteins (ATPases associated with various cellular activities), possess two nucleotide binding domains per monomer (NBD1 and NBD2), and oligomerize into hexameric ring complexes. Furthermore, Hsp104 is involved in yeast prion propagation and inheritance. It is well established that low concentrations of guanidinium chloride (GdmCl) inhibit the ATPase activity of Hsp104, leading to so called “prion curing,” the loss of prion-related phenotypes. Here, we present mechanistic details about the Hsp100 chaperone inhibition by GdmCl using the Hsp104 homolog ClpB from Thermus thermophilus. Initially, we demonstrate that NBD1 of ClpB, which was previously considered inactive as a separately expressed construct, is a fully active ATPase on its own. Next, we show that only NBD1, but not NBD2, is affected by GdmCl. We present a crystal structure of ClpB NBD1 in complex with GdmCl and ADP, showing that the Gdm⁺ ion binds specifically to the active site of NBD1. A conserved essential glutamate residue is involved in this interaction. Additionally, Gdm⁺ interacts directly with the nucleotide, thereby increasing the nucleotide binding affinity of NBD1. We propose that both the interference with the essential glutamate and the modulation of nucleotide binding properties in NBD1 is responsible for the GdmCl-specific inhibition of Hsp100 chaperones.     

J-domain Proteins form Binary Complexes with Hsp90 and Ternary Complexes with Hsp90 and Hsp70

Hsp90 and Hsp70 are highly conserved molecular chaperones that help maintain proteostasis by participating in protein folding, unfolding, remodeling and activation of proteins. Both chaperones are also important for cellular recovery following environmental stresses. Hsp90 and Hsp70 function collaboratively for the remodeling and activation of some client proteins. Previous studies using E. coli and S. cerevisiae showed that residues in the Hsp90 middle domain directly interact with a region in the Hsp70 nucleotide binding domain, in the same region known to bind J-domain proteins. Importantly, J-domain proteins facilitate and stabilize the interaction between Hsp90 and Hsp70 both in E. coli and S. cerevisiae. To further explore the role of J-domain proteins in protein reactivation, we tested the hypothesis that J-domain proteins participate in the collaboration between Hsp90 and Hsp70 by simultaneously interacting with Hsp90 and Hsp70. Using E. coli Hsp90, Hsp70 (DnaK), and a J-domain protein (CbpA), we detected a ternary complex containing all three proteins. The interaction involved the J-domain of CbpA, the DnaK binding region of E. coli Hsp90, and the J-domain protein binding region of DnaK where Hsp90 also binds. Additionally, results show that E. coli Hsp90 interacts with E. coli J-domain proteins, DnaJ and CbpA, and that yeast Hsp90, Hsp82, interacts with a yeast J-domain protein, Ydj1. Together these results suggest that the complexes may be transient intermediates in the pathway of collaborative protein remodeling by Hsp90 and Hsp70.     

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.     

Role of a conserved aspartic acid in nucleotide binding domain 1 (NBD1) of Hsp100 chaperones in their activities

Besides its beneficial role in thermotolerance, the chaperone protein Hsp104 is involved in the inheritance of yeast Saccharomyces cerevisiae prions. Guanidine hydrochloride was previously shown to interfere with Hsp104 chaperone activity in vivo, thus impairing thermotolerance and resulting in prion curing. It was also reported that guanidine inhibits Hsp104 ATPase and disaggregation activity. We show that in vitro guanidine significantly inhibits the disaggregation activity of ClpB, the bacterial orthologue of Hsp104. However, guanidine exerts opposite effects on the ATPase activities of Hsp104 and ClpB. While the ATPase activity of Hsp104 is inhibited, the analogous ClpB activity is stimulated several-fold. Mutation of the universally conserved aspartic acid residue in position 184 to serine (D184S) in HSP104 and the analogous mutation in clpB (D178S) resulted in chaperones with lower disaggregating and ATPase activities. The activities of such changed chaperones are not influenced by guanidine, which suggests the role of this residue in the interaction with guanidine.     

Physical Interaction between Bacterial Heat Shock Protein (Hsp) 90 and Hsp70 Chaperones Mediates Their Cooperative Action to Refold Denatured Proteins

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.