Dissection of Structural and Functional Requirements That Underlie the Interaction of ERdj3 Protein with Substrates in the Endoplasmic Reticulum

ERdj3, a mammalian endoplasmic reticulum (ER) Hsp40/DnaJ family member, binds unfolded proteins, transfers them to BiP, and concomitantly stimulates BiP ATPase activity. However, the requirements for ERdj3 binding to and release from substrates in cells are not well understood. We found that ERdj3 homodimers that cannot stimulate the ATPase activity of BiP (QPD mutants) bound to unfolded ER proteins under steady state conditions in much greater amounts than wild-type ERdj3. This was due to reduced release from these substrates as opposed to enhanced binding, although in both cases dimerization was strictly required for substrate binding. Conversely, heterodimers consisting of one wild-type and one mutant ERdj3 subunit bound substrates at levels comparable with wild-type ERdj3 homodimers, demonstrating that release requires only one protomer to be functional in stimulating BiP ATPase activity. Co-expressing wild-type ERdj3 and a QPD mutant, which each exclusively formed homodimers, revealed that the release rate of wild-type ERdj3 varied according to the relative half-lives of substrates, suggesting that ERdj3 release is an important step in degradation of unfolded client proteins in the ER. Furthermore, pulse-chase experiments revealed that the binding of QPD mutant homodimers remained constant as opposed to increasing, suggesting that ERdj3 does not normally undergo reiterative binding cycles with substrates.     

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.     

Modulation of the Chaperone DnaK Allosterism by the Nucleotide Exchange Factor GrpE

Hsp70 chaperones comprise two domains, the nucleotide-binding domain (Hsp70_NBD), responsible for structural and functional changes in the chaperone, and the substrate-binding domain (Hsp70_SBD), involved in substrate interaction. Substrate binding and release in Hsp70 is controlled by the nucleotide state of DnaK_NBD, with ATP inducing the open, substrate-receptive DnaK_SBD conformation, whereas ADP forces its closure. DnaK cycles between the two conformations through interaction with two cofactors, the Hsp40 co-chaperones (DnaJ in Escherichia coli) induce the ADP state, and the nucleotide exchange factors (GrpE in E. coli) induce the ATP state. X-ray crystallography showed that the GrpE dimer is a nucleotide exchange factor that works by interaction of one of its monomers with DnaK_NBD. DnaK_SBD location in this complex is debated; there is evidence that it interacts with the GrpE N-terminal disordered region, far from DnaK_NBD. Although we confirmed this interaction using biochemical and biophysical techniques, our EM-based three-dimensional reconstruction of the DnaK-GrpE complex located DnaK_SBD near DnaK_NBD. This apparent discrepancy between the functional and structural results is explained by our finding that the tail region of the GrpE dimer in the DnaK-GrpE complex bends and its tip contacts DnaK_SBD, whereas the DnaK_NBD-DnaK_SBD linker contacts the GrpE helical region. We suggest that these interactions define a more complex role for GrpE in the control of DnaK function.     

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.     

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.     

Structural Insights into the Chaperone Activity of the 40-kDa Heat Shock Protein DnaJ: BINDING AND REMODELING OF A NATIVE SUBSTRATE*

Hsp40 chaperones bind and transfer substrate proteins to Hsp70s and regulate their ATPase activity. The interaction of Hsp40s with native proteins modifies their structure and function. A good model for this function is DnaJ, the bacterial Hsp40 that interacts with RepE, the repressor/activator of plasmid F replication, and together with DnaK regulates its function. We characterize here the structure of the DnaJ-RepE complex by electron microscopy, the first described structure of a complex between an Hsp40 and a client protein. The comparison of the complexes of DnaJ with two RepE mutants reveals an intrinsic plasticity of the DnaJ dimer that allows the chaperone to adapt to different substrates. We also show that DnaJ induces conformational changes in dimeric RepE, which increase the intermonomeric distance and remodel both RepE domains enhancing its affinity for DNA.     

Multiple Molecules of Hsc70 and a Dimer of DjA1 Independently Bind to an Unfolded Protein

Protein folding is a prominent chaperone function of the Hsp70 system. Refolding of an unfolded protein is efficiently mediated by the Hsc70 system with either type 1 DnaJ protein, DjA1 or DjA2, and a nucleotide exchange factor. A surface plasmon resonance technique was applied to investigate substrate recognition by the Hsc70 system and demonstrated that multiple Hsc70 proteins and a dimer of DjA1 initially bind independently to an unfolded protein. The association rate of the Hsc70 was faster than that of DjA1 under folding-compatible conditions. The Hsc70 binding involved a conformational change, whereas the DjA1 binding was bivalent and substoichiometric. Consistently, we found that the bound ¹⁴C-labeled Hsc70 to the unfolded protein became more resistant to tryptic digestion. The gel filtration and cross-linking experiments revealed the predominant presence of the DjA1 dimer. Furthermore, the Hsc70 and DjA1 bound to distinct sets of peptide array sequences. All of these findings argue against the generality of the widely proposed hypothesis that the DnaJ-bound substrate is targeted and transferred to Hsp70. Instead, these results suggest the importance of the bivalent binding of DjA1 dimer that limits unfavorable transitions of substrate conformations in protein folding.     

Crystal structure of DnaK protein complexed with nucleotide exchange factor GrpE in DnaK chaperone system: insight into intermolecular communication

The conserved, ATP-dependent bacterial DnaK chaperones process client substrates with the aid of the co-chaperones DnaJ and GrpE. However, in the absence of structural information, how these proteins communicate with each other cannot be fully delineated. For the study reported here, we solved the crystal structure of a full-length Geobacillus kaustophilus HTA426 GrpE homodimer in complex with a nearly full-length G. kaustophilus HTA426 DnaK that contains the interdomain linker (acting as a pseudo-substrate), and the N-terminal nucleotide-binding and C-terminal substrate-binding domains at 4.1-Å resolution. Each complex contains two DnaKs and two GrpEs, which is a stoichiometry that has not been found before. The long N-terminal GrpE α-helices stabilize the linker of DnaK in the complex. Furthermore, interactions between the DnaK substrate-binding domain and the N-terminal disordered region of GrpE may accelerate substrate release from DnaK. These findings provide molecular mechanisms for substrate binding, processing, and release during the Hsp70 chaperone cycle.     

The DNAJA2 Substrate Release Mechanism Is Essential for Chaperone-mediated Folding

DNAJA1 (DJA1/Hdj2) and DNAJA2 (DJA2) are the major J domain partners of human Hsp70/Hsc70 chaperones. Although they have overall similarity with the well characterized type I co-chaperones from yeast and bacteria, they are biologically distinct, and their functional mechanisms are poorly characterized. We identified DJA2-specific activities in luciferase folding and repression of human ether-a-go-go-related gene (HERG) trafficking that depended on its expression levels in cells. Mutations in different internal domains of DJA2 abolished these effects. Using purified proteins, we addressed the mechanistic defects. A mutant lacking the region between the zinc finger motifs (DJA2-Δm2) was able to bind substrate similar to wild type but was incapable of releasing substrate during its transfer to Hsc70. The equivalent mutation in DJA1 also abolished its substrate release. A DJA2 mutant (DJA-221), which had its C-terminal dimerization region replaced by that of DJA1, was inactive but retained its ability to release substrate. The release mechanism required the J domain and ATP hydrolysis by Hsc70, although the nucleotide dependence diverged between DJA2 and DJA1. Limited proteolysis suggested further conformational differences between the two wild-type co-chaperones and the mutants. Our results demonstrate an essential role of specific DJA domains in the folding mechanism of Hsc70.     

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.     

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.     

Binding of Human Nucleotide Exchange Factors to Heat Shock Protein 70 (Hsp70) Generates Functionally Distinct Complexes in Vitro

Proteins with Bcl2-associated anthanogene (BAG) domains act as nucleotide exchange factors (NEFs) for the molecular chaperone heat shock protein 70 (Hsp70). There are six BAG family NEFs in humans, and each is thought to link Hsp70 to a distinct cellular pathway. However, little is known about how the NEFs compete for binding to Hsp70 or how they might differentially shape its biochemical activities. Toward these questions, we measured the binding of human Hsp72 (HSPA1A) to BAG1, BAG2, BAG3, and the unrelated NEF Hsp105. These studies revealed a clear hierarchy of affinities: BAG3 > BAG1 > Hsp105 ≫ BAG2. All of the NEFs competed for binding to Hsp70, and their relative affinity values predicted their potency in nucleotide and peptide release assays. Finally, we combined the Hsp70-NEF pairs with cochaperones of the J protein family (DnaJA1, DnaJA2, DnaJB1, and DnaJB4) to generate 16 permutations. The activity of the combinations in ATPase and luciferase refolding assays were dependent on the identity and stoichiometry of both the J protein and NEF so that some combinations were potent chaperones, whereas others were inactive. Given the number and diversity of cochaperones in mammals, it is likely that combinatorial assembly could generate a large number of distinct permutations.     

The 90-kDa Heat Shock Protein Hsp90 Protects Tubulin against Thermal Denaturation

Hsp90 and tubulin are among the most abundant proteins in the cytosol of eukaryotic cells. Although Hsp90 plays key roles in maintaining its client proteins in their active state, tubulin is essential for fundamental processes such as cell morphogenesis and division. Several studies have suggested a possible connection between Hsp90 and the microtubule cytoskeleton. Because tubulin is a labile protein in its soluble form, we investigated whether Hsp90 protects it against thermal denaturation. Both proteins were purified from porcine brain, and their interaction was characterized in vitro by using spectrophotometry, sedimentation assays, video-enhanced differential interference contrast light microscopy, and native polyacrylamide gel electrophoresis. Our results show that Hsp90 protects tubulin against thermal denaturation and keeps it in a state compatible with microtubule polymerization. We demonstrate that Hsp90 cannot resolve tubulin aggregates but that it likely binds early unfolding intermediates, preventing their aggregation. Protection was maximal at a stoichiometry of two molecules of Hsp90 for one of tubulin. This protection does not require ATP binding and hydrolysis by Hsp90, but it is counteracted by geldanamycin, a specific inhibitor of Hsp90.     

DnaK Chaperone-dependent Disaggregation by Caseinolytic Peptidase B (ClpB) Mutants Reveals Functional Overlap in the N-terminal Domain and Nucleotide-binding Domain-1 Pore Tyrosine

Protein disaggregation in Escherichia coli is carried out by ClpB, an AAA(+) (ATPases associated with various cellular activities) molecular chaperone, together with the DnaK chaperone system. Conformational changes in ClpB driven by ATP binding and hydrolysis promote substrate binding, unfolding, and translocation. Conserved pore tyrosines in both nucleotide-binding domain-1 (NBD-1) and -2 (NBD-2), which reside in flexible loops extending into the central pore of the ClpB hexamer, bind substrates. When the NBD-1 pore loop tyrosine is substituted with alanine (Y251A), ClpB can collaborate with the DnaK system in disaggregation, although activity is reduced. The N-domain has also been implicated in substrate binding, and like the NBD-1 pore loop tyrosine, it is not essential for disaggregation activity. To further probe the function and interplay of the ClpB N-domain and the NBD-1 pore loop, we made a double mutant with an N-domain deletion and a Y251A substitution. This ClpB double mutant is inactive in substrate disaggregation with the DnaK system, although each single mutant alone can function with DnaK. Our data suggest that this loss in activity is primarily due to a decrease in substrate engagement by ClpB prior to substrate unfolding and translocation and indicate an overlapping function for the N-domain and NBD-1 pore tyrosine. Furthermore, the functional overlap seen in the presence of the DnaK system is not observed in the absence of DnaK. For innate ClpB unfolding activity, the NBD-1 pore tyrosine is required, and the presence of the N-domain is insufficient to overcome the defect of the ClpB Y251A mutant.     

Non-native,N-terminal Hsp70 Molecular Motor Recognition Elements in Transit Peptides Support Plastid Protein Translocation

Previously, we identified the N-terminal domain of transit peptides (TPs) as a major determinant for the translocation step in plastid protein import. Analysis of Arabidopsis TP dataset revealed that this domain has two overlapping characteristics, highly uncharged and Hsp70-interacting. To investigate these two properties, we replaced the N-terminal domains of the TP of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and its reverse peptide with a series of unrelated peptides whose affinities to the chloroplast stromal Hsp70 have been determined. Bioinformatic analysis indicated that eight out of nine peptides in this series are not similar to the TP N terminus. Using in vivo and in vitro protein import assays, the majority of the precursors containing Hsp70-binding elements were targeted to plastids, whereas none of the chimeric precursors lacking an N-terminal Hsp70-binding element were targeted to the plastids. Moreover, a pulse-chase assay showed that two chimeric precursors with the most uncharged peptides failed to translocate into the stroma. The ability of multiple unrelated Hsp70-binding elements to support protein import verified that the majority of TPs utilize an N-terminal Hsp70-binding domain during translocation and expand the mechanistic view of the import process. This work also indicates that synthetic biology may be utilized to create de novo TPs that exceed the targeting activity of naturally occurring sequences.     

Hamartin-Hsp70 Interaction Is Necessary for Akt-Dependent Tuberin Phosphorylation during Heat Shock

Hamartin and tuberin interact directly to regulate cell growth negatively. In this study, far-western blotting revealed that hamartin binds directly Heat shock protein 70 (Hsp70), even in the absence of tuberin. While the hamartin-tuberin complex acts as a sensor for a variety of types of stress, it is unclear how the complex is regulated under stress conditions. We found that the hamartin-Hsp70 interaction is stabilized during heat shock. On the other hand, tuberin underwent degradation through phosphorylation in an Akt-dependent manner. Furthermore, we found that when Hsp70 expression was inhibited by N-formyl-3,4-methylenedioxy-benzylidene-γ-butyrolactam (KNK437), Akt phosphorylation on site Ser308 diminished and tuberin was not phosphorylated at Thr1462 during heat shock. We conclude that both hamartin and Hsp70 increase in response to heat shock, whereas tuberin is phosphorylated and thereafter degraded via the PI3K/Akt pathway. Through this pathway, hamartin-Hsp70 plays a crucial role as a scaffolding protein that transfers the Akt signal to tuberin.     

Chaperone-assisted excisive recombination, a solitary role for DnaJ (Hsp40) chaperone in lysogeny escape

Temperate bacteriophage lytic development is intrinsically related to the stress response in particular at the DNA replication and virion maturation steps. Alternatively, temperate phages become lysogenic and integrate their genome into the host chromosome. Under stressful conditions, the prophage resumes a lytic development program, and the phage DNA is excised before being replicated. The KplE1 defective prophage of Escherichia coli K12 constitutes a model system because it is fully competent for integrative as well as excisive recombination and presents an atypical recombination module, which is conserved in various phage genomes. In this work, we identified the host-encoded stress-responsive molecular chaperone DnaJ (Hsp40) as an active participant in KplE1 prophage excision. We first show that the recombination directionality factor TorI of KplE1 specifically interacts with DnaJ. In addition, we found that DnaJ dramatically enhances both TorI binding to its DNA target and excisive recombination in vitro. Remarkably, such stimulatory effect by DnaJ was performed independently of its DnaK chaperone partner and did not require a functional DnaJ J-domain. Taken together, our results underline a novel and unsuspected functional interaction between the generic host stress-regulated chaperone and temperate bacteriophage lysogenic development.     

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.