BAG-1 modulates the chaperone activity of Hsp70/Hsc70

The 70 kDa heat shock family of molecular chaperones is essential to a variety of cellular processes, yet it is unclear how these proteins are regulated in vivo. We present evidence that the protein BAG-1 is a potential modulator of the molecular chaperones, Hsp70 and Hsc70. BAG-1 binds to the ATPase domain of Hsp70 and Hsc70, without requirement for their carboxy-terminal peptide-binding domain, and can be co-immunoprecipitated with Hsp/Hsc70 from cell lysates. Purified BAG-1 and Hsp/Hsc70 efficiently form heteromeric complexes in vitro. BAG-1 inhibits Hsp/Hsc70-mediated in vitro refolding of an unfolded protein substrate, whereas BAG-1 mutants that fail to bind Hsp/Hsc70 do not affect chaperone activity. The binding of BAG-1 to one of its known cellular targets, Bcl-2, in cell lysates was found to be dependent on ATP, consistent with the possible involvement of Hsp/Hsc70 in complex formation. Overexpression of BAG-1 also protected certain cell lines from heat shock-induced cell death. The identification of Hsp/Hsc70 as a partner protein for BAG-1 may explain the diverse interactions observed between BAG-1 and several other proteins, including Raf-1, steroid hormone receptors and certain tyrosine kinase growth factor receptors. The inhibitory effects of BAG-1 on Hsp/Hsc70 chaperone activity suggest that BAG-1 represents a novel type of chaperone regulatory proteins and thus suggest a link between cell signaling, cell death and the stress response.     

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.     

GrpE-like regulation of the hsc70 chaperone by the anti-apoptotic protein BAG-1

The BAG-1 protein appears to inhibit cell death by binding to Bcl-2, the Raf-1 protein kinase, and certain growth factor receptors, but the mechanism of inhibition remains enigmatic. BAG-1 also interacts with several steroid hormone receptors which require the molecular chaperones Hsc70 and Hsp90 for activation. Here we show that BAG-1 is a regulator of the Hsc70 chaperone. BAG-1 binds to the ATPase domain of Hsc70 and, in cooperation with Hsp40, stimulates Hsc70's steady-state ATP hydrolysis activity approximately 40-fold. Similar to the action of the GrpE protein on bacterial Hsp70, BAG-1 accelerates the release of ADP from Hsc70. Thus, BAG-1 regulates the Hsc70 ATPase in a manner contrary to the Hsc70-interacting protein Hip, which stabilizes the ADP-bound state. Intriguingly, BAG-1 and Hip compete in binding to the ATPase domain of Hsc70. Our results reveal an unexpected diversity in the regulation of Hsc70 and raise the possibility that the observed anti-apoptotic function of BAG-1 may be exerted through a modulation of the chaperone activity of Hsc70 on specific protein folding and maturation pathways.     

An evolutionarily conserved family of Hsp70/Hsc70 molecular chaperone regulators

Heat Shock Protein 70 kDa (Hsp70) family molecular chaperones play critical roles in protein folding and trafficking in all eukaryotic cells. The mechanisms by which Hsp70 family chaperones are regulated, however, are only partly understood. BAG-1 binds the ATPase domains of Hsp70 and Hsc70, modulating their chaperone activity and functioning as a competitive antagonist of the co-chaperone Hip. We describe the identification of a family of BAG-1-related proteins from humans (BAG-2, BAG-3, BAG-4, BAG-5), the invertebrate Caenorhabditis elegans (BAG-1, BAG-2), and the fission yeast Schizosaccharomyces pombe (BAG-1A, BAG-1B). These proteins all contain a conserved approximately 45-amino acid region near their C termini (the BAG domain) that binds Hsc70/Hsp70, but they differ widely in their N-terminal domains. The human BAG-1, BAG-2, and BAG-3 proteins bind with high affinity (KD congruent with 1-10 nM) to the ATPase domain of Hsc70 and inhibit its chaperone activity in a Hip-repressible manner. The findings suggest opportunities for specification and diversification of Hsp70/Hsc70 chaperone functions through interactions with various BAG-family proteins.     

Nucleotide-induced conformational changes in the ATPase and substrate binding domains of the DnaK chaperone provide evidence for interdomain communication

Interactions of the DnaK (Hsp70) chaperone from Escherichia coli with substrates are controlled by ATP. Nucleotide-induced changes in DnaK conformation were investigated by monitoring changes in tryptic digestion pattern and tryptophan fluorescence. Using nucleotide-free DnaK preparations, not only the known ATP-induced major changes in kinetics and pattern of proteolysis but also minor ADP-induced changes were detected. Similar ATP-induced conformational changes occurred in the DnaK-T199A mutant protein defective in ATPase activity, demonstrating that they result from binding, not hydrolysis, of ATP. N-terminal sequencing and immunological mapping of tryptic fragments of DnaK identified cleavage sites that, upon ATP addition, appeared within the proposed C-terminal substrate binding region and disappeared in the N-terminal ATPase domain. They hence reflect structural alterations in DnaK correlated to substrate release and indicate ATP-dependent domain interactions. Domain interactions are a prerequisite for efficient tryptic degradation as fragments of DnaK comprising the ATPase and C-terminal domains were highly protease-resistant. Fluorescence analysis of the N-terminally located single tryptophan residue of DnaK revealed that the known ATP-induced alteration of the emission spectrum, proposed to result directly from conformational changes in the ATPase domain, requires the presence of the C-terminal domain and therefore mainly results from altered domain interaction. Analyses of the C-terminally truncated DnaK163 mutant protein revealed that nucleotide-dependent interdomain communication requires a 15-kDa segment assumed to constitute the substrate binding site.     

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.     

A mutational study of the peptide-binding domain of Hsc70 guided by secondary structure prediction

The abundant, cytoplasmic molecular chaperones of eukaryotic cells, of which mammalian Hsc70 is a member, have central roles in protein folding pathways in cells. Although substantial information is now available on substrate interactions and ATPase activity, neither the crystal structure of the intact Hsc70 molecule nor its isolated peptide-binding domain is known. Recently, the crystal structure of the isolated peptide-binding domain of an evolutionary relative of mammalian Hsc70, the DnaK protein of Escherichia coli, was solved. We have generated several rat Hsc70 mutants using site-directed and cassette mutagenesis guided by secondary structure predictions to test the hypothesis that the peptide-binding domains of mammalian Hsc70 and DnaK have similar molecular structures. Biochemical properties along with the ATPase and peptide binding activities of the resulting recombinant proteins were determined. Biochemical analyses included one- and two-dimensional gel electrophoresis, electrospray mass spectrometry, and N-terminal amino acid sequencing. The results of our study suggest that the DnaK molecular structure is a useful working model for the mammalian Hsc70 peptide-binding domain. Evidence is provided that (i) small additions to the N terminus of Hsc70 alter the function of the peptide-binding domain, (ii) alterations in the C-terminal tetrapeptide EEVD result in dramatic increases in basal ATPase activity, (iii) polyalanine substitution of a helical connector segment compensates for changes at the C terminus to restore near-normal function, (iv) specific side chain interactions involving this connector segment are not required for peptide-stimulated ATPase activity, and (v) disruption of the cap homologue region inhibits peptide binding by Hsc70.     

Oligomerization of the 17-kDa peptide-binding domain of the molecular chaperone HSC70

Crystallographic and biochemical studies have indicated that the peptide-binding site of the molecular chaperone HSC70 is located in a small subdomain comprising a beta-sheet motif followed by a helical region, and there is some evidence of the involvement of this site in oligomerization of the protein. To determine the structure of this subdomain in solution and examine its involvement in oligomerization of HSC70, a 17-kDa protein (residues 385-540 of HSC70) consisting mainly of the peptide-binding site was constructed and analyzed for oligomerization properties. This small domain was found to bind peptides and to form oligomers in solution, probably tetramers, which dissociated into monomers on peptide binding in a manner comparable with that observed for the whole protein. Furthermore, in the 60-kDa fragment of HSC70, which is composed of the 17-kDa domain and the 44-kDa ATPase domain, not only were the oligomerization properties conserved, but dissociation of multimeric species into monomers on ATP binding also occurred and peptide stimulation of ATPase activity was restored. These results indicate that the isolated 17-kDa peptide-binding domain is necessary and sufficient for oligomerization of the whole protein, suggesting that the peptide-binding site may be involved in the oligomerization process.     

The charged region of Hsp90 modulates the function of the N-terminal domain

Hsp90, an abundant heat shock protein that is highly expressed even under physiological conditions, is involved in the folding of key molecules of the cellular signal transduction system such as kinases and steroid receptors. It seems to contain two chaperone sites differing in substrate specificity. Binding of ATP or the antitumor drug geldanamycin alters the substrate affinity of the N-terminal chaperone site, whereas both substances show no influence on the C-terminal one. In wild-type Hsp90 the fragments containing the chaperone sites are connected by a highly charged linker of various lengths in different organisms. As this linker region represents the most striking difference between bacterial and eukaryotic Hsp90s, it may be involved in a gain of function of eukaryotic Hsp90s. Here, we have analyzed a fragment of yeast Hsp90 consisting of the N-terminal domain and the charged region (N272) in comparison with the isolated N-terminal domain (N210). We show that the charged region causes an increase in the affinity of the N-terminal domain for nonnative protein and establishes a crosstalk between peptide and ATP binding. Thus, the binding of peptide to N272 decreases its affinity for ATP and geldanamycin, whereas the ATP-binding properties of the monomeric N-terminal domain N210 are not influenced by peptide binding. We propose that the charged region connecting the two chaperone domains plays an important role in regulating chaperone function of Hsp90.     

Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)-domain co-chaperones

The in vivo function of the heat shock protein 90 (Hsp90) molecular chaperone is dependent on the binding and hydrolysis of ATP, and on interactions with a variety of co-chaperones containing tetratricopeptide repeat (TPR) domains. We have now analysed the interaction of the yeast TPR-domain co-chaperones Sti1 and Cpr6 with yeast Hsp90 by isothermal titration calorimetry, circular dichroism spectroscopy and analytical ultracentrifugation, and determined the effect of their binding on the inherent ATPase activity of Hsp90. Sti1 and Cpr6 both bind with sub-micromolar affinity, with Sti1 binding accompanied by a large conformational change. Two co-chaperone molecules bind per Hsp90 dimer, and Sti1 itself is found to be a dimer in free solution. The inherent ATPase activity of Hsp90 is completely inhibited by binding of Sti1, but is not affected by Cpr6, although Cpr6 can reactivate the ATPase activity by displacing Sti1 from Hsp90. Bound Sti1 makes direct contact with, and blocks access to the ATP-binding site in the N-terminal domain of Hsp90. These results reveal an important role for TPR-domain co-chaperones as regulators of the ATPase activity of Hsp90, showing that the ATP-dependent step in Hsp90-mediated protein folding occurs after the binding of the folding client protein, and suggesting that ATP hydrolysis triggers client-protein release.     

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.     

Transcription from heterologous rRNA operon promoters in chloroplasts reveals requirement for specific activating factors

The abundant molecular chaperone Hsp90 is a key regulator of protein structure in the cytosol of eukaryotic cells. Although under physiological conditions a specific subset of proteins is substrate for Hsp90, under stress conditions Hsp90 seems to perform more general functions. However, the underlying mechanism of Hsp90 remained enigmatic. Here, we analyzed the function of conserved Hsp90 domains. We show that Hsp90 possesses two chaperone sites located in the N- and C-terminal fragments, respectively. The C-terminal fragment binds to partially folded proteins in an ATP-independent way potentially regulated by cochaperones. The N-terminal domain contains a peptide binding site that seems to bind preferentially peptides longer than 10 amino acids. Peptide dissociation is induced by ATP binding. Furthermore, the antitumor drug geldanamycin both inhibits the weak ATPase of Hsp90 and stimulates peptide release. We propose that the existence of two functionally different chaperone sites together with a substrate-selecting set of cochaperones allows Hsp90 to guide the folding of a subset of target proteins and, at the same time, to exhibit general chaperone functions.     

The carboxy-terminal domain of Hsc70 provides binding sites for a distinct set of chaperone cofactors

The modulation of the chaperone activity of the heat shock cognate Hsc70 protein in mammalian cells involves cooperation with chaperone cofactors, such as Hsp40; BAG-1; the Hsc70-interacting protein, Hip; and the Hsc70-Hsp90-organizing protein, Hop. By employing the yeast two-hybrid system and in vitro interaction assays, we have provided insight into the structural basis that underlies Hsc70's cooperation with different cofactors. The carboxy-terminal domain of Hsc70, previously shown to form a lid over the peptide binding pocket of the chaperone protein, mediates the interaction of Hsc70 with Hsp40 and Hop. Remarkably, the two cofactors bind to the carboxy terminus of Hsc70 in a noncompetitive manner, revealing the existence of distinct binding sites for Hsp40 and Hop within this domain. In contrast, Hip interacts exclusively with the amino-terminal ATPase domain of Hsc70. Hence, Hsc70 possesses separate nonoverlapping binding sites for Hsp40, Hip, and Hop. This appears to enable the chaperone protein to cooperate simultaneously with multiple cofactors. On the other hand, BAG-1 and Hip have recently been shown to compete in binding to the ATPase domain. Our data thus establish the existence of a network of cooperating and competing cofactors regulating the chaperone activity of Hsc70 in the mammalian cell.     

Inhibition of Hsp70 ATPase activity and protein renaturation by a novel Hsp70-binding protein

A cDNA that codes for an Hsp70-interacting protein (HspBP1) was isolated from a human heart cDNA library using the yeast two-hybrid system. The derived amino acid sequence is unique and therefore represents a new regulator of Hsp70. Northern blots of RNA from human tissues indicate that HspBP1 mRNA has a size of approximately 1.7 kilobase pairs and is present in all tissues analyzed but is most abundant in heart and skeletal muscle. Western blot analysis revealed a protein of approximately 40 kilodaltons detected in cell extracts. The ATPase domain of Hsp70 demonstrated binding to HspBP1. Further experiments showed binding of HspBP1 to Hsp70 and Hsc70 in a total heart extract. HspBP1 (8 microM) inhibited approximately 90% of the Hsp40-activated Hsp70 ATPase activity. HspBP1 prevented ATP binding to Hsp70, and therefore this is the likely mechanism of inhibition. Hsp40-activated ATPase activity is essential for the renaturation activity of Hsp70; therefore, the effects of HspBP1 on renaturation of luciferase in a reticulocyte lysate and a defined system were examined. HspBP1 inhibited renaturation with half-maximal inhibition at 2 microM. These data indicate that we have identified a novel Hsp70-interacting protein that inhibits Hsp70 chaperone activity.     

BAG-1, a negative regulator of Hsp70 chaperone activity, uncouples nucleotide hydrolysis from substrate release

Molecular chaperones influence the process of protein folding and, under conditions of stress, recognize non-native proteins to ensure that misfolded proteins neither appear nor accumulate. BAG-1, identified as an Hsp70 associated protein, was shown to have the unique properties of a negative regulator of Hsp70. Here, we demonstrate that BAG-1 inhibits the in vitro protein refolding activity of Hsp70 by forming stable ternary complexes with non-native substrates that do not release even in the presence of nucleotide and the co-chaperone, Hdj-1. However, the substrate in the BAG-1-containing ternary complex does not aggregate and remains in a soluble intermediate folded state, indistinguishable from the refolding-competent substrate-Hsp70 complex. BAG-1 neither inhibits the Hsp70 ATPase, nor has the properties of a nucleotide exchange factor; instead, it stimulates ATPase activity, similar to that observed for Hdj-1, but with opposite consequences. In the presence of BAG-1, the conformation of Hsp70 is altered such that the substrate binding domain becomes less accessible to protease digestion, even in the presence of nucleotide and Hdj-1. These results suggest a mechanistic basis for BAG-1 as a negative regulator of the Hsp70-Hdj-1 chaperone cycle.     

Stch encodes the 'ATPase core' of a microsomal stress 70 protein

The stress70 protein chaperone family plays a central role in the processing of cytosolic and secretory proteins. We have cloned a human cDNA, designated Stch, that is conserved in rat tissues and which encodes a novel microsome-associated member of the stress70 protein chaperone family. Stch mRNA is constitutively expressed in all human cell types and is induced by incubation with the calcium ionophore A23187, but not by exposure to heat shock. Inspection of the predicted amino acid sequence reveals that the STCH product contains a unique hydrophobic leader sequence and shares homology within the amino terminal domains of the stress70 gene family, but has a 50 residue insertion within the ATP-binding domains and truncates the carboxyl terminal peptide-binding region. Immunofluorescent and subcellular analyses show that STCH migrates predominantly as a 60 kDa species and is enriched in a membrane-bound microsome fraction. In contrast to purified BiP and dnaK, however, STCH demonstrates ATPase activity that is independent of peptide stimulation. Stch, therefore, encodes a calcium-inducible, microsome-associated ATPase activity with properties similar to a proteolytically cleaved N-terminal HSC70/BiP fragment. This truncated stress70 molecule may allow increased diversity in cellular responses to protein processing requirements.     

Conformational changes of an Hsp70 molecular chaperone induced by nucleotides, polypeptides, and N-ethylmaleimide

Hsp70 molecular chaperones are highly conserved ATPases that guide the folding and assembly of proteins in many cellular pathways. They use the energy of ATP binding and hydrolysis to regulate their interactions with hydrophobic regions of unfolded proteins. The activities and the conformations of the N-terminal nucleotide- and C-terminal polypeptide-binding domains of Hsp70s are coupled. We recently reported that the sulfhydryl-modifying reagent N-ethylmaleimide (NEM) inactivates the yeast Hsp70 Ssa1p by reacting with its three cysteine residues which are located in the nucleotide-binding domain. To further characterize conformational changes associated with interdomain coupling and to determine whether NEM alters Ssa1p's conformation, the structures of Ssa1p and NEM-modified Ssa1p (NEM-Ssa1p) were compared using a variety of biophysical techniques. Size exclusion chromatography revealed that NEM-Ssa1p is more oligomeric and more resistant to nucleotide- or polypeptide-dependent depolymerization than Ssa1p. Measurement of the thermal stability indicated that NEM modification has an effect very similar to that of binding of nucleotides to the unmodified protein. Circular dichroism demonstrated small differences in the secondary structure of Ssa1p and NEM-Ssa1p, and in their complexes with nucleotides. NEM modification increased the ANS fluorescence of Ssa1p and exposed numerous trypsin-sensitive sites in its nucleotide-binding domain. The intrinsic fluorescence of Ssa1p's only tryptophan residue, which is located in a C-terminal alpha-helical region adjacent to the polypeptide-binding cleft, was quenched in the presence of ATP, but not ADP. NEM modification altered nucleotide-dependent changes in the intrinsic fluorescence of Ssa1p. Together, these results demonstrate that NEM alters the conformation of Ssa1p and disrupts, but does not eliminate, interdomain communication. Furthermore, the results provide evidence for a model in which the polypeptide-binding cleft of Hsp70s is covered by an alpha-helical lid that is open in the presence of ATP, but closed in the presence of ADP.     

Transcriptional regulation of the human proenkephalin gene by conformational switching: implications for decoy design

Molecular chaperones are essential to all living organisms. Their key role consists of mediating protein folding within the cell. Recent functional studies have provided more detailed information about the function and regulation of the chaperone network. Highlights of the past year include the crystal structure determinations of the asymmetric GroEL-GroES complex and of their isolated peptide-binding domains.