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.     

Hop as an adaptor in the heat shock protein 70 (Hsp70) and hsp90 chaperone machinery

Hop, an abundant and conserved protein of unresolved function, binds concomitantly with heat shock protein 70 (Hsp70) and Hsp90, participates with heat shock proteins at an intermediate stage of progesterone receptor assembly, and is required for efficient assembly of mature receptor complexes in vitro. A largely untested hypothesis is that Hop functions as an adaptor that targets Hsp90- to Hsp70-substrate complexes; if true, then loss of either Hsp70 binding or Hsp90 binding by Hop should equally disrupt its ability to promote assembly of mature receptor complexes. To generate Hop mutants that selectively disrupt heat shock protein interactions, highly conserved amino acids in the previously mapped Hsp70 and Hsp90 binding domains of Hop and in a conserved C-terminal domain were targeted for small substitutions and deletions. In co-precipitation assays, these mutants displayed selective loss of association with heat shock proteins. In assays using Hop-depleted rabbit reticulocyte lysate for the cell-free assembly of receptor complexes, none of the Hop mutants inhibited Hsp70 binding to receptor, but all mutants were defective in supporting Hsp90-receptor interactions. Thus, Hop has a novel role in the chaperone machinery as an adaptor that can integrate Hsp70 and Hsp90 interactions.     

Change in the therapist: the role of patient-induced inspiration

BACKGROUND: The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones, which promote protein folding and participate in many cellular functions. The Hsp70 chaperones are composed of two major domains. The N-terminal ATPase domain binds to and hydrolyzes ATP, whereas the C-terminal domain is required for polypeptide binding. Cooperation of both domains is needed for protein folding. The crystal structure of bovine Hsc70 ATPase domain (bATPase) has been determined and, more recently, the crystal structure of the peptide-binding domain of a related chaperone, DnaK, in complex with peptide substrate has been obtained. The molecular chaperone activity and conformational switch are functionally linked with ATP hydrolysis. A high-resolution structure of the ATPase domain is required to provide an understanding of the mechanism of ATP hydrolysis and how it affects communication between C- and N-terminal domains. RESULTS: The crystal structure of the human Hsp70 ATPase domain (hATPase) has been determined and refined at 1. 84 A, using synchrotron radiation at 120K. Two calcium sites were identified: the first calcium binds within the catalytic pocket, bridging ADP and inorganic phosphate, and the second calcium is tightly coordinated on the protein surface by Glu231, Asp232 and the carbonyl of His227. Overall, the structure of hATPase is similar to bATPase. Differences between them are found in the loops, the sites of amino acid substitution and the calcium-binding sites. Human Hsp70 chaperone is phosphorylated in vitro in the presence of divalent ions, calcium being the most effective. CONCLUSIONS: The structural similarity of hATPase and bATPase and the sequence similarity within the Hsp70 chaperone family suggest a universal mechanism of ATP hydrolysis among all Hsp70 molecular chaperones. Two calcium ions have been found in the hATPase structure. One corresponds to the magnesium site in bATPase and appears to be important for ATP hydrolysis and in vitro phosphorylation. Local changes in protein structure as a result of calcium binding may facilitate phosphorylation. A small, but significant, movement of metal ions and sidechains could position catalytically important threonine residues for phosphorylation. The second calcium site represents a new calcium-binding motif that can play a role in the stabilization of protein structure. We discuss how the information about catalytic events in the active site could be transmitted to the peptide-binding domain.     

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.     

Identification of a 71-kilodalton surface-associated Hsp70 homologue in Coxiella burnetii

A Coxiella burnetii Hsp70 homologue was identified by using an acid activation in vitro system in which protein synthesis has been followed by [35S]methionine labeling, autoradiography, and immunoblotting. The protein was one of those predominantly labeled, and the immunoblots revealed that it was recognized by anti-DnaK antibodies. The corresponding gene was isolated, and its nucleotide sequence was determined and analyzed. A single open reading frame (ORF) with a size of 1,968 bp was identified. The ORF encodes a protein containing 656 residues and having a molecular weight of 70, 800. The -10 promoter sequence was shown to be identical with the consensus heat shock sigma32 promoter sequence. The base composition at the presumed -35 region revealed an EcoRI site in the expected region, which is assumed to be located at the border of the cloned fragment. The gene was expressed in Escherichia coli as an intact protein. The C. burnetii 71-kDa protein sequence has a high degree of homology to sequences of the Hsp70 family. A comparison of sequences revealed that the similarity with Hsp70s from other intracellular bacteria, e.g., Legionella pneumophila and Francisella tularensis, as well as E. coli DnaK, is more than 80%. The homologous regions are found in the N-terminal and central parts of the protein sequence, and they include the signature patterns of the Hsp70 family of proteins. The presence of the 71-kDa protein in association with the cell wall as well as in the cytoplasm was demonstrated by the use of immunoelectron microscopy. The dual localization was verified by Western blot analysis of proteins in C. burnetii cell fractions, using purified antibodies directed to the 71-kDa protein.     

Structural analysis of substrate binding by the molecular chaperone DnaK

DnaK and other members of the 70-kilodalton heat-shock protein (hsp70) family promote protein folding, interaction, and translocation, both constitutively and in response to stress, by binding to unfolded polypeptide segments. These proteins have two functional units: a substrate-binding portion binds the polypeptide, and an adenosine triphosphatase portion facilitates substrate exchange. The crystal structure of a peptide complex with the substrate-binding unit of DnaK has now been determined at 2.0 angstroms resolution. The structure consists of a beta-sandwich subdomain followed by alpha-helical segments. The peptide is bound to DnaK in an extended conformation through a channel defined by loops from the beta sandwich. An alpha-helical domain stabilizes the complex, but does not contact the peptide directly. This domain is rotated in the molecules of a second crystal lattice, which suggests a model of conformation-dependent substrate binding that features a latch mechanism for maintaining long lifetime complexes.     

Binding of heptapeptides or unfolded proteins to the chimeric C-terminal domains of 70-kDa heat shock cognate protein

Seventy-kDa heat shock cognate protein (hsc70) and its homologs in bacteria, yeast and vertebrates are known to form complexes with S-carboxymethyl-alpha-lactalbumin (CMLA), an unfolded protein; and, this activity has been attributed to its C-terminal 30-kDa domain. Herein, we show that hsc70s isolated from the seeds of mung bean and peas, however, are not effective in complexing with CMLA, and that the 30-kDa domain of Arabidopsis hsc70 (At30) cannot form stable complexes with CMLA either. Moreover, chimeric 30-kDa domains, either composed of rat 18-kDa and Arabidopsis 10-kDa subdomains (R18At10) or with Arabidopsis 18-kDa and rat 10-kDa subdomains (At18R10), were prepared and tested for their ability to complex with CMLA or a heptapeptide FYQLALT. At18R10 cannot complex with both CMLA and FYQLALT. On the other hand, R18At10 is capable of forming complexes with FYQLALT at a level similar to that of the rat 30-kDa domain (R30). R18At10 also forms complexes with CMLA, but the amount of the R18At10/CMLA complexes is much less than that of R30/CMLA. The results imply that the 18-kDa subdomain dictates the binding specificity for heptapeptide, and that the C-terminal 10-kDa subdomain may also provide some selection or restriction for unfolded proteins to form complexes with hsc70.     

Aggrecan synthesis and secretion. A paradigm for molecular and cellular coordination of multiglobular protein folding and intracellular trafficking

Each globular domain of exported multiglobular proteins putatively undergoes chaperone surveillance in the endoplasmic reticulum lumen. It is difficult to visualize how surveillance of multiple globular domains might be orchestrated and regulated. Aggrecan core protein has been used as a prototype for this problem by examining transfection of informative constructs into Chinese hamster ovary cells. The salient results are as follows: 1) aggrecan's N-terminal G1 domain is minimally secreted, and its flanking Golgi reporter sites are not decorated with glycsoaminoglycan chains; in contrast, its C-terminal G3 domain is readily secreted with flanking GAG chains, and G3 also facilitates G1 secretion; 2) G3 but not G1 can be intracellularly cross-linked to chaperone Hsp25; 3) G3 and Hsp25 remain noncovalently bound and are secreted together when G3 is situated N-terminal to its normal location; 4) exon 15, which encodes the center of G3's C-lectin subdomain, is necessary and sufficient for G3 secretion. A model is proposed in which Hsp25 piggybacks onto nascent G3 in the cytosol during a translocational pause and enters the ER lumen with G3, and once G3 properly folds, Hsp25 releases G3 and recycles to the nucleus while G3 continues to the Golgi stacks, providing passage for the entire core protein.     

Differential interactions of p23 and the TPR-containing proteins Hop, Cyp40, FKBP52 and FKBP51 with Hsp90 mutants

Hsp90 is required for the normal function of steroid receptors, but its binding to steroid receptors is mediated by Hsc70 and several hsp-associated accessory proteins. An assortment of Hsp90 mutants were tested for their abilities to interact with each of the following accessories: Hop, Cyp40, FKBP52, FKBP51, and p23. Of the 11 Hsp90 mutants tested, all were defective to some extent in associating with progestin (PR) complexes. In every case, however, reduced PR binding correlated with a defect in binding of one or more accessories. Co-precipitation of mutant Hsp90 forms with individual accessories was used to map Hsp90 sequences required for accessory protein interactions. Mutation of Hsp90's highly conserved C-terminal EEVD to AAVD resulted in diminished interactions with several accessory proteins, most particularly with Hop. Deletion of amino acids 661-677 resulted in loss of Hsp90 dimerization and also caused diminished interactions with all accessory proteins. Binding of p23 mapped most strongly to the N-terminal ATP-binding domain of Hsp90 while binding of TPR proteins mapped to the C-terminal half of Hsp90. These results and others further suggest that the N- and C-terminal regions of Hsp90 maintain important conformational links through intramolecular interactions and/or intermolecular influences in homodimers.     

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.     

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

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

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.     

Modification of two distinct COOH-terminal domains is required for murine p53 activation by bacterial Hsp70

Activation of the latent DNA binding function of human p53 protein by the bacterial Hsp70, DnaK, represents a unique reaction in which a heat shock protein can interact with a native protein to affect its function. We have localized a likely DnaK interaction site on native human p53 tetramers to a motif flanking the COOH-terminal casein kinase II and protein kinase C phosphorylation sites. Murine p53 is less efficiently activated by DnaK, which has permitted a search for factors that might cooperate in p53 activation by DnaK. We show that optimal activation by DnaK may be dependent upon the phosphorylation state of murine p53, in particular, modification of p53 at the cdc2 phosphorylation site by point mutation decreases the extent of activation by DnaK. Additionally, the monoclonal antibody PAb241, binding in the vicinity of the cdc2 phosphorylation site, is able to activate the specific DNA binding function of p53. This has led us to propose a second regulatory motif flanking the tetramerization domain of p53 that cooperates with factors binding at the negative regulatory domain in the extreme COOH terminus.     

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

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

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.     

Long non-stop reading frames on the antisense strand of heat shock protein 70 genes and prion protein (PrP) genes are conserved between species

Several mammalian genes, including heat shock protein (Hsp70) and prion protein (PrP) genes, have been reported to have long open reading frames (ORFs) or non-stop reading frames (NRFs) in the antisense direction. A simple explanation would be that these long antisense reading frames, which are usually in the same triplet frame as the coding strand, are the fortuitous byproduct of a high overall [G+C] content with concomitant preference for G/C over A/T in the third codon position, a preference for RNY type codons (purine/any nucleotide/pyrimidine), and/or a bias against serine and leucine, the only amino acids with codons that can be read as stop codons in the antisense direction. The PrP genes and most heat shock genes with long antisense NRFs (aNRFs) are indeed relatively [G+C] rich but do not show a bias against serine and leucine. In several vertebrates investigated, at least one of the Hsp70 genes has a long antisense reading frame, and we found that some, though not all, putative stop codons in long Hsp70 antisense reading frames were due to sequencing errors. The PrP gene contains an extended antisense open reading frame in all 45 eutherian mammals tested, but not in a marsupial and in a bird. In the PrP gene, the long, protein-coding exon also harbors the antisense nonstop reading frame. In both Hsp70 and PrP genes, the putative antisense protein sequence is well conserved. Even though there is no clear evidence in Hsp70 or PrP genes for the existence of the respective antisense proteins, we speculate that such antisense proteins serve to regulate the genuine Hsp and PrP proteins under special circumstances. Alternatively, regulation might occur at the RNA level, and the antisense RNA would merely lack stop codons to prevent its rapid degradation by an mRNA quality control mechanism that is triggered by premature stop codons. We note that both Hsp and PrP are involved in physiological or pathological protein aggregation phenomena, that scrapie prions have been reported to modify the expression or localization of heat shock proteins, and that in yeast, propagation of a prion-like state (PSI+) depends on a heat shock (Hsp104) protein.     

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.     

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.     

The DnaJ domain of polyomavirus large T antigen is required to regulate Rb family tumor suppressor function

Tumor suppressors of the retinoblastoma susceptibility gene family regulate cell growth and differentiation. Polyomavirus large T antigens (large T) bind Rb family members and block their function. Mutations of large T sequences conserved with the DnaJ family affect large T binding to a cellular DnaK, heat shock protein 70. The same mutations abolish large T activation of E2F-containing promoters and Rb binding-dependent large T activation of cell cycle progression. Cotransfection of a cellular DnaJ domain blocks wild-type large T action, showing that the connection between the chaperone system and tumor suppressors is direct. Although they are inactive in assays dependent on Rb family binding, mutants in the J region retain the ability to associate with pRb, p107, and p130. This suggests that binding of Rb family members by large T is not sufficient for their inactivation and that a functional J domain is required as well. This work connects the DnaJ and DnaK molecular chaperones to regulation of tumor suppressors by polyomavirus large T.