Hsc62, Hsc56, and GrpE,the third Hsp70 chaperone system of Escherichia coli

Hsc62 is the third Hsp70 homolog of Escherichia coli, which we found previously. Hsc62 is structurally and biochemically similar to DnaK, but hscC gene encoding Hsc62 did not compensate for the defects in the dnaK-null mutant of E. coli MC4100 strain. We cloned the ybeV gene and purified the gene product named Hsc56, a 55,687-Da protein with a J-domain like sequence. Hsc56 stimulated the ATPase activity of only Hsc62 but not those of the other Hsp70 homologs, DnaK and Hsc66. Hsc56 contains the -His-Pro-Glu- sequence corresponding to the His-Pro-Asp motif in DnaJ, which is indispensable for DnaJ to interact with DnaK. Conversion of -His-Pro-Glu- to -Ala-Ala-Ala- abolished the ability of Hsc56 to stimulate the ATPase activity of Hsc62. GrpE, a nucleotide exchange factor for DnaK, also stimulated the ATPase activity of Hsc62 in the presence of Hsc56. Hsc62-Hsc56-GrpE is probably a new Hsp70 chaperone system of E. coli.     

Tuning of chaperone activity of Hsp70 proteins by modulation of nucleotide exchange

The Hsp70 chaperone activity in protein folding is regulated by ATP-controlled cycles of substrate binding and release. Nucleotide exchange plays a key role in these cycles by triggering substrate release. Structural searches of Hsp70 homologs revealed three structural elements within the ATPase domain: two salt bridges and an exposed loop. Mutational analysis showed that these elements control the dissociation of nucleotides, the interaction with exchange factors and chaperone activity. Sequence variations in the three elements classify the Hsp70 family members into three subfamilies, DnaK proteins, HscA proteins and Hsc70 proteins. These subfamilies show strong differences in nucleotide dissociation and interaction with the exchange factors GrpE and Bag-1.     

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

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

Complementation of an Escherichia coli DnaK defect by Hsc70-DnaK chimeric proteins

Escherichia coli DnaK and rat Hsc70 are members of the highly conserved 70-kDa heat shock protein (Hsp70) family that show strong sequence and structure similarities and comparable functional properties in terms of interactions with peptides and unfolded proteins and cooperation with cochaperones. We show here that, while the DnaK protein is, as expected, able to complement an E. coli dnaK mutant strain for growth at high temperatures and lambda phage propagation, Hsc70 protein is not. However, an Hsc70 in which the peptide-binding domain has been replaced by that of DnaK is able to complement this strain for both phenotypes, suggesting that the peptide-binding domain of DnaK is essential to fulfill the specific functions of this protein necessary for growth at high temperatures and for lambda phage replication. The implications of these findings on the functional specificities of the Hsp70s and the role of protein-protein interactions in the DnaK chaperone system are discussed.     

The in vivo and in vitro characterization of DnaK from Agrobacterium tumefaciens RUOR

Molecular chaperones of the heat shock protein 70 family (Hsp70; also called DnaK in prokaryotes) play an important role in the folding and functioning of cellular protein machinery. The dnaK gene from the plant pathogen Agrobacterium tumefaciens RUOR was amplified using the polymerase chain reaction and the DnaK protein (Agt DnaK) was over-produced as a His-tagged protein in Escherichia coli. The Agt DnaK amino acid sequence was 96% identical to the A. tumefaciens C58 DnaK sequence and 65% identical to the E. coli DnaK sequence. Agt DnaK was shown to be able to functionally replace E. coli DnaK in vivo using complementation assays with an E. coli dnaK756 mutant strain and a dnaK52 deletion strain. Over-production and purification of Agt DnaK was successful, and allowed for further characterization of the protein. Kinetic analysis of the basal ATPase activity of purified Agt DnaK revealed a Vmax of 1.3 nmol phosphate released per minute per milligram DnaK, and a Km of 62 microM ATP. Thus, this is the first study to provide both in vivo and in vitro evidence that Agt DnaK has the properties of a molecular chaperone of the Hsp70 family.     

Cloning and characterization of a new soluble murine J-domain protein that stimulates BiP, Hsc70 and DnaK ATPase activity with different efficiencies

Hsp70s perform many functions in the cell through their ATPase activity that is stimulated by a genuine partner that contains a highly conserved so called J-domain. Here we report the cloning and characterization of a new J-domain protein named MmDjC7. The complete cDNA encodes a putative soluble 22 kDa protein that contains a conserved J-domain, but lacks the G/F- and C-rich regions found in the bacterial Escherichia coli DnaJ. Northern analysis revealed that mmDjC7 mRNA (0.9 kb) is most abundant in the heart and liver tissues. Recombinant hexahistidine tagged MmDjC7 (25 kDa) was efficiently expressed in E. coli and purified to homogeneity. MmDjC7 stimulates the ATPase activity of murine BiP, Hsc70 and E. coli DnaK, albeit with very different molar ratios that vary from 1:2 (for BiP/MmDjC7) to 1:10 (for DnaK/MmDjC7). MmDjC7 thus appears to be a new J-domain protein that can possibly interact with more than one Hsp70.     

The Hsc66-Hsc20 Chaperone System in Escherichia coli: Chaperone Activity and Interactions with the DnaK-DnaJ-GrpE System

Hsc66, a stress-70 protein, and Hsc20, a J-type accessory protein, comprise a newly described Hsp70-type chaperone system in addition to DnaK-DnaJ-GrpE in Escherichia coli. Because endogenous substrates for the Hsc66-Hsc20 system have not yet been identified, we investigated chaperone-like activities of Hsc66 and Hsc20 by their ability to suppress aggregation of denatured model substrate proteins, such as rhodanese, citrate synthase, and luciferase. Hsc66 suppressed aggregation of rhodanese and citrate synthase, and ATP caused effects consistent with complex destabilization typical of other Hsp70-type chaperones. Differences in the activities of Hsc66 and DnaK, however, suggest that these chaperones have dissimilar substrate specificity profiles. Hsc20, unlike DnaJ, did not exhibit intrinsic chaperone activity and appears to function solely as a regulatory cochaperone protein for Hsc66. Possible interactions between the Hsc66-Hsc20 and DnaK-DnaJ-GrpE chaperone systems were also investigated by measuring the effects of cochaperone proteins on Hsp70 ATPase activities. The nucleotide exchange factor GrpE did not stimulate the ATPase activity of Hsc66 and thus appears to function specifically with DnaK. Cross-stimulation by the cochaperones Hsc20 and DnaJ was observed, but the requirement for supraphysiological concentrations makes it unlikely that these interactions occur significantly in vivo. Together these results suggest that Hsc66-Hsc20 and DnaK-DnaJ-GrpE comprise separate molecular chaperone systems with distinct, nonoverlapping cellular functions.     

The chaperone function of DnaK requires the coupling of ATPase activity with substrate binding through residue E171.

Central to the chaperone function of Hsp70 stress proteins including Escherichia coli DnaK is the ability of Hsp70 to bind unfolded protein substrates in an ATP-dependent manner. Mg2+/ATP dissociates bound substrates and, furthermore, substrate binding stimulates the ATPase of Hsp70. This coupling is proposed to require a glutamate residue, E175 of bovine Hsc70, that is entirely conserved within the Hsp70 family, as it contacts bound Mg2+/ATP and is part of a hinge required for a postulated ATP-dependent opening/closing movement of the nucleotide binding cleft which then triggers substrate release. We analyzed the effects of dnaK mutations which alter the corresponding glutamate-171 of DnaK to alanine, leucine or lysine. In vivo, the mutated dnaK alleles failed to complement the delta dnaK52 mutation and were dominant negative in dnaK+ cells. In vitro, all three mutant DnaK proteins were inactive in known DnaK-dependent reactions, including refolding of denatured luciferase and initiation of lambda DNA replication. The mutant proteins retained ATPase activity, as well as the capacity to bind peptide substrates. The intrinsic ATPase activities of the mutant proteins, however, did exhibit increased Km and Vmax values. More importantly, these mutant proteins showed no stimulation of ATPase activity by substrates and no substrate dissociation by Mg2+/ATP. Thus, glutamate-171 is required for coupling of ATPase activity with substrate binding, and this coupling is essential for the chaperone function of DnaK.     

Structural basis of the interspecies interaction between the chaperone DnaK(Hsp70) and the co-chaperone GrpE of archaea and bacteria

Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and eukaryotic organisms. In the archaea, which form a distinct kingdom, the Hsp70 chaperones have been found in some species only, including Methanosarcina mazei. Both the bacterial and archaeal Hsp70(DnaK) chaperones cooperate with a GrpE co-chaperone which stimulates the ATPase activity of the DnaK protein. It is currently believed that the archaeal Hsp70 system was obtained by the lateral transfer of chaperone genes from bacteria. Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the Escherichia coli GrpE and DnaK supported this hypothesis. However, the cooperation was surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%). The aim of this work was to investigate the molecular basis of the observed interspecies chaperone interaction. Infrared resolution-enhanced spectra of the M. mazei and E. coli DnaK proteins were almost identical, indicating high similarity of their secondary structures, however, some small differences in band position and in the intensity of amide I' band components were observed and discussed. Profiles of thermal denaturation of both proteins were similar, although they indicated a higher thermostability of the M. mazei DnaK compared to the E. coli DnaK. Electrophoresis under non-denaturing conditions demonstrated that purified DnaK and GrpE of E. coli and M. mazei formed mixed complexes. Protein modeling revealed high similarity of the 3-dimensional structures of the archaeal and bacterial DnaK and GrpE proteins.     

Hsc66 and Hsc20, a new heat shock cognate molecular chaperone system from Escherichia coli.

The hscA and hscB genes of Escherichia coli encode novel chaperone and co-chaperone proteins, designated Hsc66 and Hsc20, respectively. We have overproduced and purified Hsc66 and Hsc20 in high yield in E. coli and describe their initial characterization including absorbance, fluorescence, and circular dichroism spectra. Immunoblot analyses of E. coli cultures using antisera to Hsc66 and Hsc20 raised in rabbits establish that Hsc66 and Hsc20 are constitutively expressed at levels corresponding to cell concentration approximately 20 microM and approximately 10 microM, respectively. The levels do not change appreciably following heat shock (44 degrees C), but a small increase in Hsc20 is observed following a shift to 10 degrees C. Purified Hsc66 exhibits a low intrinsic ATPase activity (approximately 0.6 min-1 at 37 degrees C), and Hsc20 was found to stimulate this activity up to 3.8-fold with half-maximal stimulation at a concentration approximately 5 microM. These findings suggest that Hsc66 and Hsc20 comprise a molecular chaperone system similar to the prokaryotic DnaK/DnaJ and eukaryotic hsp70/hsp40 systems. Sequence differences between Hsc66 and Hsc20 compared to other members of this chaperone family, however, suggest that the Hsc66/Hsc20 system will display different peptide binding specificity and that it is likely to be subject to different regulatory mechanisms. The high level of constitutive expression and the lack of a major response to temperature changes suggest that Hsc66 and Hsc20 play an important cellular role(s) under non-stress conditions.     

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

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

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

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

Immunologic and structural characterization of the dominant 66- to 73-kDa antigens of Borrelia burgdorferi

The 66- to 73-kDa proteins of Borrelia burgdorferi are dominant immunogens and expressed in all strains of B. burgdorferi. The humoral response to these Ag occurs relatively early during the course of infection. Two-dimensional Western blot analysis of this group of Ag revealed them to consist of a tetrad of proteins with apparent molecular mass of 66, 68, 71, and 73 kDa. Furthermore, in this study we demonstrate the 66-kDa protein to be a potent inducer of lymphoproliferation in the patient immune to B. burgdorferi. Monospecific polyclonal antibodies and mAb demonstrate that each of these proteins was immunologically distinct. However, direct amino acid sequence of the 66- and 68-kDa Ag was almost identical and had a high level of sequence similarity to the GroEL heat-shock protein (Hsp60) of Escherichia coli and the 60-kDa immunodominant protein of Treponema pallidum. The amino terminal sequence of the 71- and 73-kDa proteins of B. burgdorferi was almost identical and these proteins had remarkable sequence similarity to the DnaK heat-shock protein of E. coli (Hsp70). It appears likely, therefore, that proteins related to the heat-shock family are potent immunogens of B. burgdorferi.     

The role of the DIF motif of the DnaJ (Hsp40) co-chaperone in the regulation of the DnaK (Hsp70) chaperone cycle

To perform effectively as a molecular chaperone, DnaK (Hsp70) necessitates the assistance of its DnaJ (Hsp40) co-chaperone partner, which efficiently stimulates its intrinsically weak ATPase activity and facilitates its interaction with polypeptide substrates. In this study, we address the function of the conserved glycine- and phenylalanine-rich (G/F-rich) region of the Escherichia coli DnaJ in the DnaK chaperone cycle. We show that the G/F-rich region is critical for DnaJ co-chaperone functions in vivo and that despite a significant degree of sequence conservation among the G/F-rich regions of Hsp40 homologs from bacteria, yeast, or humans, functional complementation in the context of the E. coli DnaJ is limited. Furthermore, we found that the deletion of the whole G/F-rich region is mirrored by mutations in the conserved Asp-Ile/Val-Phe (DIF) motif contained in this region. Further genetic and biochemical analyses revealed that this amino acid triplet plays a critical role in regulation of the DnaK chaperone cycle, possibly by modulating a crucial step subsequent to DnaK-mediated ATP hydrolysis.     

Role of the DnaK and HscA homologs of Hsp70 chaperones in protein folding in E.coli.

Folding of newly synthesized cytosolic proteins has been proposed to require assistance by Hsp70 chaperones. We investigated whether two Hsp70 homologs of Escherichia coli, DnaK and HscA, have this role in vivo. Double mutants lacking dnaK and hscA were viable and lacked defects in protein folding at intermediate temperature. After heat shock, a subpopulation of pre-existing proteins slowly aggregated in mutants lacking DnaK, but not HscA, whereas the bulk of newly synthesized proteins displayed wild-type solubility. For thermolabile firefly luciferase, DnaK was dispensable for de novo folding at 30 degrees C, but essential for aggregation prevention during heat shock and subsequent refolding. DnaK and HscA are thus not strictly essential for folding of newly synthesized proteins. DnaK instead has functions in refolding of misfolded proteins that are essential under stress.     

Connexin45 directly binds to ZO-1 and localizes to the tight junction region in epithelial MDCK cells

The molecular chaperone protein Hsp78, a member of the Clp/Hsp100 family localized in the mitochondria of Saccharomyces cerevisiae, is required for maintenance of mitochondrial functions under heat stress. To characterize the biochemical mechanisms of Hsp78 function, Hsp78 was purified to homogeneity and its role in the reactivation of chemically and heat-denatured substrate protein was analyzed in vitro. Hsp78 alone was not able to mediate reactivation of firefly luciferase. Rather, efficient refolding was dependent on the simultaneous presence of Hsp78 and the mitochondrial Hsp70 machinery, composed of Ssc1p/Mdj1p/Mge1p. Bacterial DnaK/DnaJ/GrpE, which cooperates with the Hsp78 homolog, ClpB in Escherichia coli, could not substitute for the mitochondrial Hsp70 system. However, efficient Hsp78-dependent refolding of luciferase was observed if DnaK was replaced by Ssc1p in these experiments, suggesting a specific functional interaction of both chaperone proteins. These findings establish the cooperation of Hsp78 with the Hsp70 machinery in the refolding of heat-inactivated proteins and demonstrate a conserved mode of action of ClpB homologs.     

Biochemical and Proteomic Analysis of Ubiquitination of Hsc70 and Hsp70 by the E3 Ligase CHIP

The E3 ubiquitin ligase CHIP is involved in protein triage, serving as a co-chaperone for refolding as well as catalyzing ubiquitination of substrates. CHIP functions with both the stress induced Hsp70 and constitutive Hsc70 chaperones, and also plays a role in maintaining their balance in the cell. When the chaperones carry no client proteins, CHIP catalyzes their polyubiquitination and subsequent proteasomal degradation. Although Hsp70 and Hsc70 are highly homologous in sequence and similar in structure, CHIP mediated ubiquitination promotes degradation of Hsp70 with a higher efficiency than for Hsc70. Here we report a detailed and systematic investigation to characterize if there are significant differences in the CHIP in vitro ubiquitination of human Hsp70 and Hsc70. Proteomic analysis by mass spectrometry revealed that only 12 of 39 detectable lysine residues were ubiquitinated by UbcH5a in Hsp70 and only 16 of 45 in Hsc70. The only conserved lysine identified as ubiquitinated in one but not the other heat shock protein was K159 in Hsc70. Ubiquitination assays with K-R ubiquitin mutants showed that multiple Ub chain types are formed and that the distribution is different for Hsp70 versus Hsc70. CHIP ubiquitination with the E2 enzyme Ube2W is predominantly directed to the N-terminal amine of the substrate; however, some internal lysine modifications were also detected. Together, our results provide a detailed view of the differences in CHIP ubiquitination of these two very similar proteins, and show a clear example where substantial differences in ubiquitination can be generated by a single E3 ligase in response to not only different E2 enzymes but subtle differences in the substrate.     

Conformational properties of bacterial DnaK and yeast mitochondrial Hsp70. Role of the divergent C-terminal alpha-helical subdomain

Among the eukaryotic members of the Hsp70 family, mitochondrial Hsp70 shows the highest degree of sequence identity with bacterial DnaK. Although they share a functional mechanism and homologous co-chaperones, they are highly specific and cannot be exchanged between Escherichia coli and yeast mitochondria. To provide a structural basis for this finding, we characterized both proteins, as well as two DnaK/mtHsp70 chimeras constructed by domain swapping, using biochemical and biophysical methods. Here, we show that DnaK and mtHsp70 display different conformational and biochemical properties. Replacing different regions of the DnaK peptide-binding domain with those of mtHsp70 results in chimeric proteins that: (a) are not able to support growth of an E. coli DnaK deletion strain at stress temperatures (e.g. 42 degrees C); (b) show increased accessibility and decreased thermal stability of the peptide-binding pocket; and (c) have reduced activation by bacterial, but not mitochondrial co-chaperones, as compared with DnaK. Importantly, swapping the C-terminal alpha-helical subdomain promotes a conformational change in the chimeras to an mtHsp70-like conformation. Thus, interaction with bacterial co-chaperones correlates well with the conformation that natural and chimeric Hsp70s adopt in solution. Our results support the hypothesis that a specific protein structure might regulate the interaction of Hsp70s with particular components of the cellular machinery, such as Tim44, so that they perform specific functions.     

Formation in vitro of complexes between an abnormal fusion protein and the heat shock proteins from Escherichia coli and yeast mitochondria.

Heat shock proteins (HSPs) of the Hsp70 and GroEL families associate with a variety of cell proteins in vivo. However, the formation of such complexes has not been systematically studied. A 31-kDa fusion protein (CRAG), which contains 12 residues of cro repressor, truncated protein A, and 14 residues of beta-galactosidase, when expressed in Escherichia coli, was found in complexes with DnaK, GrpE, protease La, and GroEL. When an E. coli extract not containing CRAG was applied to an affinity column containing CRAG, DnaK, GroEL, and GrpE were selectively bound. These HSPs did not bind to a normal protein A column. DnaK, GrpE, and the fraction of GroEL could be eluted from the CRAG column with ATP but not with a nonhydrolyzable ATP analog. The ATP-dependent release of DnaK and GroEL also required Mg2+, but GrpE dissociated with ATP alone. The binding and release of DnaK and GroEL were independent events, but the binding of GrpE required DnaK. Inactivation of DnaJ, GrpE, and GroES did not affect the association or dissociation of DnaK or GroEL from CRAG. The DnaK and GrpE proteins could be eluted with 10(-6) M ATP, but 10(-4) M was required for GroEL release. This approach allows a one-step purification of these proteins from E. coli and also the isolation of the DnaK and GroEL homologs from yeast mitochondria. Competition experiments with oligopeptide fragments of CRAG showed that DnaK and GroEL interact with different sites on CRAG and that the cro-derived domain of CRAG contains the DnaK-binding site.