Investigation of the interaction between DnaK and DnaJ by surface plasmon resonance spectroscopy.

Hsp70 chaperones assist protein folding through ATP-regulated transient association with substrates. Substrate binding by Hsp70 is controlled by DnaJ co-chaperones which stimulate Hsp70 to hydrolyze ATP and, consequently, to close its substrate binding cavity allowing trapping of substrates. We analyzed the interaction of the Escherichia coli Hsp70 homologue, DnaK, with DnaJ using surface plasmon resonance (SPR) spectroscopy. Resonance signals of complex kinetic characteristics were detected when DnaK was passed over a sensor chip with coupled DnaJ. This interaction was specific as it was not detected with a functionally defective DnaJ mutant protein, DnaJ259, that carries a mutation in the HPD signature motif of the conserved J-domain. Detectable DnaK-DnaJ interaction required ATP hydrolysis by DnaK and was competitively inhibited by chaperone substrates of DnaK. For DnaK mutant proteins with amino acid substitutions in the substrate binding cavity that affect substrate binding, the strength of detected interaction with DnaJ decreased proportionally with increased strength of the substrate binding defects. These findings indicate that the detected response signals resulted from DnaJ and ATP hydrolysis-dependent association of DnaJ as substrate for DnaK. Although not considered as physiologically relevant, this association allowed us to experimentally unravel the mechanism of DnaJ action. Accordingly, DnaJ stimulates ATP hydrolysis only after association of a substrate with the substrate binding cavity of DnaK. Further analysis revealed that this coupling mechanism required the J-domain of DnaJ and was also functional for natural DnaK substrates, and thus is central to the mechanism of action of the DnaK chaperone system.     

Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries.

Hsp70 chaperones assist protein folding by ATP-dependent association with linear peptide segments of a large variety of folding intermediates. The molecular basis for this ability to differentiate between native and non-native conformers was investigated for the DnaK homolog of Escherichia coli. We identified binding sites and the recognition motif in substrates by screening 4360 cellulose-bound peptides scanning the sequences of 37 biologically relevant proteins. DnaK binding sites in protein sequences occurred statistically every 36 residues. In the folded proteins these sites are mostly buried and in the majority found in beta-sheet elements. The binding motif consists of a hydrophobic core of four to five residues enriched particularly in Leu, but also in Ile, Val, Phe and Tyr, and two flanking regions enriched in basic residues. Acidic residues are excluded from the core and disfavored in flanking regions. The energetic contribution of all 20 amino acids for DnaK binding was determined. On the basis of these data an algorithm was established that predicts DnaK binding sites in protein sequences with high accuracy.     

A zinc finger-like domain of the molecular chaperone DnaJ is involved in binding to denatured protein substrates.

The Escherichia coli heat-shock protein DnaJ cooperates with the Hsp70 homolog DnaK in protein folding in vitro and in vivo. Little is known about the structural features of DnaJ that mediate its interaction with DnaK and unfolded polypeptide. DnaJ contains at least four blocks of sequence representing potential functional domains which have been conserved throughout evolution. In order to understand the role of each of these regions, we have analyzed DnaJ fragments in reactions corresponding to known functions of the intact protein. Both the N-terminal 70 amino acid 'J-domain' and a 35 amino acid glycine-phenylalanine region following it are required for interactions with DnaK. However, only complete DnaJ can cooperate with DnaK and a third protein, GrpE, in refolding denatured firefly luciferase. As demonstrated by atomic absorption and extended X-ray absorption fine structure spectroscopy (EXAFS), the 90 amino acid cysteine-rich region of DnaJ contains two Zn atoms tetrahedrally coordinated to four cysteine residues, resembling their arrangement in the C4 Zn binding domains of certain DNA binding proteins. Interestingly, binding experiments and cross-linking studies indicate that this Zn finger-like domain is required for the DnaJ molecular chaperone to specifically recognize and bind to proteins in their denatured state.     

Mechanism of the targeting action of DnaJ in the DnaK molecular chaperone system

In the DnaK (Hsp70) molecular chaperone system of Escherichia coli, the substrate polypeptide is fed into the chaperone cycle by association with the fast-binding, ATP-liganded form of the DnaK. The substrate binding properties of DnaK are controlled by its two cochaperones DnaJ (Hsp40) and GrpE. DnaJ stimulates the hydrolysis of DnaK-bound ATP, and GrpE accelerates ADP/ATP exchange. DnaJ has been described as targeting the substrate to DnaK, a concept that has remained rather obscure. Based on binding experiments with peptides and polypeptides we propose here a novel mechanism for the targeting action of DnaJ: ATP.DnaK and DnaJ with its substrate-binding domain bind to different segments of one and the same polypeptide chain forming (ATP.DnaK)m.substrate.DnaJn complexes; in these ternary complexes efficient cis-interaction of the J-domain of DnaJ with DnaK is favored by their propinquity and triggers the hydrolysis of DnaK-bound ATP, converting DnaK to its ADP-liganded high affinity state and thus locking it onto the substrate polypeptide.     

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.     

Rational mutagenesis of a 40 kDa heat shock protein from Agrobacterium tumefaciens identifies amino acid residues critical to its in vivo function

Prokaryotic DnaJ and DnaK, homologous to the eukaryotic 40 and 70kDa heat shock proteins (Hsp40 and Hsp70) respectively, play an important role as molecular chaperones in assisted protein folding under both normal and stressed conditions. DnaJ-like proteins are defined by the presence of a 70 amino acid domain termed the J domain, similar to the initial 73 amino acids of the Escherichia coli protein DnaJ. The J domain comprises four alpha-helices and a loop region containing the invariant tripeptide of histidine, proline and aspartic acid (HPD motif). This motif and Helix II have been shown previously to be important for the interaction with partner Hsp70s. Conserved amino acid residues present in the J domain were identified, and substitutions of these residues were performed to examine their effect on the in vivo functioning of the J domain of Agrobacterium tumefaciens DnaJ. Three conserved, charged residues, and three conserved, hydrophobic residues, in addition to the HPD motif, were shown to be important for the correct functioning of A. tumefaciens DnaJ. These included Arg26 located on Helix II, Arg63 and Asp59 located on Helix IV, Tyr7 and Leu10 located on Helix I, and Leu57 located on Helix III. This study has identified charged and hydrophobic residues on all the structural elements of the J domain that were critical to the structure and function of DnaJ, and in particular shown that Helix IV may have an important role in the structure and function of DnaJs in general.     

Interaction of the DnaK and DnaJ chaperone system with a native substrate,P1 RepA

DnaK, the Hsp70 chaperone of Escherichia coli interacts with protein substrates in an ATP-dependent manner, in conjunction with DnaJ and GrpE co-chaperones, to carry out protein folding, protein remodeling, and assembly and disassembly of multisubunit protein complexes. To understand how DnaJ targets specific proteins for recognition by the DnaK chaperone system, we investigated the interaction of DnaJ and DnaK with a known natural substrate, bacteriophage P1 RepA protein. By characterizing RepA deletion derivatives, we found that DnaJ interacts with a region of RepA located between amino acids 180 and 200 of the 286-amino acid protein. A peptide corresponding to amino acids 180-195 inhibited the interaction of RepA and DnaJ. Two site-directed RepA mutants with alanine substitutions in this region were about 4-fold less efficiently activated for oriP1 DNA binding by DnaJ and DnaK than wild type RepA. We also identified by deletion analysis a site in RepA, in the region of amino acids 35-49, which interacts with DnaK. An alanine substitution mutant in amino acids 36-39 was constructed and found defective in activation by DnaJ and DnaK. Taken together the results suggest that DnaJ and DnaK interact with separate sites on RepA.     

Modulation of substrate specificity of the DnaK chaperone by alteration of a hydrophobic arch

Hsp70 chaperones assist protein folding by reversible interaction with extended hydrophobic segments of substrate polypeptides. We investigated the contribution of three structural elements of the substrate- binding cavity of the Escherichia coli homologue, DnaK, to substrate specificity by investigating mutant DnaK proteins for binding to cellulose-bound peptides. Deletion of the C-terminal subdomain (Delta539-638) and blockage of the access to the hydrophobic pocket in the substrate-binding cavity (V436F) did not change the specificity, although the latter exchange reduced the affinity to all peptides investigated. Mutations (A429W, M404A/A429W) that affect the formation of a hydrophobic arch spanning over the bound substrate disfavored DnaK binding, especially to peptides with short stretches of consecutive hydrophobic residues flanked by acidic residues, while binding to most other peptides remained unchanged. The arch thus contributes to the substrate specificity of DnaK. This finding is of particular interest, since of all the residues of the substrate-binding cavity that contact bound substrate, only the arch-forming residues show significant variation within the Hsp70 family.     

D-Peptides as inhibitors of the DnaK/DnaJ/GrpE chaperone system

DnaK, a Hsp70 homolog of Escherichia coli, together with its co-chaperones DnaJ and GrpE protects denatured proteins from aggregation and promotes their refolding by an ATP-consuming mechanism. DnaJ not only stimulates the gamma-phosphate cleavage of DnaK-bound ATP but also binds polypeptide substrates on its own. Unfolded polypeptides, such as denatured luciferase, thus form ternary complexes with DnaJ and DnaK. A previous study has shown that d-peptides compete with l-peptides for the same binding site in DnaJ but do not bind to DnaK (Feifel, B., Sch?nfeld, H.-J., and Christen, P. (1998) J. Biol. Chem. 273, 11999-12002). Here we report that d-peptides efficiently inhibit the refolding of denatured luciferase by the DnaK/DnaJ/GrpE chaperone system (EC50 = 1-2 microM). The inhibition of the chaperone action is due to the binding of d-peptide to DnaJ (Kd = 1-2 microM), which seems to preclude DnaJ from forming ternary (ATP.DnaK)m.substrate.DnaJn complexes. Apparently, simultaneous binding of DnaJ and DnaK to one and the same target polypeptide is essential for effective chaperone action.     

Structural features required for the interaction of the Hsp70 molecular chaperone DnaK with its cochaperone DnaJ.

Hsp70 family members together with their Hsp40 cochaperones function as molecular chaperones, using an ATP-controlled cycle of polypeptide binding and release to mediate protein folding. Hsp40 plays a key role in the chaperone reaction by stimulating the ATPase activity and activating the substrate binding of Hsp70. We have explored the interaction between the Escherichia coli Hsp70 family member, DnaK, and its cochaperone partner DnaJ. Our data show that the binding of ATP, subsequent conformational changes in DnaK, and DnaJ-stimulated ATP hydrolysis are all required for the formation of a DnaK-DnaJ complex as monitored by Biacore analysis. In addition, our data imply that the interaction of the J-domain with DnaK depends on the substrate binding state of DnaK.     

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.     

The Escherichia coli DjlA and CbpA proteins can substitute for DnaJ in DnaK-mediated protein disaggregation

The DnaJ (Hsp40) protein of Escherichia coli serves as a cochaperone of DnaK (Hsp70), whose activity is involved in protein folding, protein targeting for degradation, and rescue of proteins from aggregates. Two other E. coli proteins, CbpA and DjlA, which exhibit homology with DnaJ, are known to interact with DnaK and to stimulate its chaperone activity. Although it has been shown that in dnaJ mutants both CbpA and DjlA are essential for growth at temperatures above 37 degrees C, their in vivo role is poorly understood. Here we show that in a dnaJ mutant both CbpA and DjlA are required for efficient protein dissaggregation at 42 degrees C.     

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.     

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.     

Scanning mutagenesis identifies amino acid residues essential for the in vivo activity of the Escherichia coli DnaJ (Hsp40) J-domain

The DnaJ (Hsp40) cochaperone regulates the DnaK (Hsp70) chaperone by accelerating ATP hydrolysis in a cycle closely linked to substrate binding and release. The J-domain, the signature motif of the Hsp40 family, orchestrates interaction with the DnaK ATPase domain. We studied the J-domain by creating 42 mutant E. coli DnaJ variants and examining their phenotypes in various separate in vivo assays, namely, bacterial growth at low and high temperatures, motility, and propagation of bacteriophage lambda. Most mutants studied behaved like wild type in all assays. In addition to the (33)HisProAsp(35) (HPD) tripeptide found in all known functional J-domains, our study uncovered three new single substitution mutations (Y25A, K26A, and F47A) that totally abolish J-domain function. Furthermore, two glycine substitution mutants in an exposed flexible loop (R36G, N37G) showed partial loss of J-domain function alone and complete loss of function as a triple (RNQ-GGG) mutant coupled with the phenotypically silent Q38G. Interestingly, all the essential residues map to a small region on the same solvent-exposed face of the J-domain. Engineered mutations in the corresponding residues of the human Hdj1 J-domain grafted in E. coli DnaJ also resulted in loss of function, suggesting an evolutionarily conserved interaction surface. We propose that these clustered residues impart critical sequence determinants necessary for J-domain catalytic activity and reversible contact interface with the DnaK ATPase domain.     

DnaJ dramatically stimulates ATP hydrolysis by DnaK: insight into targeting of Hsp70 proteins to polypeptide substrates

Most, if not all, of the cellular functions of Hsp70 proteins require the assistance of a DnaJ homologue, which accelerates the weak intrinsic ATPase activity of Hsp70 and serves as a specificity factor by binding and targeting specific polypeptide substrates for Hsp70 action. We have used pre-steady-state kinetics to investigate the interaction of the Escherichia coli DnaJ and DnaK proteins, and the effects of DnaJ on the ATPase reaction of DnaK. DnaJ accelerates hydrolysis of ATP by DnaK to such an extent that ATP binding by DnaK becomes rate-limiting for hydrolysis. At high concentrations of DnaK under single-turnover conditions, the rate-limiting step is a first-order process, apparently a change of DnaK conformation, that accompanies ATP binding and proceeds at 12-15 min-1 at 25 degrees C and 1-1.5 min-1 at 5 degrees C. By prebinding ATP to DnaK and subsequently adding DnaJ, the effects of this slow step may be bypassed, and the maximal rate-enhancement of DnaJ on the hydrolysis step is approximately 15 000-fold at 5 degrees C. The interaction of DnaJ with DnaK.ATP is likely a rapid equilibrium relative to ATP hydrolysis, and is relatively weak, with a KD of approximately 20 microM at 5 degrees C, and weaker still at 25 degrees C. In the presence of saturating DnaJ, the maximal rate of ATP hydrolysis by DnaK is similar to previously reported rates for peptide release from DnaK.ATP. This suggests that when DnaK encounters a DnaJ-bound polypeptide or protein complex, a significant fraction of such events result in ATP hydrolysis by DnaK and concomitant capture of the polypeptide substrate in a tight complex with DnaK.ADP. Furthermore, a broadly applicable kinetic mechanism for DnaJ-mediated specificity of Hsp70 action arises from these observations, in which the specificity arises largely from the acceleration of the hydrolysis step itself, rather than by DnaJ-dependent modulation of the affinity of Hsp70 for substrate polypeptides.     

cis-Effect of DnaJ on DnaK in ternary complexes with chimeric DnaK/DnaJ-binding peptides

Chimeric peptides, comprising a DnaK-binding sequence of L-amino acid residues (motif k) and an exclusive DnaJ-binding sequence of D-amino acid residues (motif j) connected through a 22-residue linker, were examined as minisubstrates for the DnaK chaperone system. The DnaJ-stimulated ATPase activity of DnaK was three times higher in the presence of the chimeric peptides pjk or pkj than in the simultaneous presence of the corresponding single-motif peptides ala-p5 (k motif) plus D-p5 (j motif). Apparently, pjk and pkj mimic unfolded proteins by forming ternary (ATP x DnaK) x peptide x DnaJ complexes which favor cis-interaction of DnaJ with DnaK. Consistent with this interpretation, the specific stimulatory effect of the chimeric peptides was abolished by either single-motif peptide in excess.     

All three J-domain proteins of the Escherichia coli DnaK chaperone machinery are DNA binding proteins

DnaJ, DjlA and CbpA are the J-domain proteins of DnaK, the major Hsp70 of Escherichia coli. CbpA was originally discovered as a DNA binding protein. Here, we show that DNA binding is a property of DnaJ and DjlA as well. Of special interest in this respect is DjlA, as this cytoplasmic protein is membrane bound and, as shown here, its affinity for DNA is extremely high. The finding that all the three J-proteins of DnaK are DNA binding proteins sheds new light on the cellular activity of these proteins.     

DnaJ recruits DnaK to protein aggregates

Thermal stress might lead to protein aggregation in the cell. Reactivation of protein aggregates depends on Hsp100 and Hsp70 chaperones. We focus in this study on the ability of DnaK, the bacterial representative of the Hsp70 family, to interact with different aggregated model substrates. Our data indicate that DnaK binding to large protein aggregates is mediated by DnaJ, and therefore it depends on its affinity for the cochaperone. Mutations in the structural region of DnaK known as the "latch" decrease the affinity of the chaperone for DnaJ, resulting in a defective activity as protein aggregate-removing agent. As expected, the chaperone activity is recovered when DnaJ concentration is raised to overcome the lower affinity of the mutant for the cochaperone, suggesting that a minimum number of aggregate-bound DnaK molecules is necessary for its efficient reactivation. Our results provide the first experimental evidence of DnaJ-mediated recruiting of ATP-DnaK molecules to the aggregate surface.