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.     

Functional analysis of CbpA, a DnaJ homolog and nucleoid-associated DNA-binding protein

DnaK/Hsp70 proteins are universally conserved ATP-dependent molecular chaperones that help proteins adopt and maintain their native conformations. DnaJ/Hsp40 and GrpE are co-chaperones that assist DnaK. CbpA is an Escherichia coli DnaJ homolog. It acts as a multicopy suppressor for dnaJ mutations and functions in vitro in combination with DnaK and GrpE in protein remodeling reactions. CbpA binds nonspecifically to DNA with preference for curved DNA and is a nucleoid-associated protein. The DNA binding and co-chaperone activities of CbpA are modulated by CbpM, a small protein that binds specifically to CbpA. To identify the regions of CbpA involved in the interaction of CbpA with CbpM and those involved in DNA binding, we constructed and characterized deletion and substitution mutants of CbpA. We discovered that CbpA interacted with CbpM through its N-terminal J-domain. We found that the region C-terminal to the J-domain was required for DNA binding. Moreover, we found that the CbpM interaction, DNA binding, and co-chaperone activities were separable; some mutants were proficient in some functions and defective in others.     

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.     

The transmembrane domain of the DnaJ-like protein DjlA is a dimerisation domain

DjlA is a bitopic inner membrane protein, which belongs to the DnaJ co-chaperone family in Escherichia coli. Overproduction of DjlA leads to the synthesis of colanic acid, resulting in mucoidy, via the activation of the two-component regulatory system RcsC/B that controls the cps (capsular polysaccharide) operon. This induction requires both the co-chaperone activity of DjlA, in cooperation with DnaK and GrpE, and its unique transmembrane (TM) domain. Here, we show that the TM segment of DjlA acts as a dimerisation domain: when fused to the N-terminal DNA-binding domain of the lambda cI repressor protein, it can substitute for the native C-terminal dimerisation domain of cI, thus generating an active cI repressor. Replacing the TM domain of DjlA by other TM domains, with or without dimerising capacity, revealed that dimerisation is not sufficient for the induction of cps expression, indicating an additional sequence- or structurally specific role for the TM domain. Finally, the conserved glycines present in the TM domain of DjlA are essential for the induction of mucoidy, but not for dimerisation.     

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.     

Structural Basis of the Regulation of the CbpA Co-chaperone by its Specific Modulator CbpM

CbpA, one of the Escherichia coli DnaJ homologues, acts as a co-chaperone in the DnaK chaperone system. Despite its extensive similarity in domain structure and function to DnaJ, CbpA has a unique and specific regulatory mechanism mediated through the small protein CbpM. Both CbpA and CbpM are highly conserved in bacteria. Earlier studies showed that CbpM interacts with the N-terminal J-domain of CbpA inhibiting its co-chaperone activity but the structural basis of this interaction is not known. Here, we have combined NMR spectroscopy, site-directed mutagenesis and surface plasmon resonance to characterize the CbpA/CbpM interaction at the molecular level. We have determined the solution structure of the CbpA J-domain and mapped the residues that are perturbed upon CbpM binding. The NMR data defined a broad region on helices α2 and α3 as involved in the interactions. Site-directed mutagenesis has been used to further delineate the CbpA J-domain/CbpM interface. We show that the binding sites of CbpM and DnaK on CbpA J-domain overlap, which suggests a competition between DnaK and CbpM for binding to CbpA as a mechanism for CbpA regulation. This study also provides the explanation for the specificity of CbpM for CbpA versus DnaJ, by identifying the key residues for differential binding.     

Structure and energetics of an allele-specific genetic interaction between dnaJ and dnaK: correlation of nuclear magnetic resonance chemical shift perturbations in the J-domain of Hsp40/DnaJ with binding affinity for the ATPase domain of Hsp70/DnaK

The molecular chaperone machine composed of Escherichia coli Hsp70/DnaK and Hsp40/DnaJ binds and releases client proteins in cycles of ATP-dependent protein folding, membrane translocation, disassembly, and degradation. The J-domain of DnaJ simultaneously stimulates ATP hydrolysis in the ATPase domain and capture of the client protein in the peptide-binding domain of DnaK. ATP-dependent binding of DnaJ to DnaK mimics DnaJ-dependent capture of a client protein. The dnaJ mutation that replaces aspartate-35 with asparagine (D35N) in the J-domain causes a defect in binding of DnaJ to DnaK. The dnaK mutation that replaces arginine-167 with alanine (R167A) in the ATPase domain of DnaK(R167A) restores binding of DnaJ(D35N). This genetic interaction was said to be allele-specific because wild-type DnaJ does not bind to DnaK(R167A). The J-domain of DnaJ binds to the ATPase domain of DnaK in its capacity as modulator of DnaK ATPase activity and conformational behavior. Surprisingly, the mutations affect the domainwise interaction in an almost opposite manner. D35N increases the affinity of the J-domain for the ATPase domain. R167A has no affect on the affinity of the ATPase domain for the D35N mutant J-domain, but it reduces the affinity for the wild-type J-domain. Previous amide ((1)H, (15)N) NMR chemical shift perturbation mapping in the J-domain suggested that the ATPase domain binds to J-domain helix II and the flanking loops. In the D35N mutant J-domain, chemical shift perturbations include additional effects at amides in the flexible loop II-III and helix III, which have been proposed to undergo an induced fit conformational change upon binding to DnaK. The integrated magnitudes of chemical shift perturbations for the various J-domain and ATPase domain pairs correlate with the free energies of binding. Thus, the J-domain structure can be described as a dynamic ensemble of conformations that is constrained by binding to the ATPase domain. J-domain helix II bends upon binding to the ATPase domain. D35N increases helix II bending, but less so in combination with R167A in the ATPase domain. Taken together, the results suggest that D35N overstabilizes an induced fit conformational change in loop II-III and helix III that is necessary for the J-domain to couple ATP hydrolysis with a conformational change in DnaK, and R167A destabilizes the induced conformation. Conclusions from this work have implications for understanding mechanisms of protein-protein interaction that are involved in allosteric regulation and genetic suppression.     

The J-domain of Hsp40 couples ATP hydrolysis to substrate capture in Hsp70

The Escherichia coli Hsp40 DnaJ uses its J-domain to target substrate polypeptides for binding to the Hsp70 DnaK, but the mechanism of J-domain function has been obscured by a substrate-like interaction between DnaJ and DnaK. ATP hydrolysis in DnaK is associated with a conformational change that captures the substrate, and both DnaJ and substrate can stimulate ATP hydrolysis. However, substrates cannot trigger capture by DnaK in the presence of ATP, and substrates stimulate a DnaK conformational change that is uncoupled from ATP hydrolysis. The role of the J-domain was examined using the fluorescent derivative of a fusion protein composed of the J-domain and a DnaK-binding peptide. In the absence of ATP, DnaK-binding affinity of the fusion protein is similar to that of the unfused peptide. However, in the presence of ATP, the affinity of the fusion protein is dramatically increased, which is opposite to the decrease in DnaK affinity typically exhibited by peptides. Binding of a fusion protein that contains a defective J-domain is insensitive to ATP. According to results from isothermal titration calorimetry, the J-domain binds to the DnaK ATPase domain with weak affinity (K(D) = 23 microM at 20 degrees C). The interaction is characterized by a positive enthalpy, small heat capacity change (DeltaC(p)= -33 kcal mol(-1)), and increasing binding affinity for increasing temperatures in the physiological range. In conditions that support binding of the J-domain to the ATPase domain, the J-domain accelerates ATP hydrolysis and a simultaneous conformational change in DnaK that is associated with peptide capture. The defective J-domain is inactive, despite the fact that it binds to the DnaK ATPase domain with higher than wild-type affinity. The results are most consistent with an allosteric mechanism of J-domain action in which the J-domain couples ATP hydrolysis to peptide capture by accelerating ATP hydrolysis and delaying DnaK closure until ATP is hydrolyzed.     

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.     

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.     

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.     

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.     

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.     

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.     

The roles of the two zinc binding sites in DnaJ

All type I DnaJ (Hsp40) homologues share the presence of two highly conserved zinc centers. To elucidate their function, we constructed DnaJ mutants that separately replaced cysteines of either zinc center I or zinc center II with serine residues. We found that in the absence of zinc center I, the autonomous, DnaK-independent chaperone activity of DnaJ is dramatically reduced. Surprisingly, this only slightly impaired the in vivo function of DnaJ, and its ability to function as a co-chaperone in the DnaK/DnaJ/GrpE foldase machine. The DnaJ zinc center II, on the other hand, was found to be absolutely essential for the in vivo and in vitro function of DnaJ. This did not seem to be caused by a lack of substrate binding affinity or an inability to work as an ATPase-stimulating factor. Rather it appears that zinc center II mutant proteins lack a necessary additional interaction site with DnaK, which seems to be crucial for locking-in substrate proteins onto DnaK. These findings led us to a model in which ATP hydrolysis in DnaK is only the first step in converting DnaK into its high affinity binding state. Additional interactions between DnaK and DnaJ are required to make the DnaK/DnaJ/GrpE foldase machinery catalytically active.     

J-domain Proteins form Binary Complexes with Hsp90 and Ternary Complexes with Hsp90 and Hsp70

Hsp90 and Hsp70 are highly conserved molecular chaperones that help maintain proteostasis by participating in protein folding, unfolding, remodeling and activation of proteins. Both chaperones are also important for cellular recovery following environmental stresses. Hsp90 and Hsp70 function collaboratively for the remodeling and activation of some client proteins. Previous studies using E. coli and S. cerevisiae showed that residues in the Hsp90 middle domain directly interact with a region in the Hsp70 nucleotide binding domain, in the same region known to bind J-domain proteins. Importantly, J-domain proteins facilitate and stabilize the interaction between Hsp90 and Hsp70 both in E. coli and S. cerevisiae. To further explore the role of J-domain proteins in protein reactivation, we tested the hypothesis that J-domain proteins participate in the collaboration between Hsp90 and Hsp70 by simultaneously interacting with Hsp90 and Hsp70. Using E. coli Hsp90, Hsp70 (DnaK), and a J-domain protein (CbpA), we detected a ternary complex containing all three proteins. The interaction involved the J-domain of CbpA, the DnaK binding region of E. coli Hsp90, and the J-domain protein binding region of DnaK where Hsp90 also binds. Additionally, results show that E. coli Hsp90 interacts with E. coli J-domain proteins, DnaJ and CbpA, and that yeast Hsp90, Hsp82, interacts with a yeast J-domain protein, Ydj1. Together these results suggest that the complexes may be transient intermediates in the pathway of collaborative protein remodeling by Hsp90 and Hsp70.     

The Hsp40 J-domain Stimulates Hsp70 When Tethered by the Client to the ATPase Domain

The Escherichia coli Hsp40 DnaJ uses its J-domain (Jd) to couple ATP hydrolysis and client protein capture in Hsp70 DnaK. Fusion of the Jd to peptide p5 (as in Jdp5) dramatically increases the apparent affinity of the p5 moiety for DnaK in the presence of ATP, and Jdp5 stimulates ATP hydrolysis in DnaK by several orders of magnitude. NMR experiments with [¹⁵N]Jdp5 demonstrated that the peptide tethers the Jd to the ATPase domain. Thus, ATP hydrolysis and client protein binding in DnaK are coupled principally through the association of the client with DnaJ. Overexpression of a recombinant Jd was specifically toxic to cells that simultaneously expressed DnaK. No toxicity was observed when overexpressing Jdp5 or mutant Jd or when co-overexpressing the Jd and the nucleotide exchange factor GrpE. The results suggest that the Jd shifts DnaK to a client-bound form by stimulating the DnaK ATPase but only when the Jd is brought to DnaK by a client-Hsp40 complex.     

The djlA Gene Acts Synergistically with dnaJ in Promoting Escherichia coli Growth

The DnaK chaperone of Escherichia coli is known to interact with the J domains of DnaJ, CbpA, and DjlA. By constructing multiple mutants, we found that the djlA gene was essential for bacterial growth above 37 degrees C in the absence of dnaJ. This essentiality depended upon the J domain of DjlA but not upon the normal membrane location of DjlA.