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 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.     

CbpA, a DnaJ homolog, is a DnaK co-chaperone, and its activity is modulated by CbpM

The DnaK chaperone system, consisting of DnaK, DnaJ, and GrpE, remodels and refolds proteins during both normal growth and stress conditions. CbpA, one of several DnaJ analogs in Escherichia coli, is known to function as a multicopy suppressor for dnaJ mutations and to bind nonspecifically to DNA and preferentially to curved DNA. We found that CbpA functions as a DnaJ-like co-chaperone in vitro. CbpA acted in an ATP-dependent reaction with DnaK and GrpE to remodel inactive dimers of plasmid P1 RepA into monomers active in P1 DNA binding. Additionally, CbpA participated with DnaK in an ATP-dependent reaction to prevent aggregation of denatured rhodanese. The cbpA gene is in an operon with an open reading frame, yccD, which encodes a protein that has some homology to DafA of Thermus thermophilus. DafA is a protein required for the assembly of ring-like particles that contain trimers each of T. thermophilus DnaK, DnaJ, and DafA. The E. coli YccD was isolated because of its potential functional relationship to CbpA. Purified YccD specifically inhibited both the co-chaperone activity and the DNA binding activity of CbpA, suggesting that YccD modulates the activity of CbpA. We named the product of the yccD gene CbpM for "CbpA modulator."     

DjlA is a third DnaK co-chaperone of Escherichia coli, and DjlA-mediated induction of colanic acid capsule requires DjlA-DnaK interaction

DjlA is a 30-kDa type III membrane protein of Escherichia coli with the majority, including an extreme C-terminal putative J-domain, oriented toward the cytoplasm. No other regions of sequence similarity aside from the J-domain exist between DjlA and the known DnaK (Hsp70) co-chaperones DnaJ (Hsp40) and CbpA. In this study, we explored whether and to what extent DjlA possesses DnaK co-chaperone activity and under what conditions a DjlA-DnaK interaction could be important to the cell. We found that the DjlA J-domain can substitute fully for the J-domain of DnaJ using various in vivo functional complementation assays. In addition, the purified cytoplasmic fragment of DjlA was shown to be capable of stimulating DnaK ATPase in a manner indistinguishable from DnaJ, and, furthermore, DjlA could act as a DnaK co-chaperone in the reactivation of chemically denatured luciferase in vitro. DjlA expression in the cell is tightly controlled, and even its mild overexpression leads to induction of mucoid capsule. Previous analysis showed that DjlA-mediated induction of the wca capsule operon required the RcsC/RcsB two-component signaling system and that wca induction by DjlA was lost when cells contained mutations in either the dnaK or grpE gene. We now show using allele-specific genetic suppression analysis that DjlA must interact with DnaK for DjlA-mediated stimulation of capsule synthesis. Collectively, these results demonstrate that DjlA is a co-chaperone for DnaK and that this chaperone-co-chaperone pair is implicated directly, or indirectly, in the regulation of colanic acid capsule.     

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.     

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.     

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.     

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.     

Its substrate specificity characterizes the DnaJ co-chaperone as a scanning factor for the DnaK chaperone

The evolutionarily conserved DnaJ proteins are essential components of Hsp70 chaperone systems. The DnaJ homologue of Escherichia coli associates with chaperone substrates and mediates their ATP hydrolysis-dependent locking into the binding cavity of its Hsp70 partner, DnaK. To determine the substrate specificity of DnaJ proteins, we screened 1633 peptides derived from 14 protein sequences for binding to E.coli DnaJ. The binding motif of DnaJ consists of a hydrophobic core of approximately eight residues enriched for aromatic and large aliphatic hydrophobic residues and arginine. The hydrophobicity of this motif explains why DnaJ itself can prevent protein aggregation. Although this motif shows differences from DnaK's binding motif, DnaJ and DnaK share the majority of binding peptides. In contrast to DnaK, DnaJ binds peptides consisting of L- and D-amino acids, and therefore is not restricted by backbone contacts. These features allow DnaJ to scan hydrophobic protein surfaces and initiate the functional cycle of the DnaK system by associating with hydrophobic exposed patches and subsequent targeting of DnaK to these or to hydrophobic patches in spatial neighbourhood.     

Molecular chaperones DnaK and DnaJ share predicted binding sites on most proteins in the E. coli proteome

In Escherichia coli, the molecular chaperones DnaK and DnaJ cooperate to assist the folding of newly synthesized or unfolded polypeptides. DnaK and DnaJ bind to hydrophobic motifs in these proteins and they also bind to each other. Together, this system is thought to be sufficiently versatile to act on the entire proteome, which creates interesting challenges in understanding the interactions between DnaK, DnaJ and their thousands of potential substrates. To address this question, we computationally predicted the number and frequency of DnaK- and DnaJ-binding motifs in the E. coli proteome, guided by free energy-based binding consensus motifs. This analysis revealed that nearly every protein is predicted to contain multiple DnaK- and DnaJ-binding sites, with the DnaJ sites occurring approximately twice as often. Further, we found that an overwhelming majority of the DnaK sites partially or completely overlapped with the DnaJ-binding motifs. It is well known that high concentrations of DnaJ inhibit DnaK-DnaJ-mediated refolding. The observed overlapping binding sites suggest that this phenomenon may be explained by an important balance in the relative stoichiometry of DnaK and DnaJ. To test this idea, we measured the chaperone-assisted folding of two denatured substrates and found that the distribution of predicted DnaK- and DnaJ-binding sites was indeed a good predictor of the optimal stoichiometry required for folding. These studies provide insight into how DnaK and DnaJ might cooperate to maintain global protein homeostasis.     

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.     

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.     

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.     

A novel DnaJ-like protein in Escherichia coli inserts into the cytoplasmic membrane with a Type III topology

We describe a novel Escherichia coli protein, DjlA, containing a highly conserved J-region motif, which is present in the DnaJ protein chaperone family and required for interaction with DnaK. Remarkably, DjlA is shown to be a membrane protein, localized to the inner membrane with the unusual Type III topology (N-out, C-in). Thus, DjlA appears to present an extremely short N-terminus to the periplasm and has a single transmembrane domain (TMD) and a large cytoplasmic domain containing the C-terminal J-region. Analysis of the TMD of DjIA and recently identified homologues in Coxiella burnetti and Haemophilus influenzae revealed a striking pattern of conserved glycines (or rarely alanine), with a four-residue spacing. This motif, predicted to form a spiral groove in the TMD, is more marked than a repeating glycine motif, implicated in the dimerization of TMDs of some eukaryotic proteins. This feature of DjlA could represent a promiscuous docking mechanism for interaction with a variety of membrane proteins. DjlA null mutants can be isolated but these appear rapidly to accumulate suppressors to correct envelope and growth defects. Moderate (10-fold) overproduction of DjlA suppresses a mutation in FtsZ but markedly perturbs cell division and cell-envelope growth in minimal medium. We propose that DjlA plays a role in the correct assembly, activity and/or maintenance of a number of membrane proteins, including two-component signal-transduction systems.     

Heat shock protein-mediated disassembly of nucleoprotein structures is required for the initiation of bacteriophage lambda DNA replication

Three Escherichia coli heat shock proteins, DnaJ, DnaK, and GrpE, are required for replication of the bacteriophage lambda chromosome in vivo. We show that the GrpE heat shock protein is not required for initiation of lambda DNA replication in vitro when the concentration of DnaK is sufficiently high. GrpE does, however, greatly potentiate the action of DnaK in the initiation process when the DnaK concentration is reduced to a subsaturating level. We demonstrate in the accompanying articles (Alfano, C. and McMacken, R. (1989) J. Biol. Chem. 264, 10699-10708; Dodson, M., McMacken, R., and Echols, H. (1989) J. Biol. Chem. 264, 10719-10725) that DnaJ and DnaK bind to prepriming nucleoprotein structures that are assembled at the lambda replication origin (ori lambda). Binding of DnaJ and DnaK completes the ordered assembly of an ori lambda initiation complex that also contains the lambda O and P initiators and the E. coli DnaB helicase. With the addition of ATP, the DnaJ and DnaK heat shock proteins mediate the partial disassembly of the initiation complex, and the P and DnaJ proteins are largely removed from the template. Concomitantly, on supercoiled ori lambda plasmid templates, the intrinsic helicase activity of DnaB is activated and DnaB initiates localized unwinding of the DNA duplex, thereby preparing the template for priming and DNA chain elongation. We infer from our results that DnaK and DnaJ function in normal E. coli metabolism to promote ATP-dependent protein unfolding and disassembly reactions. We also provide evidence that neither the lambda O and P initiators nor the E. coli DnaJ and DnaK heat shock proteins play a direct role in the propagation of lambda replication forks in vitro.     

Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda. Protein association and disassociation reactions responsible for localized initiation of replication

Binding of the O protein of phage lambda to the replication origin (ori lambda) results in the formation of an organized nucleoprotein structure termed the O-some. The O-some serves to localize and initiate a six-protein sequential reaction that provides for localized unwinding of the origin region, the critical prepriming step for precise initiation of DNA replication. By the use of electron microscopy of gold-tagged antibody complexes, we have defined four stages of protein association and dissociation reactions that are involved in the prepriming pathway. First, as defined previously, O protein binds to multiple DNA sites and self-associates to form the O-some. Second, lambda P and host DnaB proteins add to the O-some to generate an O.P.DnaB.ori lambda complex. Addition of the DnaK and DnaJ proteins yields a third stage complex containing DnaK, DnaJ, O, P, and DnaB. With the addition of ATP and single-strand binding protein (SSB), the P protein is largely removed, and the DnaB acts as a helicase to generate locally unwound, SSB-coated single strand DNA. Thus, the initiation of lambda DNA replication requires ordered assembly and partial disassembly of specialized nucleoprotein structures. The disassembly activity of DnaK and DnaJ may be their general role in the heat shock response.     

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.     

In vivo modulation of a DnaJ homolog, CbpA, by CbpM

CbpA, an Escherichia coli DnaJ homolog, can function as a cochaperone for the DnaK/Hsp70 chaperone system, and its in vitro activity can be modulated by CbpM. We discovered that CbpM specifically inhibits the in vivo activity of CbpA, preventing it from functioning in cell growth and division. Furthermore, we have shown that CbpM interacts with CbpA in vivo during stationary phase, suggesting that the inhibition of activity is a result of the interaction. These results reveal that the activity of the E. coli DnaK system can be regulated in vivo by a specific inhibitor.