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.     

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.     

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.     

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.     

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 influence of C-terminal extension on the structure of the "J-domain" in E. coli DnaJ

Two different recombinant constructs of the N-terminal domain in Escherichia coli DnaJ were uniformly labeled with nitrogen-15 and carbon-13. One, DnaJ(1-78), contains the complete "J-domain," and the other, DnaJ(1-104), contains both the "J-domain" and a conserved "G/F" extension at the C-terminus. The three-dimensional structures of these proteins have been determined by heteronuclear NMR experiments. In both proteins the "J-domain" adopts a compact structure consisting of a helix-turn-helix-loop-helix-turn-helix motif. In contrast, the "G/F" region in DnaJ(1-104) does not fold into a well-defined structure. Nevertheless, the "G/F" region has been found to have an effect on the packing of the helices in the "J-domain" in DnaJ(1-104). Particularly, the interhelical angles between Helix IV and other helices are significantly different in the two structures. In addition, there are some local conformational changes in the loop region connecting the two central helices. These structural differences in the "J-domain" in the presence of the "G/F" region may be related to the observation that DnaJ (1-78) is incapable of stimulating the ATPase activity of the molecular chaperone protein DnaK despite evidence that sites mediating the binding of DnaJ to DnaK are located in the 1-78 segment.     

Role of DnaJ G/F-rich Domain in Conformational Recognition and Binding of Protein Substrates

DnaJ from Escherichia coli is a Type I Hsp40 that functions as a cochaperone of DnaK (Hsp70), stimulating its ATPase activity and delivering protein substrates. How DnaJ binds protein substrates is still poorly understood. Here we have studied the role of DnaJ G/F-rich domain in binding of several substrates with different conformational properties (folded, partially (un)folded and unfolded). Using partial proteolysis we find that RepE, a folded substrate, contacts a wide DnaJ area that involves part of the G/F-rich region and Zn-binding domain. Deletion of G/F-rich region hampers binding of native RepE and reduced the affinity for partially (un)folded substrates. However, binding of completely unfolded substrates is independent on the G/F-rich region. These data indicate that DnaJ distinguishes the substrate conformation and is able to adapt the use of the G/F-rich region to form stable substrate complexes.     

Genetic analysis of the polyomavirus DnaJ domain

Polyomavirus T antigens share a common N-terminal sequence that comprises a DnaJ domain. DnaJ domains activate DnaK molecular chaperones. The functions of J domains have primarily been tested by mutation of their conserved HPD residues. Here, we report detailed mutagenesis of the polyomavirus J domain in both large T (63 mutants) and middle T (51 mutants) backgrounds. As expected, some J mutants were defective in binding DnaK (Hsc70); other mutants retained the ability to bind Hsc70 but were defective in stimulating its ATPase activity. Moreover, the J domain behaves differently in large T and middle T. A given mutation was twice as likely to render large T unstable as it was to affect middle T stability. This apparently arose from middle T's ability to bind stabilizing proteins such as protein phosphatase 2A (PP2A), since introduction of a second mutation preventing PP2A binding rendered some middle T J-domain mutants unstable. In large T, the HPD residues are critical for Rb-dependent effects on the host cell. Residues Q32, A33, Y34, H49, M52, and N56 within helix 2 and helix 3 of the large T J domain were also found to be required for Rb-dependent transactivation. Cyclin A promoter assays showed that J domain function also contributes to large T transactivation that is independent of Rb. Single point mutations in middle T were generally without effect. However, residue Q37 is critical for middle T's ability to form active signaling complexes. The Q37A middle T mutant was defective in association with pp60(c-src) and in transformation.     

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.     

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.     

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.     

Severe oxidative stress causes inactivation of DnaK and activation of the redox-regulated chaperone Hsp33

DnaK/DnaJ/GrpE constitutes the primary chaperone machinery in E. coli that functions to protect proteins against heat-induced protein aggregation. Surprisingly, upon exposure of cells to reactive oxygen species at elevated temperature, proteins are no longer protected by the DnaK system. Instead, they bind now to the redox-regulated chaperone Hsp33, which is activated by the same conditions that inactivate DnaK. The inactivation of DnaK seems to be induced by the dramatic decrease in intracellular ATP levels that occurs upon exposure of cells to reactive oxygen species. This appears to render DnaK's N-terminal ATPase domain nucleotide depleted and thermolabile. DnaK's N terminus reversibly unfolds in vivo, and DnaK loses its ability to protect proteins against stress-induced aggregation. Now, the ATP-independent chaperone holdase Hsp33 binds to a large number of cellular proteins and prevents their irreversible aggregation. Upon return to nonstress conditions, Hsp33 becomes inactivated while DnaK reactivates and resumes its task to support protein folding.     

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.     

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.     

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.     

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.     

Hsp70 chaperone ligands control domain association via an allosteric mechanism mediated by the interdomain linker

Hsp70 chaperones assist in protein folding, disaggregation, and membrane translocation by binding to substrate proteins with an ATP-regulated affinity that relies on allosteric coupling between ATP-binding and substrate-binding domains. We have studied single- and two-domain versions of the E. coli Hsp70, DnaK, to explore the mechanism of interdomain communication. We show that the interdomain linker controls ATPase activity by binding to a hydrophobic cleft between subdomains IA and IIA. Furthermore, the domains of DnaK dock only when ATP binds and behave independently when ADP is bound. Major conformational changes in both domains accompany ATP-induced docking: of particular importance, some regions of the substrate-binding domain are stabilized, while those near the substrate-binding site become destabilized. Thus, the energy of ATP binding is used to form a stable interface between the nucleotide- and substrate-binding domains, which results in destabilization of regions of the latter domain and consequent weaker substrate binding.     

Physical Interaction between Bacterial Heat Shock Protein (Hsp) 90 and Hsp70 Chaperones Mediates Their Cooperative Action to Refold Denatured Proteins

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.     

Backbone dynamics of the N-terminal domain in E. coli DnaJ determined by 15N- and 13CO-relaxation measurements.

The backbone dynamics of the N-terminal domain of the chaperone protein Escherichia coli DnaJ have been investigated using steady-state 1H-15N NOEs, 15N T1, T2, and T1 rho relaxation times, steady-state 13C alpha-13CO NOEs, and 13CO T1 relaxation times. Two recombinant constructs of the N-terminal domain of DnaJ have been studied. One, DnaJ(1-78), contains the most conserved "J-domain" of DnaJ, and the other, DnaJ(1-104), includes a glycine/phenylalanine rich region ("G/F" region) in addition to the "J-domain". DnaJ(1-78) is not capable of stimulating ATP hydrolysis by DnaK, despite the fact that all currently identified sites responsible for DnaJ-DnaK interaction are located in this region. DnaJ(1-104), on the other hand, retains nearly the full ATPase stimulatory activity of full length DnaJ. Recently, a structural analysis of these two molecules was presented in an effort to elucidate the origin of their functional differences [Huang, K., Flanagan, J. M., and Prestegard, J. H. (1999) Protein Science 8, 203-214]. Herein, an analysis of dynamic properties is presented in a similar effort. A generalized model-free approach with a full treatment of the anisotropic overall rotation of the proteins is used in the analysis of measured relaxation parameters. Our results show that internal motions on pico- to nanosecond time scales in the backbone of DnaJ(1-78) are reduced on the inclusion of the "G/F" region, while conformational exchange on micro- to millisecond time scales increases. We speculate that the enhanced flexibility of residues on the slow time scale upon the inclusion of the "G/F" region could be relevant to the ATPase stimulatory activity of DnaJ if an "induced-fit" mechanism applies to DnaJ-DnaK interactions.     

Visualization and functional analysis of the oligomeric states of <Emphasis Type="Italic">Escherichia coli</Emphasis> heat shock protein 70 (Hsp70/DnaK)

The molecular chaperone DnaK binds to exposed hydrophobic segments in proteins, protecting them from aggregation. DnaK interacts with protein substrates via its substrate-binding domain, and the affinity of this interaction is allosterically regulated by its nucleotide-binding domain. In addition to regulating interdomain allostery, the nucleotide state has been found to influence homo-oligomerization of DnaK. However, the architecture of oligomeric DnaK and its potential functional relevance in the chaperone cycle remain undefined. Towards that goal, we examined the structures of DnaK by negative stain electron microscopy. We found that DnaK samples contain an ensemble of monomers, dimers, and other small, defined multimers. To better understand the function of these oligomers, we stabilized them by cross-linking and found that they retained ATPase activity and protected a model substrate from denaturation. However, these oligomers had a greatly reduced ability to refold substrate and did not respond to stimulation by DnaJ. Finally, we observed oligomeric DnaK in Escherichia coli cellular lysates by native gel electrophoresis and found that these structures became noticeably more prevalent in cells exposed to heat shock. Together, these studies suggest that DnaK oligomers are composed of ordered multimers that are functionally distinct from monomeric DnaK. Thus, oligomerization of DnaK might be an important step in chaperone cycling.