Structural basis for inhibition of a response regulator of σS stability by a ClpXP antiadaptor.

The stationary phase promoter specificity subunit σS (RpoS) is delivered to the ClpXP machinery for degradation dependent on the adaptor RssB. This adaptor-specific degradation of σS provides a major point for regulation and transcriptional reprogramming during the general stress response. RssB is an atypical response regulator and the only known ClpXP adaptor that is inhibited by multiple but dissimilar antiadaptors (IraD, IraP, and IraM). These are induced by distinct stress signals and bind to RssB in poorly understood manners to achieve stress-specific inhibition of σS turnover. Here we present the first crystal structure of RssB bound to an antiadaptor, the DNA damage-inducible IraD. The structure reveals that RssB adopts a compact closed architecture with extensive interactions between its N-terminal and C-terminal domains. The structural data, together with mechanistic studies, suggest that RssB plasticity, conferred by an interdomain glutamate-rich flexible linker, is critical for regulation of σS degradation. Structural modulation of interdomain linkers may thus constitute a general strategy for tuning response regulators.

Structure of phosphorylated-like RssB, the adaptor delivering σs to the ClpXP proteolytic machinery, reveals an interface switch for activation.

In enterobacteria such as Escherichia coli, the general stress response is mediated by σs, the stationary phase dissociable promoter specificity subunit of RNA polymerase. σs is degraded by ClpXP during active growth in a process dependent on the RssB adaptor, which is thought to be stimulated by the phosphorylation of a conserved aspartate in its N-terminal receiver domain. Here we present the crystal structure of full-length RssB bound to a beryllofluoride phosphomimic. Compared to the structure of RssB bound to the IraD anti-adaptor, our new RssB structure with bound beryllofluoride reveals conformational differences and coil-to-helix transitions in the C-terminal region of the RssB receiver domain and in the interdomain segmented helical linker. These are accompanied by masking of the α4-ß5-α5 (4-5-5) "signaling" face of the RssB receiver domain by its C-terminal domain. Critically, using hydrogen-deuterium exchange mass spectrometry, we identify σs-binding determinants on the 4-5-5 face, implying that this surface needs to be unmasked to effect an interdomain interface switch and enable full σs engagement and hand-off to ClpXP. In activated receiver domains, the 4-5-5 face is often the locus of intermolecular interactions, but its masking by intramolecular contacts upon phosphorylation is unusual, emphasizing that RssB is a response regulator that undergoes atypical regulation.

Hsp90 of E. coli modulates assembly of FtsZ, the bacterial tubulin homolog.

Heat shock protein 90 (Hsp90) is a highly conserved molecular chaperone involved in ATP-dependent client protein remodeling and activation. It also functions as a protein holdase, binding and stabilizing clients in an ATP-independent process. Hsp90 remodels over 300 client proteins and is essential for cell survival in eukaryotes. In bacteria, Hsp90 is a highly abundant protein, although very few clients have been identified and it is not essential for growth in many bacterial species. We previously demonstrated that in Escherichia coli, Hsp90 causes cell filamentation when expressed at high levels. Here, we have explored the cause of filamentation and identified a potentially important client of E. coli Hsp90 (Hsp90Ec), FtsZ. We observed that FtsZ, a bacterial tubulin homolog essential for cell division, fails to assemble into FtsZ rings (divisomes) in cells overexpressing Hsp90Ec Additionally, Hsp90Ec interacts with FtsZ and inhibits polymerization of FtsZ in vitro, in an ATP-independent holding reaction. The FtsZ-Hsp90Ec interaction involves residues in the client-binding region of Hsp90Ec and in the C-terminal tail of FtsZ, where many cell-division proteins and regulators interact. We observed that E. coli deleted for the Hsp90Ec gene htpG turn over FtsZ more rapidly than wild-type cells. Additionally, the length of ΔhtpG cells is reduced compared to wild-type cells. Altogether, these results suggest that Hsp90Ec is a modulator of cell division, and imply that the polypeptide-holding function of Hsp90 may be a biologically important chaperone activity.

Uncovering a region of heat shock protein 90 important for client binding in E. coli and chaperone function in yeast.

The heat shock protein 90 (Hsp90) family of heat shock proteins is an abundantly expressed and highly conserved family of ATP-dependent molecular chaperones. Hsp90 facilitates remodeling and activation of hundreds of proteins. In this study, we developed a screen to identify Hsp90-defective mutants in E. coli. The mutations obtained define a region incorporating residues from the middle and C-terminal domains of E. coli Hsp90. The mutant proteins are defective in chaperone activity and client binding in vitro. We constructed homologous mutations in S. cerevisiae Hsp82 and identified several that caused defects in chaperone activity in vivo and in vitro. However, the Hsp82 mutant proteins were less severely defective in client binding to a model substrate than the corresponding E. coli mutant proteins. Our results identify a region in Hsp90 important for client binding in E. coli Hsp90 and suggest an evolutionary divergence in the mechanism of client interaction by bacterial and yeast Hsp90.

Functional and physical interaction between yeast Hsp90 and Hsp70.

Heat shock protein 90 (Hsp90) is a highly conserved ATP-dependent molecular chaperone that is essential in eukaryotes. It is required for the activation and stabilization of more than 200 client proteins, including many kinases and steroid hormone receptors involved in cell-signaling pathways. Hsp90 chaperone activity requires collaboration with a subset of the many Hsp90 cochaperones, including the Hsp70 chaperone. In higher eukaryotes, the collaboration between Hsp90 and Hsp70 is indirect and involves Hop, a cochaperone that interacts with both Hsp90 and Hsp70. Here we show that yeast Hsp90 (Hsp82) and yeast Hsp70 (Ssa1), directly interact in vitro in the absence of the yeast Hop homolog (Sti1), and identify a region in the middle domain of yeast Hsp90 that is required for the interaction. In vivo results using Hsp90 substitution mutants showed that several residues in this region were important or essential for growth at high temperature. Moreover, mutants in this region were defective in interaction with Hsp70 in cell lysates. In vitro, the purified Hsp82 mutant proteins were defective in direct physical interaction with Ssa1 and in protein remodeling in collaboration with Ssa1 and cochaperones. This region of Hsp90 is also important for interactions with several Hsp90 cochaperones and client proteins, suggesting that collaboration between Hsp70 and Hsp90 in protein remodeling may be modulated through competition between Hsp70 and Hsp90 cochaperones for the interaction surface.

Anti-adaptors provide multiple modes for regulation of the RssB adaptor protein.

RpoS, an RNA polymerase σ factor, controls the response of Escherichia coli and related bacteria to multiple stress responses. During nonstress conditions, RpoS is rapidly degraded by ClpXP, mediated by the adaptor protein RssB, a member of the response regulator family. In response to stress, RpoS degradation ceases. Small anti-adaptor proteins--IraP, IraM, and IraD, each made under a different stress condition--block RpoS degradation. RssB mutants resistant to either IraP or IraM were isolated and analyzed in vivo and in vitro. Each of the anti-adaptors is unique in its interaction with RssB and sensitivity to RssB mutants. One class of mutants defined an RssB N-terminal region close to the phosphorylation site and critical for interaction with IraP but unnecessary for IraM and IraD function. A second class, in the RssB C-terminal PP2C-like domain, led to activation of RssB function. These mutants allowed the response regulator to act in the absence of phosphorylation but did not abolish interaction with anti-adaptors. This class of mutants is broadly resistant to the anti-adaptors and bears similarity to constitutively activated mutants found in a very different PP2C protein. The mutants provide insight into how the anti-adaptors perturb RssB response regulator function and activation.

Heat shock protein 90 from Escherichia coli collaborates with the DnaK chaperone system in client protein remodeling.

Molecular chaperones are proteins that assist the folding, unfolding, and remodeling of other proteins. In eukaryotes, heat shock protein 90 (Hsp90) proteins are essential ATP-dependent molecular chaperones that remodel and activate hundreds of client proteins with the assistance of cochaperones. In Escherichia coli, the activity of the Hsp90 homolog, HtpG, has remained elusive. To explore the mechanism of action of E. coli Hsp90, we used in vitro protein reactivation assays. We found that E. coli Hsp90 promotes reactivation of heat-inactivated luciferase in a reaction that requires the prokaryotic Hsp70 chaperone system, known as the DnaK system. An Hsp90 ATPase inhibitor, geldanamycin, inhibits luciferase reactivation demonstrating the importance of the ATP-dependent chaperone activity of E. coli Hsp90 during client protein remodeling. Reactivation also depends upon the ATP-dependent chaperone activity of the DnaK system. Our results suggest that the DnaK system acts first on the client protein, and then E. coli Hsp90 and the DnaK system collaborate synergistically to complete remodeling of the client protein. Results indicate that E. coli Hsp90 and DnaK interact in vivo and in vitro, providing additional evidence to suggest that E. coli Hsp90 and the DnaK system function together.

Species-specific collaboration of heat shock proteins (Hsp) 70 and 100 in thermotolerance and protein disaggregation.

Yeast Hsp104 and its bacterial homolog, ClpB, are Clp/Hsp100 molecular chaperones and AAA+ ATPases. Hsp104 and ClpB collaborate with the Hsp70 and DnaK chaperone systems, respectively, to retrieve and reactivate stress-denatured proteins from aggregates. The action of Hsp104 and ClpB in promoting cell survival following heat stress is species-specific: Hsp104 cannot function in bacteria and ClpB cannot act in yeast. To determine the regions of Hsp104 and ClpB necessary for this specificity, we tested chimeras of Hsp104 and ClpB in vivo and in vitro. We show that the Hsp104 and ClpB middle domains dictate the species-specificity of Hsp104 and ClpB for cell survival at high temperature. In protein reactivation assays in vitro, chimeras containing the Hsp104 middle domain collaborate with Hsp70 and those with the ClpB middle domain function with DnaK. The region responsible for the specificity is within helix 2 and helix 3 of the middle domain. Additionally, several mutants containing amino acid substitutions in helix 2 of the ClpB middle domain are defective in protein disaggregation in collaboration with DnaK. In a bacterial two-hybrid assay, DnaK interacts with ClpB and with chimeras that have the ClpB middle domain, implying that species-specificity is due to an interaction between DnaK and the middle domain of ClpB. Our results suggest that the interaction between Hsp70/DnaK and helix 2 of the middle domain of Hsp104/ClpB determines the specificity required for protein disaggregation both in vivo and in vitro, as well as for cellular thermotolerance.

Acetylation of the yeast Hsp40 chaperone protein Ydj1 fine-tunes proteostasis and translational fidelity.

Proteostasis, the maintenance of cellular protein balance, is essential for cell viability and is highly conserved across all organisms. Newly synthesized proteins, or "clients," undergo sequential processing by Hsp40, Hsp70, and Hsp90 chaperones to achieve proper folding and functionality. Despite extensive characterization of post-translational modifications (PTMs) on Hsp70 and Hsp90, the modifications on Hsp40 remain less understood. This study aims to elucidate the role of lysine acetylation on the yeast Hsp40, Ydj1. By mutating acetylation sites on Ydj1's J-domain to either abolish or mimic constitutive acetylation, we observed that preventing acetylation had no noticeable phenotypic impact, whereas acetyl-mimic mutants exhibited various defects indicative of impaired Ydj1 function. Proteomic analysis revealed several Ydj1 interactions affected by J-domain acetylation, notably with proteins involved in translation. Further investigation uncovered a novel role for Ydj1 acetylation in stabilizing ribosomal subunits and ensuring translational fidelity. Our data suggest that acetylation may facilitate the transfer of Ydj1 between Ssa1 and Hsp82. Collectively, this work highlights the critical role of Ydj1 acetylation in proteostasis and translational fidelity. Author Summary: Cells require a suite of chaperone and co-chaperone proteins to maintain a healthy balance of functional proteins. A large number of modifications on chaperone and co-chaperone proteins have been identified, but their functional importance has not been fully explored. In this study, we identify acetylation sites on the yeast co-chaperone Ydj1 that impact its interactions with major chaperones and client proteins including those involved in protein synthesis. This work sheds light on how modifications on co-chaperones can also play an important role in the health of the proteome.

DnaK chaperone-dependent disaggregation by caseinolytic peptidase B (ClpB) mutants reveals functional overlap in the N-terminal domain and nucleotide-binding domain-1 pore tyrosine.

Protein disaggregation in Escherichia coli is carried out by ClpB, an AAA(+) (ATPases associated with various cellular activities) molecular chaperone, together with the DnaK chaperone system. Conformational changes in ClpB driven by ATP binding and hydrolysis promote substrate binding, unfolding, and translocation. Conserved pore tyrosines in both nucleotide-binding domain-1 (NBD-1) and -2 (NBD-2), which reside in flexible loops extending into the central pore of the ClpB hexamer, bind substrates. When the NBD-1 pore loop tyrosine is substituted with alanine (Y251A), ClpB can collaborate with the DnaK system in disaggregation, although activity is reduced. The N-domain has also been implicated in substrate binding, and like the NBD-1 pore loop tyrosine, it is not essential for disaggregation activity. To further probe the function and interplay of the ClpB N-domain and the NBD-1 pore loop, we made a double mutant with an N-domain deletion and a Y251A substitution. This ClpB double mutant is inactive in substrate disaggregation with the DnaK system, although each single mutant alone can function with DnaK. Our data suggest that this loss in activity is primarily due to a decrease in substrate engagement by ClpB prior to substrate unfolding and translocation and indicate an overlapping function for the N-domain and NBD-1 pore tyrosine. Furthermore, the functional overlap seen in the presence of the DnaK system is not observed in the absence of DnaK. For innate ClpB unfolding activity, the NBD-1 pore tyrosine is required, and the presence of the N-domain is insufficient to overcome the defect of the ClpB Y251A mutant.

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.

Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity.

Two members of the AAA+ superfamily, ClpB and Hsp104, collaborate with Hsp70 and Hsp40 to rescue aggregated proteins. However, the mechanisms that elicit and underlie their protein-remodeling activities remain unclear. We report that for both Hsp104 and ClpB, mixtures of ATP and ATP-gammaS unexpectedly unleash activation, disaggregation and unfolding activities independent of cochaperones. Mutations reveal how remodeling activities are elicited by impaired hydrolysis at individual nucleotide-binding domains. However, for some substrates, mixtures of ATP and ATP-gammaS abolish remodeling, whereas for others, ATP binding without hydrolysis is sufficient. Remodeling of different substrates necessitates a diverse balance of polypeptide 'holding' (which requires ATP binding but not hydrolysis) and unfolding (which requires ATP hydrolysis). We suggest that this versatility in reaction mechanism enables ClpB and Hsp104 to reactivate the entire aggregated proteome after stress and enables Hsp104 to control prion inheritance.

Coupling ATP utilization to protein remodeling by ClpB, a hexameric AAA+ protein.

ClpB and Hsp104 are members of the AAA+ (ATPases associated with various cellular activities) family of proteins and are molecular machines involved in thermotolerance. They are hexameric proteins containing 12 ATP binding sites with two sites per protomer. ClpB and Hsp104 possess some innate protein remodeling activities; however, they require the collaboration of the DnaK/Hsp70 chaperone system to disaggregate and reactivate insoluble aggregated proteins. We investigated the mechanism by which ClpB couples ATP utilization to protein remodeling with and without the DnaK system. When wild-type ClpB, which is unable to remodel proteins alone in the presence of ATP, was mixed with a ClpB mutant that is unable to hydrolyze ATP, the heterohexamers surprisingly gained protein remodeling activity. Optimal protein remodeling by the heterohexamers in the absence of the DnaK system required approximately three active and three inactive protomers. In addition, the location of the active and inactive ATP binding sites in the hexamer was not important. The results suggest that in the absence of the DnaK system, ClpB acts by a probabilistic mechanism. However, when we measured protein disaggregation by ClpB heterohexamers in conjunction with the DnaK system, incorporation of a single inactive ClpB subunit blocked activity, supporting a sequential mechanism of ATP utilization. Taken together, the results suggest that the mechanism of ATP utilization by ClpB is adaptable and can vary depending on the specific substrate and the presence of the DnaK system.

ClpXP protease degrades the cytoskeletal protein, FtsZ, and modulates FtsZ polymer dynamics.

FtsZ is the major cytoskeletal protein in bacteria and a tubulin homologue. It polymerizes and forms a ring where constriction occurs to divide the cell. We found that FtsZ is degraded by E. coli ClpXP, an ATP-dependent protease. In vitro, ClpXP degrades both FtsZ protomers and polymers; however, polymerized FtsZ is degraded more rapidly than the monomer. Deletion analysis shows that the N-terminal domain of ClpX is important for polymer recognition and that the FtsZ C terminus contains a ClpX recognition signal. In vivo, FtsZ is turned over slower in a clpX deletion mutant compared with a WT strain. Overexpression of ClpXP results in increased FtsZ degradation and filamentation of cells. These results suggest that ClpXP may participate in cell division by modulating the equilibrium between free and polymeric FtsZ via degradation of FtsZ filaments and protomers.

The interplay of ClpXP with the cell division machinery in Escherichia coli.

ClpXP is a two-component protease composed of ClpX, an ATP-dependent chaperone that recognizes and unfolds specific substrates, and ClpP, a serine protease. One ClpXP substrate in Escherichia coli is FtsZ, which is essential for cell division. FtsZ polymerizes and forms the FtsZ ring at midcell, where division occurs. To investigate the role of ClpXP in cell division, we examined the effects of clpX and clpP deletions in several strains that are defective for cell division. Together, our results suggested that ClpXP modulates cell division through degradation of FtsZ and possibly other cell division components that function downstream of FtsZ ring assembly. In the ftsZ84 strain, which is temperature sensitive for filamentation due to a mutation in ftsZ, we observed that deletion of clpX or clpP suppresses filamentation and reduces FtsZ84 degradation. These results are consistent with ClpXP playing a role in cell division by modulating the level of FtsZ through degradation. In another division-defective strain, ΔminC, the additional deletion of clpX or clpP delays cell division and exacerbates filamentation. Our results demonstrate that ClpXP modulates division in cells lacking MinC by a mechanism that requires ATP-dependent degradation. However, antibiotic chase experiments in vivo indicate that FtsZ degradation is slower in the ΔminC strain than in the wild type, suggesting there may be another cell division component degraded by ClpXP. Taken together these studies suggest that ClpXP may degrade multiple cell division proteins, thereby modulating the precise balance of the components required for division.

Intermolecular Interactions between Hsp90 and Hsp70.

Members of the Hsp90 and Hsp70 families of molecular chaperones are imp\ortant for the maintenance of protein homeostasis and cellular recovery following environmental stresses, such as heat and oxidative stress. Moreover, the two chaperones can collaborate in protein remodeling and activation. In higher eukaryotes, Hsp90 and Hsp70 form a functionally active complex with Hop (Hsp90-Hsp70 organizing protein) acting as a bridge between the two chaperones. In bacteria, which do not contain a Hop homolog, Hsp90 and Hsp70, DnaK, directly interact during protein remodeling. Although yeast possesses a Hop-like protein, Sti1, Hsp90, and Hsp70 can directly interact in yeast in the absence of Sti1. Previous studies showed that residues in the middle domain of Escherichia coli Hsp90 are important for interaction with the J-protein binding region of DnaK. The results did not distinguish between the possibility that (i) these sites were involved in direct interaction and (ii) the residues in these sites participate in conformational changes which are transduced to other sites on Hsp90 and DnaK that are involved in the direct interaction. Here we show by crosslinking experiments that the direct interaction is between a site in the middle domain of Hsp90 and the J-protein binding site of Hsp70 in both E. coli and yeast. Moreover, J-protein promotes the Hsp70-Hsp90 interaction in the presence of ATP, likely by converting Hsp70 into the ADP-bound conformation. The identification of the protein-protein interaction site is anticipated to lead to a better understanding of the collaboration between the two chaperones in protein remodeling.

Bacterial Hsp90 ATPase Assays.

Bacterial Hsp90 is an ATP-dependent molecular chaperone involved in protein remodeling and activation. The E. coli Hsp90, Hsp90Ec, collaborates in protein remodeling with another ATP-dependent chaperone, DnaK, the E. coli Hsp70. Both Hsp90Ec and DnaK hydrolyze ATP and client (substrate) proteins stimulate the hydrolysis. Additionally, ATP hydrolysis by the combination of Hsp90Ec and DnaK is synergistically stimulated in the presence of client (substrate). Here, we describe two steady-state ATPase assays used to monitor ATP hydrolysis by Hsp90Ec and DnaK as well as the synergistic stimulation of ATP hydrolysis by the combination of Hsp90Ec and DnaK in the presence of a client (substrate). The first assay is a spectrophotometric assay based on enzyme-coupled reactions that utilize the ADP formed during ATP hydrolysis to oxidize NADH. The second assay is a more sensitive method that directly quantifies the radioactive inorganic phosphate released following the hydrolysis of [γ-33P] ATP or [γ-32P] ATP.

Substrate Discrimination by ClpB and Hsp104.

ClpB of E. coli and yeast Hsp104 are homologous molecular chaperones and members of the AAA+ (ATPases Associated with various cellular Activities) superfamily of ATPases. They are required for thermotolerance and function in disaggregation and reactivation of aggregated proteins that form during severe stress conditions. ClpB and Hsp104 collaborate with the DnaK or Hsp70 chaperone system, respectively, to dissolve protein aggregates both in vivo and in vitro. In yeast, the propagation of prions depends upon Hsp104. Since protein aggregation and amyloid formation are associated with many diseases, including neurodegenerative diseases and cancer, understanding how disaggregases function is important. In this study, we have explored the innate substrate preferences of ClpB and Hsp104 in the absence of the DnaK and Hsp70 chaperone system. The results suggest that substrate specificity is determined by nucleotide binding domain-1.

Interaction of E. coli Hsp90 with DnaK Involves the DnaJ Binding Region of DnaK.

The 90-kDa heat shock protein (Hsp90) is a widely conserved and ubiquitous molecular chaperone that participates in ATP-dependent protein remodeling in both eukaryotes and prokaryotes. It functions in conjunction with Hsp70 and the Hsp70 cochaperones, an Hsp40 (J-protein) and a nucleotide exchange factor. In Escherichia coli, the functional collaboration between Hsp90Ec and Hsp70, DnaK, requires that the two chaperones directly interact. We used molecular docking to model the interaction of Hsp90Ec and DnaK. The top-ranked docked model predicted that a region in the nucleotide-binding domain (NBD) of DnaK interacted with a region in the middle domain of Hsp90Ec. We then made substitution mutants in DnaK residues suggested by the model to interact with Hsp90Ec. Of the 12 mutants tested, 11 were defective or partially defective in their ability to interact with Hsp90Ecin vivo in a bacterial two-hybrid assay and in vitro in a bio-layer interferometry assay. These DnaK mutants were also defective in their ability to function collaboratively in protein remodeling with Hsp90Ec but retained the ability to act with DnaK cochaperones. Taken together, these results suggest that a specific region in the NBD of DnaK is involved in the interaction with Hsp90Ec, and this interaction is functionally important. Moreover, the region of DnaK that we found to be necessary for Hsp90Ec binding includes residues that are also involved in J-protein binding, suggesting a functional interplay among DnaK, DnaK cochaperones, and Hsp90Ec.

Hsp70 and Hsp90 of E. coli Directly Interact for Collaboration in Protein Remodeling.

Hsp90 is a highly conserved molecular chaperone that remodels hundreds of client proteins, many involved in the progression of cancer and other diseases. It functions with the Hsp70 chaperone and numerous cochaperones. The bacterial Hsp90 functions with an Hsp70 chaperone, DnaK, but is independent of Hsp90 cochaperones. We explored the collaboration between Escherichia coli Hsp90 and DnaK and found that the two chaperones form a complex that is stabilized by client protein binding. A J-domain protein, CbpA, facilitates assembly of the Hsp90Ec-DnaK-client complex. We identified E. coli Hsp90 mutants defective in DnaK interaction in vivo and show that the purified mutant proteins are defective in physical and functional interaction with DnaK. Understanding how Hsp90 and Hsp70 collaborate in protein remodeling will provide the groundwork for the development of new therapeutic strategies targeting multiple chaperones and cochaperones.