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.

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.

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.

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.

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.

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.

Interplay between E. coli DnaK, ClpB and GrpE during protein disaggregation.

The DnaK/Hsp70 chaperone system and ClpB/Hsp104 collaboratively disaggregate protein aggregates and reactivate inactive proteins. The teamwork is specific: Escherichia coli DnaK interacts with E. coli ClpB and yeast Hsp70, Ssa1, interacts with yeast Hsp104. This interaction is between the middle domains of hexameric ClpB/Hsp104 and the DnaK/Hsp70 nucleotide-binding domain (NBD). To identify the site on E. coli DnaK that interacts with ClpB, we substituted amino acid residues throughout the DnaK NBD. We found that several variants with substitutions in subdomains IB and IIB of the DnaK NBD were defective in ClpB interaction in vivo in a bacterial two-hybrid assay and in vitro in a fluorescence anisotropy assay. The DnaK subdomain IIB mutants were also defective in the ability to disaggregate protein aggregates with ClpB, DnaJ and GrpE, although they retained some ability to reactivate proteins with DnaJ and GrpE in the absence of ClpB. We observed that GrpE, which also interacts with subdomains IB and IIB, inhibited the interaction between ClpB and DnaK in vitro, suggesting competition between ClpB and GrpE for binding DnaK. Computational modeling of the DnaK-ClpB hexamer complex indicated that one DnaK monomer contacts two adjacent ClpB protomers simultaneously. The model and the experiments support a common and mutually exclusive GrpE and ClpB interaction region on DnaK. Additionally, homologous substitutions in subdomains IB and IIB of Ssa1 caused defects in collaboration between Ssa1 and Hsp104. Altogether, these results provide insight into the molecular mechanism of collaboration between the DnaK/Hsp70 system and ClpB/Hsp104 for protein disaggregation.

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.

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.

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.

Collaboration between the ClpB AAA+ remodeling protein and the DnaK chaperone system.

ClpB and Hsp104, members of the AAA+ superfamily of proteins, protect cells from the devastating effects of protein inactivation and aggregation that arise after extreme heat stress. They exist as a hexameric ring and contain two nucleotide-binding sites per monomer. ClpB and Hsp104 are able to dissolve protein aggregates in conjunction with the DnaK/Hsp70 chaperone system, although the roles of the individual chaperones in disaggregation are not well understood. In the absence of the DnaK/Hsp70 system, ClpB and Hsp104 alone are able to perform protein remodeling when their ATPase activity is asymmetrically slowed either by providing a mixture of ATP and ATP gamma S, a nonphysiological and slowly hydrolyzed ATP analog, or by inactivating one of the two nucleotide-binding domains by mutation. To gain insight into the roles of ClpB and the DnaK system in protein remodeling, we tested whether there was a further stimulation by the DnaK chaperone system under conditions that elicited remodeling activity by ClpB alone. Our results demonstrate that ClpB and the DnaK system act synergistically to remodel proteins and dissolve aggregates. The results further show that ATP is required and that both nucleotide-binding sites of ClpB must be able to hydrolyze ATP to permit functional collaboration between ClpB and the DnaK system.

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.

Two peptide sequences can function cooperatively to facilitate binding and unfolding by ClpA and degradation by ClpAP.

Clp/Hsp100 proteins comprise a large family of AAA(+) ATPases. Some Clp proteins function alone as molecular chaperones, whereas others act in conjunction with peptidases, forming ATP-dependent proteasome-like compartmentalized proteases. Protein degradation by Clp proteases is regulated primarily by substrate recognition by the Clp ATPase component. The ClpA and ClpX ATPases of Escherichia coli generally recognize short amino acid sequences that are located near the N or C terminus of a substrate. However, both ClpAP and ClpXP are able to degrade proteins in which the end containing the recognition signal is fused to GFP such that the signal is in the interior of the primary sequence of the substrate. Here, we tested whether the internal ClpA recognition signal was the sole element required for targeting the substrate to ClpA. The results show that, in the absence of a high-affinity peptide recognition signal at the terminus, two elements are important for recognition of GFP-RepA fusion proteins by ClpA. One element is the natural ClpA recognition signal located at the junction of GFP and RepA in the fusion protein. The second element is the C-terminal peptide of the fusion protein. Together, these two elements facilitate binding and unfolding by ClpA and degradation by ClpAP. The internal site appears to function similarly to Clp adaptor proteins but, in this case, is covalently attached to the polypeptide containing the terminal tag and both the "adaptor" and "substrate" modules are degraded.

Binding and degradation of heterodimeric substrates by ClpAP and ClpXP.

ClpA and ClpX function both as molecular chaperones and as the regulatory components of ClpAP and ClpXP proteases, respectively. ClpA and ClpX bind substrate proteins through specific recognition signals, catalyze ATP-dependent protein unfolding of the substrate, and when in complexes with ClpP translocate the unfolded polypeptide into the cavity of the ClpP peptidase for degradation. To examine the mechanism of interaction of ClpAP with dimeric substrates, single round binding and degradation experiments were performed, revealing that ClpAP degraded both subunits of a RepA homodimer in one cycle of binding. Furthermore, ClpAP was able to degrade both protomers of a RepA heterodimer in which only one subunit contained the ClpA recognition signal. In contrast, ClpXP degraded both subunits of a dimeric substrate only when both protomers contained a recognition signal. These data suggest that ClpAP and ClpXP may recognize and bind substrates in significantly different ways.

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."

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.

ClpAP and ClpXP degrade proteins with tags located in the interior of the primary sequence.

Clp/Hsp100 ATPases comprise a large family of ATP-dependent chaperones, some of which are regulatory components of two-component proteases. Substrate specificity resides in the Clp protein and the current thinking is that Clp proteins recognize motifs located near one or the other end of the substrate. We tested whether or not ClpA and ClpX can recognize tags when they are located in the interior of the primary sequence of the substrate. A protein with an NH2-terminal ClpA recognition tag, plasmid P1 RepA, was fused to the COOH terminus of green fluorescent protein (GFP). GFP is not recognized by ClpA or ClpX and is not degraded by ClpAP or ClpXP. We found that ClpA binds and unfolds the fusion protein and ClpAP degrades the protein. Both the GFP and RepA portions of the fusion protein are degraded. A protein with a COOH-terminal ClpX tag, MuA, was fused to the NH2 terminus of GFP. ClpXP degrades MuA-GFP, however, the rate is 10-fold slower than that of GFP-MuA. The MuA portion but not the GFP portion of MuA-GFP is degraded. Thus, a substrate with an internal ClpA recognition motif can be unfolded by ClpA and degraded by ClpAP. Similarly, although less efficiently, ClpXP degrades a substrate with an internal ClpX recognition motif. We also found that ClpA recognizes the NH2-terminal 15 aa RepA tag, when it is fused to the COOH terminus of GFP. Moreover, ClpA recognizes the RepA tag in either the authentic or inverse orientation.