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A GrpE Mutant Containing the NH2-Terminal “Tail” Region Is Able to Displace Bound Polypeptide Substrate from DnaK
Abstract
A key feature to the dimeric structure for the GrpE heat shock protein is the pair of long helices at the NH(2)-terminal end followed by a presumable extended segment of about 30 amino acids from each monomer. We have constructed a GrpE deletion mutant protein that contains only the unique tail portion (GrpE1-89) and another that is missing this region (GrpE88-197). Circular dichroism analysis shows that the GrpE1-89 mutant still contains one-third percent alpha-helical secondary structure. Using an assay that measures bound peptide to DnaK we show that the GrpE1-89 is able to lower the amount of bound peptide, whereas GrpE88-197 has no effect. Additionally, when the same peptide binding assay is carried out with the COOH-terminal domain of DnaK, the full-length GrpE and the two GrpE deletion mutants show little to no effect on peptide release. Furthermore, the GrpE88-197 mutant is able to enhance the off-rate of nucleotide from DnaK and the 1-89 mutant has no effect on the nucleotide release. Similar results of nucleotide release are observed with the NH(2)-terminal ATPase domain mutant of DnaK. The results presented show directly that there is interaction between the GrpE protein's "tail" region and the substrate COOH-terminal peptide binding domain of DnaK, although the effect is only fully manifest with an intact full-length DnaK molecule.
... We were unsuccessful in creating a clone for GrpE88-138, presumably due to the small size of the insert in the cloning process. All the mutant proteins were purified by similar means based on the purification of full-length GrpE (Mehl et al. 2001). The purity level was sufficient for the physical characterization experiments described in this work. ...
... Protein secondary structural content of full-length GrpE and the four deletion mutants was probed for by circular dichroism (CD) spectroscopy. Note that CD analysis for fulllength GrpE, have been reported (Mehl et al. 2001) and are reproduced here for comparison purposes. As shown in Figure 2, full-length GrpE gives a spectrum that is characteristic of a protein with significant ␣-helical secondary structure, showing negative dips in the spectrum at 208 and 222 nm. ...
... When analyzed for amount of ␣-helical content using SELCON3 software (Sreerama et al. 1999), the GrpE1-88 showed 22% ␣-helical content ( Table 1). The percentage of ␣-helical content reported previously (Mehl et al. 2001) was done incorrectly. Clearly, with the GrpE1-88 mutant the "native" structure for the tail region is lost. ...
The GrpE heat shock protein from Escherichia coli has a homodimeric structure. The dimer interface encompasses two long alpha-helices at the NH(2)-terminal end from each monomer (forming a "tail"), which lead into a small four-helix bundle from which each monomer contributes two short sequential alpha-helices in an antiparallel topological arrangement. We have created a number of different deletion mutants of GrpE that have portions of the dimer interface to investigate requirements for dimerization and to study four-helix bundle formation. Using chemical crosslinking and analytical ultracentrifugation techniques to probe for multimeric states, we find that a mutant containing only the long alpha-helical tail portion (GrpE1-88) is unable to form a dimer, most likely due to a decrease in alpha-helical content as determined by circular dichroism spectroscopy, thus one reason for a dimeric structure for the GrpE protein is to support the tail region. Mutants containing both of the short alpha-helices (GrpE1-138 and GrpE88-197) are able to form a dimer and presumably the four-helix bundle at the dimer interface. These two mutants have equilibrium constants for the monomer-dimer equilibrium that are very similar to the full-length protein suggesting that the tail region does not contribute significantly to the stability of the dimer. Interestingly, one mutant that contains just one of the short alpha-helices (GrpE1-112) exists as a tetrameric species, which presumably is forming a four-helix bundle structure. A proposed model is discussed for this mutant and its relevance for factors influencing four-helix bundle formation.
... A slightly modified protocol from the Quick Change™ Site-directed mutagenesis method developed by Stratagene (La Jolla, CA, USA) was utilized to create the point mutants. Briefly, a PCR reaction containing appropriate primers (R57A: 5′-GCTGAAGCCCAGACCGCTGAACGTGACGGC-3′/ 5′-GCCGTCACGTTCAGCGGTCTGGGCTTCAGC-3′; E94A: 5′-GAAATTCATCAACGCATTGCTGCCGGTGATTG-3′/ 5′-CAATCACCGGCAGCAATGCGTTGATGAATTTC-3′) from IDT, Inc. (Coralville, IA, USA) at 125 ng each, plasmid DNA (pRLM159) (Mehl et al., 2001) at 50 ng, dNTP mix (Perkin Elmer, Branchburg, NJ, USA) at 0.01 mM, and Pfu DNA polymerase (New England Biolabs Inc., Beverly, MA, USA) at 2.5 units was run followed by addition of Dpn 1 (New England Biolabs Inc., Beverly, MA, USA) (40 units) at 37°C for 1.5 h. A 10 μl aliquot was used to transform RLM569 cells (Mehl et al., 2001). ...
... Briefly, a PCR reaction containing appropriate primers (R57A: 5′-GCTGAAGCCCAGACCGCTGAACGTGACGGC-3′/ 5′-GCCGTCACGTTCAGCGGTCTGGGCTTCAGC-3′; E94A: 5′-GAAATTCATCAACGCATTGCTGCCGGTGATTG-3′/ 5′-CAATCACCGGCAGCAATGCGTTGATGAATTTC-3′) from IDT, Inc. (Coralville, IA, USA) at 125 ng each, plasmid DNA (pRLM159) (Mehl et al., 2001) at 50 ng, dNTP mix (Perkin Elmer, Branchburg, NJ, USA) at 0.01 mM, and Pfu DNA polymerase (New England Biolabs Inc., Beverly, MA, USA) at 2.5 units was run followed by addition of Dpn 1 (New England Biolabs Inc., Beverly, MA, USA) (40 units) at 37°C for 1.5 h. A 10 μl aliquot was used to transform RLM569 cells (Mehl et al., 2001). Plasmids were isolated and purified using Qiagen Midi Plasmid Kits (Valencia, CA, USA) and the correct plasmid DNA sequence was verified (Protein and Nucleic Acid Chemistry Laboratory, Washington University School of Medicine, St. Louis, MO, USA). ...
... The resulting plasmids were named pAFM49 for R57A and pAFM23 for E94A. The wild-type protein was purified as described previously (Mehl et al., 2001) and both mutant proteins were purified using the same protocol utilized for that of the wild-type. ...
The GrpE protein from E. coli is a homodimer with an unusual structure of two long paired alpha-helices from each monomer interacting in a parallel arrangement to form a "tail" at the N-terminal end. Using site-directed mutagenesis, we show that there is a key electrostatic interaction involving R57 (mediated by a water molecule) that provides thermal stability to this "tail" region. The R57A mutant showed a drop in T (m) of 8.5 degrees C and a smaller DeltaH (u) (unfolding) compared to wild-type for the first unfolding transition, but no significant decrease in dimer stability as shown through equilibrium analytical ultracentrifugation studies. Another mutant (E94A) at the dimer interface showed a decrease in DeltaH (u )but no drop in T (m) for the second unfolding transition and a slight increase in dimer stability.
... The DnaK R151A mutant was expressed and purified following the protocol developed by Taneva et al. (21). GrpE was cloned, expressed, and purified as described (22). The GrpE (34 -197) mutant was generated by digestion of wtGrpE with papain (125:1 (w:w), 90 min, 25°C), followed by chromatography purification in a HiLoad Superdex 75 16/60 column (GE Healthcare) (7). ...
... We suggest that the DnaK-GrpE complex cycles between at least two conformations (Fig. 6A), one with the tail in a straighter conformation and the other with the tip interacting with DnaK SBD . This interaction could provide the structural basis for the competition observed between substrates and GrpE residues 1-33 for the DnaK substrate-binding site (7,22,34). ...
Hsp70 chaperones comprise two domains, the nucleotide-binding domain (Hsp70NBD), responsible for structural and functional changes in the chaperone, and the substrate-binding domain (Hsp70SBD), involved in substrate interaction. Substrate binding and release in Hsp70 is controlled by the nucleotide state of DnaKNBD, with ATP inducing the open, substrate-receptive DnaKSBD, conformation, whereas ADP forces its closure. DnaK cycles between the two conformations through interaction with two cofactors, the Hsp40 co-chaperones (DnaJ in E. coli) induce the ADP state, and the nucleotide exchange factors (NEF; GrpE in E. coli) induce the ATP state. X-ray crystallography showed that the GrpE dimer is a NEF that works by interaction of one of its monomers with DnaKNBD. DnaKSBD location in this complex is debated; there is evidence that it interacts with the GrpE N-terminal disordered region, far from DnaKNBD. Although we confirmed this interaction using biochemical and biophysical techniques, our EM-based 3D reconstruction of the DnaK:GrpE complex located DnaKSBD near DnaKNBD. This apparent discrepancy between the functional and structural results is explained by our finding that the tail region of the GrpE dimer in the DnaK:GrpE complex bends and its tip contacts DnaKSBD, while the DnaKNBD-DnaKSBD linker contacts the GrpE helical region. We suggest that these interactions define a more complex role for GrpE in the control of DnaK function.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
... This plasmid is the same as pRLM76 [15] except for the fact that the polylinker region is more versatile. Plasmid pRLM 159 [16]carrying the grpE gene was used for the template in the Polymerase Chain Reaction (PCR) mediated cloning of the IDM's. All the IDM's were created using the same procedure; to summarize the approach: two segments of DNA retaining the code for the wanted protein regions, each containing the code for a truncated α-helix, along with a common restriction enzyme site (Ava 1) were PCR amplified using the grpe gene as a template. ...
... The expression and purification of wild-type GrpE protein has been described previously [16]. The expression and purification of IDM1.0 and IDM2.0 utilized the same exact protocol as that of the wild-type GrpE protein, namely a three step purification method involving DEAE cellulose, Affi-gel Blue, and Mono-Q resins was employed. ...
Insight into protein stability and folding remains an important area for protein research, in particular protein-protein interactions and the self-assembly of homodimers. The GrpE protein from Escherichia coli is a homodimer with a four-helix bundle at the dimer interface. Each monomer contributes a helix-loop-helix to the bundle. To probe the interface stabilization requirements, in terms of the amount of buried residues in the bundle necessary for dimer formation, internal deletion mutants (IDMs) were created that sequentially truncate each of the two helices in the helix-loop-helix region. Circular dichroism (CD) spectroscopy showed that all IDM's still contained a significant amount of α-helical secondary structure. IDM's that contained 11 or fewer of 22 residues originally present in the helices, or those that lost at least 50% of residues with less than 20% the solvent accessible surfaces (that is, hydrophobic residues) were unable to form a significant amount of dimer species as shown by chemical cross-linking. Gel filtration studies of IDM3.0 (one that retains 10 residues in each helix) show this variant to be mainly monomeric.
... meric GrpE has a striking and asymmetric appearance (seeFig 1) . A monomer of GrpE consists of an unstructured region not present in the crystal structure (Mehl et al 2001; Gelinas et al 2002), a long -helix followed by a short -helix, and then a compact -sheet domain at the C terminus. In the dimer, the 2 long -helices appear to be almost directly parallel and do not bury side chains in the same fashion as coiled-coils (neither ''knobs into holes'' nor ''ridges into grooves''). ...
... For the 2 peptides used in the study, the researchers concluded that GrpE contributed more to the rates of peptide release from the DnaK substrate-binding domain than did the addition of ATP to the reactions and noted that GrpE did not promote substrate release from the isolated DnaK substrate-binding domain. It was also observed that GrpE 89–197, a fragment that lacks the unstructured N terminus and the long -helices, could promote nucleotide dissociation but not substrate dissociation (Mehl et al 2001). ...
The cochaperone GrpE functions as a nucleotide exchange factor to promote dissociation of adenosine 5'-diphosphate (ADP) from the nucleotide-binding cleft of DnaK. GrpE and the DnaJ cochaperone act in concert to control the flux of unfolded polypeptides into and out of the substrate-binding domain of DnaK by regulating the nucleotide-bound state of DnaK. DnaJ stimulates nucleotide hydrolysis, and GrpE promotes the exchange of ADP for adenosine triphosphate (ATP) and also augments peptide release from the DnaK substrate-binding domain in an ATP-independent manner. The eukaryotic cytosol does not contain GrpE per se because GrpE-like function is provided by the BAG1 protein, which acts as a nucleotide exchange factor for cytosolic Hsp70s. GrpE, which plays a prominent role in mitochondria, chloroplasts, and bacterial cytoplasms, is a fascinating molecule with an unusual quaternary structure. The long alpha-helices of GrpE have been hypothesized to act as a thermosensor and to be involved in the decrease in GrpE-dependent nucleotide exchange that is observed in vitro at temperatures relevant to heat shock. This review describes the molecular biology of GrpE and focuses on the structural and kinetic aspects of nucleotide exchange, peptide release, and the thermosensor hypothesis.
... GrpE est capable de stimuler la dissociation de l'ADP et de l'ATP de manière non discriminante. Il stimule également la dissociation du substrat de DnaK via son domaine N-terminal non structuré (Han and Christen, 2003;Mally and Witt, 2001 ;Mehl et al., 2001 ). ...
Hsp70 are a highly conserved family of ubiquitous molecular chaperones that play essential roles in protein folding, transport or degradation. The cytosol of most eukaryotic cells contains multiple highly conserved Hsp70 orthologs that differ mainly by their spatio-temporal expression patterns. While several reports suggest that specialized functions of Hsp70 orthologs were selected through evolution, few studies addressed systematically this issue. First, we compared the ability of Ssa1-Ssa4 from Saccharomyces cerevisiae and Ssa5-Ssa8 from the evolutionary distant yeast Yarrowia lipolytica to perform Hsp70-dependent tasks when expressed as the sole Hsp70 for S. cerevisiae in vivo. We showed that Hsp70 isoforms: 1) supported yeast viability yet with markedly different growth rates; 2) influenced the propagation and stability of the [PSI+] and [URE3] prions; but 3) did not significantly affect the proteasomal degradation rate of CFTR. Second, we showed that biofilm formation in yeast depends on the Hsp70 machinery that controls, through distinct pathways, the expression, maturation and recycling of a cell-surface adhesin (Flo11) required for this process. Finally, we constructed and analyzed Y. lipolytica mutants bearing one or multiple deletion(s) in genes encoding molecular chaperones and others proteostasis modulators (e.g. Hsp70, Hsp104, CHIP). Despite very high homology and overlapping functions, the different Hsp70 orthologs have evolved to possess distinct activities that are required to cope with different types of substrates or stress situations.
... DnaK, DnaJ, GrpE, ClpB, and the GroELD87K variant were obtained as previously reported. [52][53][54] Purified IBs (0.2 μM) were resuspended in buffer [50 mM Tris-HCl (pH 7.5), 20 mM MgCl 2 , 150 mM KCl, and 10 mM DTT] containing DnaK (3.5 μM), DnaJ (0.7 μM), and GrpE (0.35 μM) in the absence or in the presence of ClpB (5 μM or 10 μM monomer). Reactivation was started by adding 4 mM ATP to the reaction mixture that also contained an ATP regenerating system (15 mM phosphoenolpyruvate and 20 ng/mL pyruvate kinase). ...
The formation of aggregates by misfolded proteins is thought to be inherently toxic, affecting cell fitness. This observation has led to the suggestion that selection against protein aggregation might be a major constraint on protein evolution. The precise fitness cost associated with protein aggregation has been traditionally difficult to evaluate. Moreover, it is not known if the detrimental effect of aggregates on cell physiology is generic or depends on the specific structural features of the protein deposit. In bacteria, the accumulation of intracellular protein aggregates reduces cell reproductive ability, promoting cellular aging. Here, we exploit the cell division defects promoted by the intracellular aggregation of Alzheimer's-disease-related amyloid β peptide in bacteria to demonstrate that the fitness cost associated with protein misfolding and aggregation is connected to the protein sequence, which controls both the in vivo aggregation rates and the conformational properties of the aggregates. We also show that the deleterious impact of protein aggregation on bacterial division can be buffered by molecular chaperones, likely broadening the sequential space on which natural selection can act. Overall, the results in the present work have potential implications for the evolution of proteins and provide a robust system to experimentally model and quantify the impact of protein aggregation on cell fitness.
... DnaK was expressed, purified, and extensively dialyzed to obtain nucleotidefree samples [20]. DnaJ, GrpE were obtained as previously reported [21,22]. Protein concentration was determined by the colorimetric Bradford assay (Bio-Rad), except for DnaK, that was determined spectrophotometrically using e 280 = 15.8 ...
ClpB is a member of the AAA+ superfamily that forms a ring-shaped homohexamer. Each protomer contains two nucleotide binding domains arranged in two rings that hydrolyze ATP. We extend here previous studies on ClpB nucleotide utilization requirements by using an experimental approach that maximizes random incorporation of different subunits into the protein hexamer. Incorporation of one subunit unable to bind or hydrolyze ATP knocks down the chaperone activity, while the wt hexamer can accommodate two mutant subunits that hydrolyze ATP in only one protein ring. Four subunits seem to build the functional cooperative unit, provided that one of the protein rings contains active nucleotide binding sites.
... Eventuell beschleunigen gebundene Peptide die Nukleotidfreisetzung von DnaK und ermöglichen so einen funktionellen Chaperonzyklus (Theyssen et al., 1996). Der negative Effekt von sehr hohen GrpE-Konzentrationen kann durch Interaktionen von GrpE mit der Substratbindestelle von DnaK erklärt werden (Mehl et al., 2001). (Diamant et al., 2000). ...
ClpB aus T. thermophilus kooperiert mit dem DnaK-Chaperonsystem in der Reaktivierung von Proteinaggregaten. ClpB gehört zur Gruppe der AAA+-Proteine, die durch die konservierte AAA-Kassette charakterisiert werden. Diese Domänen sind für die Nukleotidbindung und Hydrolyse der Proteine verantwortlich. Anhand von Sequenzvergleichen kann ClpB in vier Domänen eingeteilt werden: Der N-terminalen Domäne folgt die erste AAA-Kassette, die durch eine Linkerdomäne mit der zweiten AAA-Kassette verbunden ist. Ziel dieser Arbeit war es die Funktionen der einzelnen Domänen in der Deaggregationsreaktion genauer zu charakterisieren. Für diese Untersuchungen mussten zunächst neue Substratproteine zur Messung der Chaperonaktivität etabliert werden. Es konnte gezeigt werden, dass die N-terminale Domäne nicht essentiell für die Oligomerisierung, ATPase-Aktivität und Chaperonfunktion von ClpB ist. Sie ist an der Bindung von Casein beteiligt, interagiert jedoch nicht mit anderen Substraten.Die Linkerdomäne bildet eine funktionelle Einheit mit der ersten AAA-Kassette des Proteins. Daher kann ClpB in zwei Hälften mit jeweils einer AAA-Kassette unterteilt werden. In die erste AAA-Kassette mit der Linkerdomäne und in die zweite AAA-Kassette. Die beiden AAA-Kassetten sind nicht mehr funktionell aktiv und in der Oligomerisierung stark eingeschränkt. Ihre ATPase-Aktivität ist jedoch unterschiedlich. Die erste AAA-Kassette ist inaktiv, die zweite AAA-Kassette sehr aktiv in ATPase-Aktivitätsmessungen. Die beiden AAA-Kassetten können einen Komplex ausbilden, der biochemische Eigenschaften wie ClpB besitzt und über Chaperonaktivität verfügt. Dies zeigt, dass für die Chaperonaktivität die kovalente Verknüpfung der AAA-Kassetten von ClpB nicht notwendig ist. Die Komplexbildung führt zudem zu einer reduzierten ATPase-Aktivität der zweiten AAA-Kassette, was auf eine regulative Funktion der ersten AAA-Kassette und eine katalytische Funktion der AAA-Kassette im ATPase-Zyklus hinweist.
... DnaK was expressed, purified, and extensively dialyzed to obtain nucleotide-free samples [11]. DnaJ, GrpE and ClpB were obtained as previously reported [12][13][14]. G6PDH and a-glucosidase were purchased from Sigma, AdhE was expressed and purified as previously described [15]. Protein concentration was determined by the colorimetric Bradford assay (Bio-Rad), except for DnaK, that was determined spectrophotometrically using e 280 = 15.8 ...
Intracellular protein aggregates formed under severe thermal stress can be reactivated by the concerted action of the Hsp70 system and Hsp100 chaperones. We analyzed here the interaction of DnaJ/DnaK and ClpB with protein aggregates. We show that aggregate properties modulate chaperone binding, which in turn determines aggregate reactivation efficiency. ClpB binding strictly depends on previous DnaK association with the aggregate. The affinity of ClpB for the aggregate-DnaK complex is low (K(d)=5-10 microM), indicating a weak interaction. Therefore, formation of the DnaK-ClpB bichaperone network is a three step process. After initial DnaJ binding, the cochaperone drives association of DnaK to aggregates, and in the third step, as shown here, DnaK mediates ClpB interaction with the aggregate surface.
... It is imperative to conduct presteady-state kinetic studies to determine the effect of DnaJ on the kinetic constants for peptide binding to ATP·DnaK. It is intriguing that GrpE also promotes peptide release from DnaK-peptide complexes, although the functional significance of this is not yet clear (Harrison et al., 1997;Mally and Witt, 2001;Mehl et al., 2001). ...
We discuss recent experiments that have illuminated individual steps in the reaction cycle of the Escherichia coli Hsp70 molecular chaperone DnaK. Using this new information, we compare two distinctly different global mechanisms of action--holding versus unfolding--and argue that the available evidence suggests that DnaK is an unfoldase.
... All proteins were extensively dialyzed against 20 mm imidazole, pH 7.2, 2 mm EDTA, 10% glycerol to remove the bound nucleotide. DnaJ and GrpE were expressed in BL21 cells and purified as described elsewhere [52,53]. Recombinant his-tagged versions of Mdj1p and Mge1p, where the mitochondrial presequences were removed, were expressed in E. coli and purified as described elsewhere [17]. ...
Among the eukaryotic members of the Hsp70 family, mitochondrial Hsp70 shows the highest degree of sequence identity with bacterial DnaK. Although they share a functional mechanism and homologous co-chaperones, they are highly specific and cannot be exchanged between Escherichia coli and yeast mitochondria. To provide a structural basis for this finding, we characterized both proteins, as well as two DnaK/mtHsp70 chimeras constructed by domain swapping, using biochemical and biophysical methods. Here, we show that DnaK and mtHsp70 display different conformational and biochemical properties. Replacing different regions of the DnaK peptide-binding domain with those of mtHsp70 results in chimeric proteins that: (a) are not able to support growth of an E. coli DnaK deletion strain at stress temperatures (e.g. 42 degrees C); (b) show increased accessibility and decreased thermal stability of the peptide-binding pocket; and (c) have reduced activation by bacterial, but not mitochondrial co-chaperones, as compared with DnaK. Importantly, swapping the C-terminal alpha-helical subdomain promotes a conformational change in the chimeras to an mtHsp70-like conformation. Thus, interaction with bacterial co-chaperones correlates well with the conformation that natural and chimeric Hsp70s adopt in solution. Our results support the hypothesis that a specific protein structure might regulate the interaction of Hsp70s with particular components of the cellular machinery, such as Tim44, so that they perform specific functions.
Chaperone proteins and their cochaperones are perhaps one of the most intriguing systems for investigation. Ubiquitous in nature, they can be found in every organism and that perhaps is the reason that their sequence shows amazingly high homology and similarity. Allosteric mechanisms govern their functions and that makes them very interesting and hard to investigate at the same time. In medicine, chaperone proteins are of extreme interest since they are related to various diseases including neurodegerative diseases such as Alzheimers disease, Pick???s disease and Parkinsons disease. Various forms of cancer also relate to the chaperone proteins. Hence the investigation of various small molecules that could up/down-regulate this system is of great biomedical relevance.
In this dissertation we explored a member of the chaperone family that is constitutively expressed in every mammalian cell, the cognate Hsp70 (Hsc70). More specifically, we investigate the interactions of its ATPase domain with various small molecules that either act as inhibitors or enhancers of the protein???s activity. For example, we found that the compound MKT-077 blocks the Hsc70 ATPase activity by binding in a negatively charged cleft with its positive charge. This was identified with the use of chemical shift mapping experiments, NOE experiments and AUTODOCK simulations. MKT-077 binds only to the ADP form of the protein elucidating an amazing mechanism for the inhibition of the protein. Another inhibitor, methylene blue, binds to another locus of the ATPase. Interestingly, both of these molecules reduce the levels of tau in cell-based models of Alzheimers disease. This would suggest that the use of compounds that bind to Hsc70 at two separate sites could modulate Hsp70 activity in cells in a synergistic fashion.
DnaK, the prokaryotic Hsp70 molecular chaperone, requires the nucleotide exchange factor and heat shock protein GrpE to release ADP. GrpE and DnaK are tightly associated molecules with an extensive protein–protein interface, and in the absence of ADP, the dissociation constant for GrpE and DnaK is in the low nanomolar range. GrpE reduces the affinity of DnaK for ADP, and the reciprocal linkage is also true: ADP reduces the affinity of DnaK for GrpE. The energetic contributions of GrpE side-chains to GrpE–DnaK binding were probed by alanine-scanning mutagenesis. Sedimentation velocity (SV) analytical ultracentrifugation (AUC) was used to measure the equilibrium constants (Keq) for GrpE binding to the ATPase domain of DnaK in the presence of ADP. ADP-bound DnaK is the natural target of GrpE, and the addition of ADP (final concentration of 5 μM) to the preformed GrpE–DnaKATPase complexes allowed the equilibrium association constants to be brought into an experimentally accessible range. Under these experimental conditions, the substitution of one single GrpE amino acid residue, arginine 183 with alanine, resulted in a GrpE–DnaKATPase complex that was weakly associated (Keq=9.4×104 M). This residue has been previously shown to be part of a thermodynamic linkage between two structural domains of GrpE: the thermosensing long helices and the C-terminal β-domains. Several other GrpE side-chains were found to have a significant change in the free energy of binding (ΔΔG∼1.5 to 1.7 kcal mol−1), compared to wild-type GrpE·DnaKATPase in the same experimental conditions. Overall, the strong interactions between GrpE and DnaK appear to be dominated by electrostatics, not unlike barnase and barstar, another well-characterized protein–protein interaction. GrpE, an inherent thermosensor, exhibits non-Arrhenius behavior with respect to its nucleotide exchange function at bacterial heat shock temperatures, and mutation of several solvent-exposed side-chains located along the thermosensing indicated that these residues are indeed important for GrpE–DnaK interactions.
Hexameric AAA+ chaperone ClpB reactivates protein aggregates in collaboration with the DnaK system. An intriguing aspect of ClpB function is that the active hexamer is unstable and therefore questions how this chaperone uses multiple rounds of ATP hydrolysis to translocate substrates through its central channel. Here we report the use of biochemical and fluorescence tools to explore ClpB dynamics under different experimental conditions. The analysis of the chaperone activity and the kinetics of subunit exchange between protein hexamers labelled at different protein domains indicates, in contrast to the current view, that i) ATP favors assembly and ADP dissociation of the hexameric assembly; ii) subunit exchange kinetics is at least one order of magnitude slower that the ATP hydrolysis rate; iii) ClpB dynamics and activity are related proccesses; and iv) DnaK and substrate proteins regulate the ATPase activity and dynamics of ClpB. These data suggest that ClpB hexamers remain associated during several ATP hydrolysis events required to partially or completely translocate substrates through the protein central channel, and that ClpB dynamics is tuned by DnaK and substrate proteins.
A protein hydrogel system based on the assembly of a four-helix bundle motif was proposed and synthesized by genetic engineering methods. This new polypeptide, named GBH1, consists of identical amphipathic helices of 22 residues in length oriented in opposite fashion to one another at each end of a polypeptide with a total length of 227 amino acids. The middle portion of the polypeptide (residues 79-147) is an unstructured random coil. The region between the amphipathic and unstructured segment is an α-helical stretch (23-78 and 148-204) not possessing a sequence compatible with a coiled-coil conformation, but rather possesses regions that have overwinding of the helix. The thermal unfolding of GBH1 shows more than one inflection point (T(m1) = 30.5 °C, T(m2) = 64.6 °C), indicative of a partially unfolded intermediate and, thus, multiple interactions in the folded state. A qualitative assessment of hydrogel formation with varying pH showed that acidic conditions did not support the gel state, indirectly indicating that the proposed four-helix bundle is the major cross-linking structure and not a leucine zipper motif. Scanning electron microscopy reveals a network of interacting protein molecules forming a spongelike matrix with numerous pores that would be occupied with water molecules.
ClpB is a hexameric chaperone that solubilizes and reactivates protein aggregates in cooperation with the Hsp70/DnaK chaperone system. Each of the identical protein monomers contains two nucleotide binding domains (NBD), whose ATPase activity must be coupled to exert on the substrate the mechanical work required for its reactivation. However, how communication between these sites occurs is at present poorly understood. We have studied herein the affinity of each of the NBDs for nucleotides in WT ClpB and protein variants in which one or both sites are mutated to selectively impair nucleotide binding or hydrolysis. Our data show that the affinity of NBD2 for nucleotides (K(d) = 3-7 μm) is significantly higher than that of NBD1. Interestingly, the affinity of NBD1 depends on nucleotide binding to NBD2. Binding of ATP, but not ADP, to NBD2 increases the affinity of NBD1 (the K(d) decreases from ≈160-300 to 50-60 μm) for the corresponding nucleotide. Moreover, filling of the NBD2 ring with ATP allows the cooperative binding of this nucleotide and substrates to the NBD1 ring. Data also suggest that a minimum of four subunits cooperate to bind and reactivate two different aggregated protein substrates.
ClpB is a hexameric molecular chaperone that, together with the DnaK system, has the ability to disaggregate stress-denatured proteins. The hexamer is a highly dynamic complex, able to reshuffle subunits. To further characterize the biological implications of the ClpB oligomerization state, the association equilibrium of the wild-type (wt) protein and of two deletion mutants, which lack part or the whole M domain, was quantitatively analyzed under different experimental conditions, using several biophysical [analytical ultracentrifugation, composition-gradient (CG) static light scattering, and circular dichroism] and biochemical (ATPase and chaperone activity) methods. We have found that (i) ClpB self-associates from monomers to form hexamers and higher-order oligomers that have been tentatively assigned to dodecamers, (ii) oligomer dissociation is not accompanied by modifications of the protein secondary structure, (iii) the M domain is engaged in intersubunit interactions that stabilize the protein hexamer, and (iv) the nucleotide-induced rearrangement of ClpB affects the protein oligomeric core, in addition to the proposed radial extension of the M domain. The difference in the stability of the ATP- and ADP-bound states [ΔΔG(ATP-ADP) = -10 kJ/mol] might explain how nucleotide exchange promotes the conformational change of the protein particle that drives its functional cycle.
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.
A DNA fragment encoding Bacillus licheniformis GrpE (BlGrpE) with double mutations at codons 52 and 134 was obtained during PCR cloning. Leu52 and Leu134 in BlGrpE were individually replaced with Pro and His to generate BlGrpE-L52P and BlGrpE-L134H. BlGrpE and BlGrpE-L52P synergistically stimulated the ATPase activity of B. licheniformis DnaK (BlDnaK); however, BlGrpE-L134H and the double-mutated protein (BlGrpE-L52P/L134H) had no co-chaperone function. BlGrpE, BlGrpE-L52P, and BlGrpE-L134H mainly interacted with the monomer of BlDnaK but non-specific interaction was observed for BlGrpE-L52P/L134H. Measurement of intrinsic fluorescence revealed a significant alteration of the microenvironment of aromatic acid residues in the mutant proteins. As compared with BlGrpE, quenching of 208-nm and 222-nm signals were observed in the mutant BlGrpEs and the single-mutated proteins were more sensitive to thermal denaturation.
GrpE is the nucleotide exchange factor for the Escherichia coli molecular chaperone DnaK, the prokaryotic homologue of Hsp70. Thermodynamic properties of GrpE structural domains were characterized by examining a number of structural and point mutants using circular dichroism, differential scanning calorimetry and analytical ultracentrifugation. These structural domains are the long paired N-terminal helices, the central four-helix bundle, and the C-terminal compact beta-domains. We show that the central four-helix bundle (t(m) approximately 75 degrees C) provides a stable platform for the association of the long paired N-terminal helices (t(m) approximately 50 degrees C), which can then function as a temperature sensor. The stability of the N-terminal helices is linked to the presence of the C-terminal compact beta-domains of GrpE, providing a potential mechanism for coupling of DnaK-binding activity of GrpE with temperature. On the basis of our thermodynamic analysis of E.coli GrpE, we present a structure-based model for the melting properties of the nucleotide exchange factor, wherein the long paired helices function as a molecular thermocouple.
GrpE is the nucleotide exchange factor for the Escherichia coli molecular chaperone DnaK, the bacterial homologue of Hsp70. In the temperature range of the bacterial heat shock response, the long helices of GrpE undergo a helix-to-coil transition, and GrpE exhibits non-Arrhenius behavior with respect to its nucleotide exchange function. It is hypothesized that GrpE acts as a thermosensor and that unwinding of the long helices of E. coli GrpE reduces its activity as a nucleotide exchange factor. In turn, it was proposed that temperature-dependent down-regulation of the activity of GrpE may increase the time in which DnaK binds its substrates at higher temperatures. A combination of thermodynamic and hydrodynamic techniques, in concert with the luciferase refolding assay, were used to characterize a molecular mechanism in which the long helices of GrpE are thermodynamically linked with the beta-domains via an intramolecular contact between Phe86 and Arg183. These "thermosensing" long helices were found to be necessary for full activity as a nucleotide exchange factor in the luciferase refolding assay. Point mutations in the beta-domains and in the long helices of GrpE destabilized the beta-domains. Engineered disulfide bonds in the long helices alternately stabilized the long helices and the four-helix bundle. This allowed the previously reported 75 degrees C thermal transition seen in the excess heat capacity function as monitored by differential scanning calorimetry to be further characterized. The observed thermal transition represents the unfolding of the four-helix bundle and the beta-domains. The thermal transitions for these two domains are superimposed but are not thermodynamically linked.
In this study, we have used surface plasmon resonance (SPR) and isothermal microtitration calorimetry (ITC) to study the mechanism of complex formation between the Hsp70 molecular chaperone, DnaK, and its cochaperone, GrpE, which is a nucleotide exchange factor. Experiments were geared toward understanding the influence of DnaK's three domains, the ATPase (residues 1-388), substrate-binding (residues 393-507), and lid (residues 508-638) domains, on complex formation with GrpE. We show that the equilibrium dissociation constants for the interaction of GrpE with wtDnaK, lidless DnaK(2-517), the ATPase domain (2-388), and the substrate-binding fragment (393-507) are 64 (+/-16) nM, 4.0 (+/-1.5) nM, 35 (+/-10) nM, and 67 (+/-11) microM, respectively, and that the on-rate constant for the different reactions varies by over 4 orders of magnitude. SPR experiments revealed that GrpE-DnaK(393-507) complex formation is inhibited by added peptide and abolished when the 33-residue flexible "tail" of GrpE is deleted. Such results strongly suggest that the 33-residue flexible N-terminal tail of GrpE binds in the substrate-binding pocket of DnaK. This unique mode of binding between GrpE's tail and DnaK contributes to, but does not fully explain, the decrease in K(d) from 64 to 4 nM upon deletion of DnaK's lid. The possibility that deletion of DnaK's lid creates a more symmetrically shaped molecule, with enhanced affinity to GrpE, is also discussed. Our results reveal a complex set of molecular interactions between DnaK and its cochaperone GrpE. We discuss the impact of each domain on complex formation and dissociation.
DnaK, the prokaryotic Hsp70 molecular chaperone, requires the nucleotide exchange factor and heat shock protein GrpE to release ADP. GrpE and DnaK are tightly associated molecules with an extensive protein-protein interface, and in the absence of ADP, the dissociation constant for GrpE and DnaK is in the low nanomolar range. GrpE reduces the affinity of DnaK for ADP, and the reciprocal linkage is also true: ADP reduces the affinity of DnaK for GrpE. The energetic contributions of GrpE side-chains to GrpE-DnaK binding were probed by alanine-scanning mutagenesis. Sedimentation velocity (SV) analytical ultracentrifugation (AUC) was used to measure the equilibrium constants (Keq) for GrpE binding to the ATPase domain of DnaK in the presence of ADP. ADP-bound DnaK is the natural target of GrpE, and the addition of ADP (final concentration of 5 microM) to the preformed GrpE-DnaK(ATPase) complexes allowed the equilibrium association constants to be brought into an experimentally accessible range. Under these experimental conditions, the substitution of one single GrpE amino acid residue, arginine 183 with alanine, resulted in a GrpE-DnaK(ATPase) complex that was weakly associated (Keq =9.4 x 10(4) M). This residue has been previously shown to be part of a thermodynamic linkage between two structural domains of GrpE: the thermosensing long helices and the C-terminal beta-domains. Several other GrpE side-chains were found to have a significant change in the free energy of binding (DeltaDeltaG approximately 1.5 to 1.7 kcal mol(-1)), compared to wild-type GrpE.DnaK(ATPase) in the same experimental conditions. Overall, the strong interactions between GrpE and DnaK appear to be dominated by electrostatics, not unlike barnase and barstar, another well-characterized protein-protein interaction. GrpE, an inherent thermosensor, exhibits non-Arrhenius behavior with respect to its nucleotide exchange function at bacterial heat shock temperatures, and mutation of several solvent-exposed side-chains located along the thermosensing indicated that these residues are indeed important for GrpE-DnaK interactions.
To gain further insight into the interactions involved in the allosteric transition of DnaK we have characterized wild-type
(wt) protein and three mutants in which ionic interactions at the interface between the two subdomains of the substrate binding
domain, and within the lid subdomain have been disrupted. Our data show that ionic contacts, most likely forming an electrically
charged network, between the N-terminal region of helix B and an inner loop of the β-sandwich are involved in maintaining
the position of the lid relative to the β-subdomain in the ADP state but not in the ATP state of the protein. Disruption of
the ionic interactions between the C-terminal region of helix B and the outer loops of the β-sandwich, known as the latch,
does not have the same conformational consequences but results equally in an inactive protein. This indicates that a variety
of mechanisms can inactivate this complex allosteric machine. Our results identify the ionic contacts at the subdomain and
interdomain interfaces that are part of the hinge region involved in the ATP-induced allosteric displacement of the lid away
from the peptide binding site. These interactions also stabilize peptide-Hsp70 complexes at physiological (37 °C) and stress
(42 °C) temperatures, a requirement for productive substrate (re)folding.
GrpE acts as a nucleotide exchange factor for the Hsp70 chaperone system. Only one GrpE isoform is present in Escherichia coli, but for reasons not yet well understood, two GrpE isoforms have been found in mammalian mitochondria.Therefore, studies aimed at evaluating the physico-chemical characteristics of these proteins are important for the comprehension of the function of the Hsp70 chaperone system in different organisms. Here we report biophysical studies on human mitochondrial GrpE isoform 2. Small angle X-ray scattering measurements of human GrpE isoform 2 showed that this protein has a quaternary structure which is similar to those of human GrpE isoform 1 and E. coli GrpE: a dimer with a cruciform elongated shape. However, mitochondrial isoforms differed from each other regarding chemical and thermal denaturation profiles. This fact, combined with results of distinct expression patterns previously reported, point out that these proteins may have different response to external stimuli. Our results also indicate that human GrpE isoform 2 is more similar to the GrpE from E. coli than to human GrpE isoform 1. These results are relevant because differences in the conformation of Hsp70 co-chaperones are considered to be one of the reasons for functional diversity of this system.
The co-chaperone GrpE is essential for the activities of the Hsp70 system, which assists protein folding. GrpE is present in several organisms, and characterization of homologous GrpEs is important for developing structure-function relationships. Cloning, producing, and conformational studies of the recombinant human mitochondrial GrpE are reported here. Circular dichroism measurements demonstrate that the purified protein is folded. Thermal unfolding of human GrpE measured both by circular dichroism and differential scanning calorimetry differs from that of prokaryotic GrpE. Analytical ultracentrifugation data indicate that human GrpE is a dimer, and the sedimentation coefficient agrees with an elongated shape model. Small angle x-ray scattering analysis shows that the protein possesses an elongated shape in solution and demonstrates that its envelope, determined by an ab initio method, is similar to the high resolution envelope of Escherichia coli GrpE bound to DnaK obtained from single crystal x-ray diffraction. However, in these conditions, the E. coli GrpE dimer is asymmetric because the monomer that binds DnaK adopts an open conformation. It is of considerable importance for structural GrpE research to answer the question of whether the GrpE dimer is only asymmetric while bound to DnaK or also as a free dimer in solution. The low resolution structure of human GrpE presented here suggests that GrpE is a symmetric dimer when not bound to DnaK. This information is important for understanding the conformational changes GrpE undergoes on binding to DnaK.
GrpE proteins function as nucleotide exchange factors for DnaK-type Hsp70s. We have previously identified a chloroplast homolog
of GrpE in Chlamydomonas reinhardtii, termed CGE1. CGE1 exists as two isoforms, CGE1a and CGE1b, which are generated by temperature-dependent alternative splicing.
CGE1b contains additional valine and glutamine residues in its extreme NH2-terminal region. Here we show that CGE1a is predominant at lower temperatures but that CGE1b becomes as abundant as CGE1a
at elevated temperatures. Coimmunoprecipitation experiments revealed that CGE1b had a ∼25% higher affinity for its chloroplast
chaperone partner HSP70B than CGE1a. Modeling of the structure of CGE1b revealed that the extended α-helix formed by GrpE
NH2 termini is 34 amino acids longer in CGE1 than in Escherichia coli GrpE and appears to contain a coiled coil motif. Progressive deletions of this coiled coil increasingly impaired the ability
of CGE1 to form dimers, to interact with DnaK at elevated temperatures, and to complement temperature-sensitive growth of
a ΔgrpE E. coli strain. In contrast, deletion of the four-helix bundle required for dimerization of E. coli GrpE did not affect CGE1 dimer formation. Circular dichroism measurements revealed that CGE1, like GrpE, undergoes two thermal
transitions, the first of which is in the physiologically relevant temperature range (midpoint ∼45 °C). Truncating the NH2-terminal coiled coil shifted the second transition to lower temperatures, whereas removal of the four-helix bundle abolished
the first transition. Our data suggest that bacterial GrpE and chloroplast CGE1 share similar structural and biochemical properties,
but some of these, like dimerization, are realized by different domains.
GrpE acts as a nucleotide exchange factor for DnaK, the main Hsp70 protein in bacteria, accelerating ADP/ATP exchange by several orders of magnitude. GrpE is a homodimer, each subunit containing three structural domains: a N-terminal unordered segment, two long coils and a C-terminal globular domain formed by a four-helix bundle, and a beta-subdomain. GrpE association to DnaK nucleotide-binding domain involves side-chain and backbone interactions located within the "headpiece" of the cochaperone, which consists of the C-terminal half of the coils, the four-helix bundle and the beta-subdomain. However, the role of the GrpE N-terminal region in the interaction with DnaK and the activity of the cochaperone remain controversial. In this study we explore the contribution of this domain to the binding reaction, using the wild-type proteins, two deletion mutants of GrpE (GrpE(34-197) and GrpE(69-197)) and the isolated DnaK nucleotide-binding domain. Analysis of the thermodynamic binding parameters obtained by isothermal titration calorimetry shows that both GrpE N-terminal segments, 1-33 and 34-68, contribute to the binding reaction. Partial proteolysis and substrate dissociation kinetics also suggest that the N-terminal half of GrpE coils (residues 34-68) interacts with DnaK interdomain linker, regulates the nucleotide exchange activity of the cochaperone and is required to stabilize DnaK-substrate complexes in the ADP-bound conformation.
Thermal stress might lead to protein aggregation in the cell. Reactivation of protein aggregates depends on Hsp100 and Hsp70
chaperones. We focus in this study on the ability of DnaK, the bacterial representative of the Hsp70 family, to interact with
different aggregated model substrates. Our data indicate that DnaK binding to large protein aggregates is mediated by DnaJ,
and therefore it depends on its affinity for the cochaperone. Mutations in the structural region of DnaK known as the “latch”
decrease the affinity of the chaperone for DnaJ, resulting in a defective activity as protein aggregate-removing agent. As
expected, the chaperone activity is recovered when DnaJ concentration is raised to overcome the lower affinity of the mutant
for the cochaperone, suggesting that a minimum number of aggregate-bound DnaK molecules is necessary for its efficient reactivation.
Our results provide the first experimental evidence of DnaJ-mediated recruiting of ATP-DnaK molecules to the aggregate surface.
A simple approach to estimate the number of α-helical and β-strand segments from protein circular dichroism spectra is described. The α-helix and β-sheet conformations in globular protein structures, assigned by DSSP and STRIDE algorithms, were divided into regular and distorted fractions by considering a certain number of terminal residues in a given α-helix or β-strand segment to be distorted. The resulting secondary structure fractions for 29 reference proteins were used in the analyses of circular dichroism spectra by the SELCON method. From the performance indices of the analyses, we determined that, on an average, four residues per α-helix and two residues per β-strand may be considered distorted in proteins. The number of α-helical and β-strand segments and their average length in a given protein were estimated from the fraction of distorted α-helix and β-strand conformations determined from the analysis of circular dichroism spectra. The statistical test for the reference protein set shows the high reliability of such a classification of protein secondary structure. The method was used to analyze the circular dichroism spectra of four additional proteins and the predicted structural characteristics agree with the crystal structure data.
The polypeptide binding and release cycle of the molecular chaperone DnaK (Hsp70) of Escherichia coli is regulated by the two co-chaperones DnaJ and GrpE. Here, we show that the DnaJ-triggered conversion of DnaK.ATP (T state) to DnaK.ADP.Pi (R state), as monitored by intrinsic protein fluorescence, is monophasic and occurs simultaneously with ATP hydrolysis. This is in contrast with the T-->R conversion in the absence of DnaJ which is biphasic, the first phase occurring simultaneously with the hydrolysis of ATP (Theyssen, H., Schuster, H.-P., Packschies, L., Bukau, B., and Reinstein, J. (1996) J. Mol. Biol. 263, 657-670). Apparently, DnaJ not only stimulates ATP hydrolysis but also couples it with conformational changes of DnaK. In the absence of GrpE, DnaJ forms a tight ternary complex with peptide.DnaK.ADP.Pi (Kd = 0.14 microM). However, by monitoring complex formation between DnaK (1 microM) and a fluorophore-labeled peptide in the presence of ATP (1 mM), DnaJ (1 microM), and varying concentrations of the ADP/ATP exchange factor GrpE (0.1-3 microM), substoichiometric concentrations of GrpE were found to shift the equilibrium from the slowly binding and releasing, high-affinity R state of DnaK completely to the fast binding and releasing, low-affinity T state and thus to prevent the formation of a long lived ternary DnaJ. substrate.DnaK.ADP.Pi complex. Under in vivo conditions with an estimated chaperone ratio of DnaK:DnaJ:GrpE = 10:1:3, both DnaJ and GrpE appear to control the chaperone cycle by transient interactions with DnaK.
Replication of the chromosome of bacteriophage lambda depends on the cooperative action of two phage-coded proteins and seven replication and heat shock proteins from its Escherichia coli host. As previously described, the first stage in this process is the binding of multiple copies of the lambda O initiator to the lambda replication origin (ori lambda) to form the nucleosomelike O-some. The O-some serves to localize subsequent protein-protein and protein-DNA interactions involved in the initiation of lambda DNA replication to ori lambda. To study these interactions, we have developed a sensitive immunoblotting protocol that permits the protein constituents of complex nucleoprotein structures to be identified. Using this approach, we have defined a series of sequential protein assembly and protein disassembly events that occur at ori lambda during the initiation of lambda DNA replication. A second-stage ori lambda.O (lambda O protein).P (lambda P protein).DnaB nucleoprotein structure is formed when O, P, and E. coli DnaB helicase are incubated with ori lambda DNA. In a third-stage reaction the E. coli DnaJ heat shock protein specifically binds to the second-stage structure to form an ori lambda.O.P.DnaB.DnaJ complex. Each of the nucleoprotein structures formed in the first three stages was isolated and shown to be a physiological intermediate in the initiation of lambda DNA replication. The E. coli DnaK heat shock protein can bind to any of these early stage nucleoprotein structures, and in a fourth-stage reaction a complete ori lambda.O.P.DnaB.DnaJ.DnaK initiation complex is assembled. Addition of ATP to the reaction enables the DnaK and DnaJ heat shock proteins to mediate a partial disassembly of the fourth-stage complex. These protein disassembly reactions activate the intrinsic helicase activity of DnaB and result in localized unwinding of the ori lambda template. The protein disassembly reactions are described in the accompanying articles.
Previous studies have demonstrated that the Escherichia coli DnaK, DnaJ, and GrpE heat shock proteins participate in the initiation of bacteriophage lambda DNA replication by mediating the required disassembly of a preinitiation nucleoprotein structure that is formed at the phage replication origin. To gain some understanding in a simpler system of how the DnaJ and GrpE cochaperonins influence the activity of DnaK, we have examined the effect of the cochaperonins on the weak intrinsic ATPase activity of the molecular chaperone DnaK in the presence and absence of peptide effectors. We have found that random sequence peptide chains of 8 or 9 amino acid residues in length yield optimal (10-fold) activation of the DnaK ATPase, whereas peptides with 5 or fewer residues fail to stimulate the ATPase of this bacterial hsp70 homologue. Furthermore, we have discovered that those peptides that interact best with DnaK, as judged by their KA as activators of ATP hydrolysis by DnaK, also act as strong inhibitors of lambda DNA replication in vitro. The inhibitory effect of peptides on lambda DNA replication was overcome by increasing the concentration of DnaK in the replication system. Diminished inhibition was also found when the replication system was supplemented with GrpE cochaperonin, a protein known to increase the effectiveness of DnaK action in lambda DNA replication. These and other results suggest that the peptide-binding site of DnaK is required for its function in lambda DNA replication. Apparently, peptides sequester free DnaK protein and block lambda DNA replication by reducing the amount of DnaK that is free to mediate disassembly of nucleoprotein preinitiation structures. In related studies, we have found that DnaJ, like short peptides, activates the intrinsic ATPase activity of DnaK. DnaJ, however, is substantially more potent in this regard, since it activates DnaK at concentrations 1000-fold below those required for a peptide of random sequence. By itself, the GrpE cochaperonin has no effect on the peptide-independent ATPase activity of DnaK, but GrpE does vigorously stimulate the peptide-dependent ATPase of the DnaK chaperone. Under steady-state conditions, the Vmax of ATP hydrolysis by DnaK was elevated approximately 40-fold by the presence of GrpE and saturating levels of peptides.
Molecular chaperones of the Hsp70 class bind unfolded polypeptide chains and are thought to be involved in the cellular folding pathway of many proteins. DnaK, the Hsp70 protein of Escherichia coli, is regulated by the chaperone protein DnaJ and the cofactor GrpE. To gain a biologically relevant understanding of the mechanism of Hsp70 action, we have analyzed a model reaction in which DnaK, DnaJ, and GrpE mediate the folding of denatured firefly luciferase. The binding and release of substrate protein for folding involves the following ATP hydrolysis-dependent cycle: (i) unfolded luciferase binds initially to DnaJ; (ii) upon interaction with luciferase-DnaJ, DnaK hydrolyzes its bound ATP, resulting in the formation of a stable luciferase-DnaK-DnaJ complex; (iii) GrpE releases ADP from DnaK; and (iv) ATP binding to DnaK triggers the release of substrate protein, thus completing the reaction cycle. A single cycle of binding and release leads to folding of only a fraction of luciferase molecules. Several rounds of ATP-dependent interaction with DnaK and DnaJ are required for fully efficient folding.
Applying stopped-flow fluorescence spectroscopy for measuring conformational changes of the DnaK molecular chaperone (bacterial Hsp70 homologue) and its binding to target peptide, we found that after ATP hydrolysis, DnaK is converted to the DnaK*(ADP) conformation, which possesses limited affinity for peptide substrates and the GrpE cochaperone but efficiently binds the DnaJ chaperone. In the presence of DnaJ (bacterial Hsp40 homologue), the DnaK*(ADP) form is converted back to the DnaK conformation, and the resulting DnaJ-DnaK(ADP) complex binds to peptide substrates more tightly. Formation of the DnaJ(substrate-DnaK(ADP)) complex is a rate-limiting reaction. The presence of GrpE and ATP hydrolysis promotes the fast release of the peptide substrate from the chaperone complex and converts DnaK to the DnaK*(ADP) conformation. We conclude that in the presence of DnaJ and GrpE, the binding-release cycle of DnaK is stoichiometrically coupled to the adenosine triphosphatase activity of DnaK.
The DnaK and DnaJ heat shock proteins function as the primary Hsp70 and Hsp40 homologues, respectively, of Escherichia coli. Intensive studies of various Hsp70 and DnaJ-like proteins over the past decade have led to the suggestion that interactions between specific pairs of these two types of proteins permit them to serve as molecular chaperones in a diverse array of protein metabolic events, including protein folding, protein trafficking, and assembly and disassembly of multisubunit protein complexes. To further our understanding of the nature of Hsp70-DnaJ interactions, we have sought to define the minimal sequence elements of DnaJ required for stimulation of the intrinsic ATPase activity of DnaK. As judged by proteolysis sensitivity, DnaJ is composed of three separate regions, a 9-kDa NH2-terminal domain, a 30-kDa COOH-terminal domain, and a protease-sensitive glycine- and phenylalanine-rich (G/F-rich) segment of 30 amino acids that serves as a flexible linker between the two domains. The stable 9-kDa proteolytic fragment was identified as the highly conserved J-region found in all DnaJ homologues. Using this structural information as a guide, we constructed, expressed, purified, and characterized several mutant DnaJ proteins that contained either NH2-terminal or COOH-terminal deletions. At variance with current models of DnaJ action, DnaJ1-75, a polypeptide containing an intact J-region, was found to be incapable of stimulating ATP hydrolysis by DnaK protein. We found, instead, that two sequence elements of DnaJ, the J-region and the G/F-rich linker segment, are each required for activation of DnaK-mediated ATP hydrolysis and for minimal DnaJ function in the initiation of bacteriophage lambda DNA replication. Further analysis indicated that maximal activation of ATP hydrolysis by DnaK requires two independent but simultaneous protein-protein interactions: (i) interaction of DnaK with the J-region of DnaJ and (ii) binding of a peptide or polypeptide to the polypeptide-binding site associated with the COOH-terminal domain of DnaK. This dual signaling process required for activation of DnaK function has mechanistic implications for those protein metabolic events, such as polypeptide translocation into the endoplasmic reticulum in eukaryotic cells, that are dependent on interactions between Hsp70-like and DnaJ-like proteins.
We used depletion studies designed to further investigate the role of the DnaK, DnaJ, and GrpE heat shock proteins in the SecB-dependent and SecB-independent secretion pathways. Our previous finding that SecB-deficient strains containing the grpE280 mutation were still secretion proficient raised the possibility that GrpE was not involved in this secretory pathway. Using depletion studies, we now demonstrate a requirement for GrpE in this pathway. In addition, depletion studies demonstrate that while DnaK, DnaJ, and GrpE are involved in the secretion of the SecB-independent proteins (alkaline phosphatase, ribose-binding protein, and beta-lactamase), they are not the primary chaperones in this process.
DnaK and other members of the 70-kilodalton heat-shock protein (hsp70) family promote protein folding, interaction, and translocation,
both constitutively and in response to stress, by binding to unfolded polypeptide segments. These proteins have two functional
units: a substrate-binding portion binds the polypeptide, and an adenosine triphosphatase portion facilitates substrate exchange.
The crystal structure of a peptide complex with the substrate-binding unit of DnaK has now been determined at 2.0 Å resolution.
The structure consists of a β-sandwich subdomain followed by α-helical segments. The peptide is bound to DnaK in an extended
conformation through a channel defined by loops from the β sandwich. An α-helical domain stabilizes the complex, but does
not contact the peptide directly. This domain is rotated in the molecules of a second crystal lattice, which suggests a model
of conformation-dependent substrate binding that features a latch mechanism for maintaining long lifetime complexes.
We have previously shown that the molecular chaperone HSC70 self-associates in solution into dimers, trimers, and probably high order oligomers, according to a slow temperature- and concentration-dependent equilibrium that is shifted toward the monomer upon binding of ATP peptides or unfolded proteins. To determine the structural basis of HSC70 self-association, the oligomerization properties of the isolated amino- and carboxyl-terminal domains of this protein have been analyzed by gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Whereas the amino-terminal ATPase domain (residues 1-384) was found to be monomeric in solution even at high concentrations, the carboxyl-terminal peptide binding domain (residues 385-646) exists as a slow temperature- and concentration-dependent equilibrium involving monomers, dimers, and trimers. The association equilibrium constant obtained for this domain alone is on the order of 10(5) M-1, very close to that determined previously for the entire protein, suggesting that self-association of HSC70 is determined solely by its carboxyl-terminal domain. Furthermore, oligomerization of the isolated carboxyl-terminal peptide binding domain is, like that of the entire protein, reversed by peptide binding, indicating that self-association of the protein may be mediated by the peptide binding site and, as such, should play a role in the regulation of HSC70 chaperone function. A general model for self-association of HSP70 is proposed in which the protein is in equilibrium between two states differing by the conformation of their carboxyl-terminal domain and their self-association properties.
The crystal structure of the adenine nucleotide exchange factor GrpE in complex with the adenosine triphosphatase (ATPase) domain of Escherichia coli DnaK [heat shock protein 70 (Hsp70)] was determined at 2.8 angstrom resolution. A dimer of GrpE binds asymmetrically to a single molecule of DnaK. The structure of the nucleotide-free ATPase domain in complex with GrpE resembles closely that of the nucleotide-bound mammalian Hsp70 homolog, except for an outward rotation of one of the subdomains of the protein. This conformational change is not consistent with tight nucleotide binding. Two long alpha helices extend away from the GrpE dimer and suggest a role for GrpE in peptide release from DnaK.
We previously reported the cDNA cloning and characterization of a mammalian mitochondrial GrpE protein ( approximately 21 kDa, mt-GrpE#1) and now provide evidence for the presence of distinct cytosolic ( approximately 40 kDa), microsomal ( approximately 50 kDa), and additional mitochondrial ( approximately 22 kDa, mt-GrpE#2) GrpE-like members. While a cytosolic GrpE-like protein has recently been identified, the demonstration of both a microsomal and a second mitochondrial GrpE-like member represents the first in any biological system. Investigation of the microsomal and two mitochondrial GrpE-like proteins revealed that they bound specifically to Escherichia coli DnaK, and the complexes formed were not disrupted in the presence of 0.5 M salt but were readily dissociated in the presence of 5 mM ATP. The functional integrity of mt-GrpE#1 and #2 was verified by their ability to specifically interact with and stimulate the ATPase activity of mammalian mitochondrial Hsp70 (mt-Hsp70). Analysis of the cDNA sequences encoding the two mammalian mitochondrial GrpE-like proteins revealed approximately 47% positional identity at the amino acid level, the presence of a highly conserved mitochondrial leader sequence, and putative destabilization elements within the 3'-untranslated region of the mt-GrpE#2 transcript which are not present in the mt-GrpE#1 transcript. A constitutive expression of both mitochondrial GrpE-like transcripts in 22 distinct mouse tissues was observed but possible different post-transcriptional regulation of the mt-GrpE#1 and #2 transcripts may confer a different expression pattern of the encoded proteins.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
The crystal structure of the adenine nucleotide exchange factor GrpE in complex with the adenosine triphosphatase (ATPase)
domain ofEscherichia coli DnaK [heat shock protein 70 (Hsp70)] was determined at 2.8 angstrom resolution. A dimer of GrpE binds asymmetrically to a
single molecule of DnaK. The structure of the nucleotide-free ATPase domain in complex with GrpE resembles closely that of
the nucleotide-bound mammalian Hsp70 homolog, except for an outward rotation of one of the subdomains of the protein. This
conformational change is not consistent with tight nucleotide binding. Two long α helices extend away from the GrpE dimer
and suggest a role for GrpE in peptide release from DnaK.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
To probe the mechanism of chaperone substrate selection, we have investigated the kinetics of complex formation and dissociation between the molecular chaperone DnaK and a short peptide (Cro, representing amino acids 1-12 of the cro repressor protein). The Cro protein was N-terminally labeled with the environmentally sensitive fluorophore dansyl chloride (Cro*), and steady-state and stopped-flow fluorescence spectroscopies and fluorescence-detected high-performance size exclusion chromatography (HPSEC) were used to monitor complex formation and dissociation over a range of temperatures in the absence of ATP. The results are summarized as follows: (i) Cro* binds to DnaK with a second-order rate constant, k(on), which varies from 8 to 200 M-1 s-1 between 15 and 37 degrees C. The slow on-rate is a consequence of a large activation energy barrier. The activation enthalpy (delta H*) and the prefactor [omega exp delta S*/R)] are 26 kcal mol-1 and 7 x 10(20) M-1 s-1, respectively. (ii) Once formed, DnaK-Cro* complexes are long-lived, especially at low temperatures (T < 15 degrees C). The off-rate is unusually temperature-sensitive, for example, there is a 478-fold increase in k(off) from 2.3 x 10(6) to 1.1 x 10(-3) s-1 over a range of only 30 degrees C (5-35 degrees C). The steep temperature-dependence of the off-rate is a consequence of a very large activation energy barrier to DnaK-Cro* complex dissociation [delta H* = 34.6 kcal mol-1 and omega exp (delta S*/R) = 2 x 10(21) s-1]. The relatively low affinity of the Cro* peptide for DnaK is due to a large kinetic barrier to binding. We discuss possible causes for these large kinetic barriers.
The activity of DnaK (Hsp70) chaperones in assisting protein folding relies on DnaK binding and ATP-controlled release of protein substrates. The ATPase activity of DnaK is tightly controlled by the nucleotide exchange factor GrpE. We find that GrpE interacts stably with the amino-terminal ATPase domain of DnaK. Analysis of the mutant DnaK756 protein, which has a lower affinity for GrpE, reveals a role for residue Gly 32 in GrpE binding. Gly 32 is located in an exposed loop near the nucleotide binding site of DnaK. Deletion of this loop prevents stable GrpE binding, ATPase stimulation by GrpE, and DnaK chaperone activity. Conservation of this loop within the Hsp70 family suggests that cooperation between Hsp70 and GrpE-like proteins may be a general feature of this class of chaperone.
The folding of many newly synthesized proteins in the cell depends on a set of conserved proteins known as molecular chaperones. These prevent the formation of misfolded protein structures, both under normal conditions and when cells are exposed to stresses such as high temperature. Significant progress has been made in the understanding of the ATP-dependent mechanisms used by the Hsp70 and chaperonin families of molecular chaperones, which can cooperate to assist in folding new polypeptide chains.
GroEL and DnaK with their cofactors constitute the major chaperone systems promoting protein folding in the Escherichia coli cytosol. The ability of GroEL to bind and promote folding of a substrate released from DnaK led to the proposal that the DnaK and GroEL systems act successively along a protein folding pathway. Here we have investigated the role of both systems in preventing aggregation and assisting refolding of firefly luciferase denatured by guanidinium chloride and heat. We find that DnaK and GroEL compete with each other for binding to non-native luciferase. Addition of ATP and co-operating proteins results in release of luciferase from either chaperone in a non-native conformation. Only a small fraction of luciferase molecules released from GroEL can reach the native state. Instead, the released luciferase must bind repeatedly to the DnaK system, and only then is it able to fold to the native state. Thus, during a folding reaction, DnaK and GroEL do not obligatorily act in succession by promoting earlier and later protein folding steps, respectively. Rather, the two chaperone systems and perhaps others can form a lateral network of co-operating proteins. This chaperone network is proposed to be of particular importance for the assisted refolding of proteins that are unfolded by stress treatment such as heat shock and whose size is too large to allow folding inside the substrate binding cavity of the GroEL ring underneath GroES.
The folding and assembly of proteins in cells often requires the assistance of molecular chaperones such as the Hsp70 and Hsp60 heat shock proteins. Hsp70 chaperones cooperate with DnaJ and GrpE homologues to ensure a productive folding cycle. In this study we describe the gene for the first chloroplast localized DnaJ homologue and present evidence that the gene product is at least partially associated with the inner envelope membrane. Immunoblot analysis also provides evidence for the presence of a GrpE homologue in plastids.
The ATP hydrolysis and protein-binding and release cycle of the molecular chaperone DnaK is regulated by the accessory proteins GrpE and DnaJ. Here we describe a study of the formation of complexes between the molecular chaperone DnaK, its nucleotide exchange factor GrpE, and the fluorescent ADP analog N8-[4-[(N'-methylanthraniloyl)amino]butyl]-8-aminoadenosine 5'-diphosphate (MABA-ADP) by equilibrium and stopped flow kinetic experiments. The catalytic cycle of the GrpE-stimulated nucleotide exchange involves a ternary DnaK x GrpE x ADP complex as well as the binary DnaK x GrpE and DnaK x ADP complexes. The equilibrium data of the interaction of GrpE with DnaK x ADP and the nucleotide-free DnaK can be described by a simple equilibrium system where GrpE reduces the affinity of ADP for DnaK 200-fold. However, transient kinetic studies revealed that the functional cycle of GrpE in addition includes at least two distinct ternary DnaK x GrpE x ADP complexes. Our data indicate that the initial weak binding of GrpE to DnaK x ADP is followed by an isomerization of the ternary complex which leads to weakening of nucleotide binding and finally to its rapid dissociation. The maximal stimulation for nucleotide exchange brought about by GrpE was found to be 5000-fold. We propose that this kinetically observed isomerization represents a structural change (opening) of the nucleotide binding pocket of DnaK that allows for fast nucleotide exchange.
The molecular chaperone DnaK, the Hsp70 homolog of Escherichia coli, acts in concert with the co-chaperones DnaJ and GrpE. The aim of this study was to identify the particular phase of the peptide binding-release cycle of the DnaK/DnaJ/GrpE system that is directly responsible for the chaperone effects. By real-time kinetic measurements of changes in the intrinsic fluorescence of DnaK and in the fluorescence of dansyl-labeled peptide ligands, the rates of the following steps in the chaperone cycle were determined: (1) binding of target peptide to fast-binding-and-releasing, low-affinity DnaK ATP; (2) DnaJ-triggered conversion of peptide x DnaK x ATP (T state) to slowly-acting, high-affinity peptide x DnaK x ADP x P(i) (R state); (3) switch from R to T state induced by GrpE-facilitated ADP/ATP exchange; (4) release of peptide. Under conditions approximating those in the cell, the apparent rate constants for the T --> R and R --> T conversion were 0.04 s(-1) and 1.0 s, respectively. The clearly rate-limiting T --> R conversion renders the R state a minor form of DnaK that cannot account for the chaperone effects. Because DnaK in the absence of the co-chaperones is chaperone-ineffective, the T state has also to be excluded. Apparently, the slow, ATP-driven conformational change T --> R is the key step in the DnaK/DnaJ/GrpE chaperone cycle underlying the chaperone effects such as the prevention of protein aggregation, disentangling of polypeptide chains and, in the case of eukaryotic Hsp70 homologs, protein translocation through membranes or uncoating of clathrin-coated vesicles.
DnaK, the prototype Hsp70 protein of Escherichia coli, functions as a molecular chaperone in protein folding disassembly reactions through cycles of polypeptides binding and release that are coupled to its intrinsic ATPase activity. To further our understanding of these processes, we sought to obtain a quantitative description of the basic ATPase cycle of DnaK. To this end, we have performed steady-state and pre-steady-state kinetics experiments and have determined rate constants corresponding to individual steps in the DnaK ATPase cycle at 25 °C. Hydrolysis of ATP proceeds very slowly with a rate constant (k(hyd) ≃ 0.02 min-1) at least 10-fold smaller than the rate constant for any other first-order step in the forward reaction pathway. The ATP hydrolysis step has an activation energy of 26.2 ± 0.4 kcal/mol and is rate limiting in the steady-state under typical in vitro conditions. ATP binds with unusual strength to DnaK, with a measured K(D) ≃ 1 nM. ADP binds considerably less tightly than ATP). However, in the presence of physiologically relevant concentrations of inorganic phosphate (P(i)), the release of ADP from DnaK is greatly slowed, approximately to the rate of ATP hydrolysis. Under these conditions, the ADP-bound form of DnaK, the form that binds substrate polypeptidase most tightly, was found to represent a significant fraction of the DnaK population. The slowing of ADP release by exogenous P(i) is due to thermodynamic coupling of the binding of the two ligands, which produces a coupling energy of ~1.6 kcal/mol. This result implies that product release is not strictly ordered. In the absence of exogenous inorganic phosphate, P(i) product, by virtue of its higher k(off), is released prior to ADP. However, at physiological concentrations of inorganic phosphate, the alternate product release pathway, whereby ADP dissociates from a ternary DnaK·ADP·P(i) complex, become more prominent.
The solution structure of the 21 kDa substrate-binding domain of the Escherichia coli Hsp70-chaperone protein DnaK (DnaK 386-561) has been determined to a precision of 1.00 A (backbone of the beta-domain) from 1075 experimental restraints obtained from multinuclear, multidimensional NMR experiments. The domain is observed to bind to its own C-terminus and offers a preview of the interaction of this chaperone with other proteins. The bound protein region is tightly held at a single amino acid position (a leucyl residue) that is buried in a deep pocket lined with conserved hydrophobic residues. A second hydrophobic binding site was identified using paramagnetically labeled peptides. It is located in a region close to the N-terminus of the domain and may constitute the allosteric region that links substrate-binding affinity with nucleotide binding in the Hsp70 chaperones.
Heat-shock proteins DnaK, DnaJ, and GrpE (KJE) from Escherichia coli constitute a three-component chaperone system that prevents aggregation of denatured proteins and assists the refolding of proteins in an ATP-dependent manner. We found that the rate of KJE-mediated refolding of heat- and chemically denatured proteins is decreased at high temperatures. The efficiency and reversibility of protein-folding arrest during and after heat shock depended on the stability of the complex between KJE and the denatured proteins. Whereas a thermostable protein was released and partially refolded during heat shock, a thermolabile protein remained bound to the chaperone. The apparent affinity of GrpE and DnaJ for DnaK was decreased at high temperatures, thereby decreasing futile consumption of ATP during folding arrest. The coupling of ATP hydrolysis and protein folding was restored after the stress. This strongly indicates that KJE chaperones are heat-regulated heat-shock proteins which can specifically arrest the folding of aggregation-prone proteins during stress and preferentially resume refolding under conditions that allow individual proteins to reach and maintain a stable native conformation.
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.
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