Direct observation of chemo-mechanical coupling in DnaK by single-molecule force experiments

Protein allostery requires a communication channel for functional regulation between distal sites within a protein. In the molecular chaperone Hsp70, a two-domain enzyme, the ATP/ADP status of an N-terminal nucleotide-binding domain regulates the substrate affinity of a C-terminal substrate-binding domain. Recently available three-dimensional structures of Hsp70 in ATP/ADP states have provided deep insights into molecular pathways of allosteric signals. However, direct mechanical probing of long-range allosteric coupling between the ATP hydrolysis step and domain states is missing. Using laser optical tweezers, we examined the mechanical properties of a truncated two-domain DnaK(1–552ye) in apo/ADP/ATP- and peptide-bound states. We find that in the apo and ADP states, DnaK domains are mechanically stable and rigid. However, in the ATP state, substrate-binding domain (SBD)¹ye is mechanically destabilized as the result of interdomain docking followed by the unfolding of the α-helical lid. By observing the folding state of the SBD, we could observe the continuous ATP/ADP cycling of the enzyme in real time with a single molecule. The SBD lid closure is strictly coupled to the chemical steps of the ATP hydrolysis cycle even in the presence of peptide substrate.     

Allosteric regulation of Hsp70 chaperones involves a conserved interdomain linker

The 70-kDa heat shock proteins (Hsp70) are essential members of the cellular chaperone machinery that assists protein-folding processes. To perform their functions Hsp70 chaperones toggle between two nucleotide-controlled conformational states. ATP binding to the ATPase domain triggers the transition to the low affinity state of the substrate-binding domain, while substrate binding to the substrate-binding domain in synergism with the action of a J-domain-containing cochaperone stimulates ATP hydrolysis and thereby transition to the high affinity state. Thus, ATPase and substrate-binding domains mutually affect each other through an allosteric control mechanism, the basis of which is largely unknown. In this study we identified two positively charged, surface-exposed residues in the ATPase domain and a negatively charged residue in the linker connecting both domains that are important for interdomain communication. Furthermore, we demonstrate that the linker alone is sufficient to stimulate the ATPase activity, an ability that is lost upon amino acid replacement. The linker therefore is most likely the lever that is wielded by the substrate-binding domain and the cochaperone onto the ATPase domain to induce a conformation favorable for ATP hydrolysis. Based on our results we propose a mechanism of interdomain communication.     

Defining the structure of the substrate-free state of the DnaK molecular chaperone

Members of the Hsp70 (heat-shock protein of 70 kDa) family of molecular chaperones bind to exposed hydrophobic stretches on substrate proteins in order to dissociate molecular complexes and prevent aggregation in the cell. Substrate affinity for the C-terminal domain of the Hsp70 is regulated by ATP binding to the N-terminal domain utilizing an allosteric mechanism. Our multi-dimensional NMR studies of a substrate-binding domain fragment (amino acids 387-552) from an Escherichia coli Hsp70, DnaK(387-552), have uncovered a pH-dependent conformational change, which we propose to be relevant for the full-length protein also. At pH 7, the C-terminus of DnaK(387-552) mimics substrate by binding to its own substrate-binding site, as has been observed previously for truncated Hsp70 constructs. At pH 5, the C-terminus is released from the binding site, such that DnaK is in the substrate-free state 10-20% of the time. We propose that the mechanism for the release of the tail is a loss of affinity for substrate at low pH. The pH-dependent fluorescence changes at a tryptophan residue near the substrate-binding pocket in full-length DnaK lead us to extend these conclusions to the full-length DnaK as well. In the context of the DnaK substrate-binding domain fragment, the release of the C-terminus from the substrate-binding site provides our first glimpse of the empty conformation of an Hsp70 substrate-binding domain containing a portion of the helical subdomain.     

Conserved,Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

The 70-kDa heat shock proteins (Hsp70s) function as molecular chaperones through the allosteric coupling of their nucleotide- and substrate-binding domains, the structures of which are highly conserved. In contrast, the roles of the poorly structured, variable length C-terminal regions present on Hsp70s remain unclear. In many eukaryotic Hsp70s, the extreme C-terminal EEVD tetrapeptide sequence associates with co-chaperones via binding to tetratricopeptide repeat domains. It is not known whether this is the only function for this region in eukaryotic Hsp70s and what roles this region performs in Hsp70s that do not form complexes with tetratricopeptide repeat domains. We compared C-terminal sequences of 730 Hsp70 family members and identified a novel conservation pattern in a diverse subset of 165 bacterial and organellar Hsp70s. Mutation of conserved C-terminal sequence in DnaK, the predominant Hsp70 in Escherichia coli, results in significant impairment of its protein refolding activity in vitro without affecting interdomain allostery, interaction with co-chaperones DnaJ and GrpE, or the binding of a peptide substrate, defying classical explanations for the chaperoning mechanism of Hsp70. Moreover, mutation of specific conserved sites within the DnaK C terminus reduces the capacity of the cell to withstand stresses on protein folding caused by elevated temperature or the absence of other chaperones. These features of the C-terminal region support a model in which it acts as a disordered tether linked to a conserved, weak substrate-binding motif and that this enhances chaperone function by transiently interacting with folding clients.     

Crystal structure of DnaK protein complexed with nucleotide exchange factor GrpE in DnaK chaperone system: insight into intermolecular communication

The conserved, ATP-dependent bacterial DnaK chaperones process client substrates with the aid of the co-chaperones DnaJ and GrpE. However, in the absence of structural information, how these proteins communicate with each other cannot be fully delineated. For the study reported here, we solved the crystal structure of a full-length Geobacillus kaustophilus HTA426 GrpE homodimer in complex with a nearly full-length G. kaustophilus HTA426 DnaK that contains the interdomain linker (acting as a pseudo-substrate), and the N-terminal nucleotide-binding and C-terminal substrate-binding domains at 4.1-Å resolution. Each complex contains two DnaKs and two GrpEs, which is a stoichiometry that has not been found before. The long N-terminal GrpE α-helices stabilize the linker of DnaK in the complex. Furthermore, interactions between the DnaK substrate-binding domain and the N-terminal disordered region of GrpE may accelerate substrate release from DnaK. These findings provide molecular mechanisms for substrate binding, processing, and release during the Hsp70 chaperone cycle.     

Effect of evolution of the C-terminal region on chaperone activity of Hsp70

Dynamic interdomain interactions within the Hsp70 protein is the basis for the allosteric and functional properties of Hsp70s. While Hsp70s are generally conserved in terms of structure, allosteric behavior, and some overlapping functions, Hsp70s also contain variable sequence regions which are related to distinct functions. In the Hsp70 sequence, the part with the greatest sequence variation is the C-terminal α-helical lid subdomain of substrate-binding domain (SBDα) together with the intrinsically disordered region. Dynamic interactions between the SBDα and β-sandwich substrate-binding subdomain (SBDβ) contribute to the chaperone functions of Hsp70s by tuning kinetics of substrate binding. To investigate how the C-terminal region of Hsp70 has evolved from prokaryotic to eukaryotic organisms, we tested whether this region can be exchanged among different Hsp70 members to support basic chaperone functions. We found that this region from eukaryotic Hsp70 members cannot substitute for the same region in Escherichia coli DnaK to facilitate normal chaperone activity of DnaK. In contrast, this region from E. coli DnaK and Saccharomyces cerevisiae Hsp70 (Ssa1 and Ssa4) can partially support some roles of human stress inducible Hsp70 (hHsp70) and human cognate Hsp70 (hHsc70). Our results indicate that the C-terminal region from eukaryotic Hsp70 members cannot properly support SBDα–SBDβ interactions in DnaK, but this region from DnaK/Ssa1/Ssa4 can still form some SBDα–SBDβ interactions in hHsp70 or hHsc70, which suggests that the mode for SBDα–SBDβ interactions is different in prokaryotic and eukaryotic Hsp70 members. This study provides new insight in the divergency among different Hsp70 homologs and the evolution of Hsp70s.     

Mechanism of the targeting action of DnaJ in the DnaK molecular chaperone system

In the DnaK (Hsp70) molecular chaperone system of Escherichia coli, the substrate polypeptide is fed into the chaperone cycle by association with the fast-binding, ATP-liganded form of the DnaK. The substrate binding properties of DnaK are controlled by its two cochaperones DnaJ (Hsp40) and GrpE. DnaJ stimulates the hydrolysis of DnaK-bound ATP, and GrpE accelerates ADP/ATP exchange. DnaJ has been described as targeting the substrate to DnaK, a concept that has remained rather obscure. Based on binding experiments with peptides and polypeptides we propose here a novel mechanism for the targeting action of DnaJ: ATP.DnaK and DnaJ with its substrate-binding domain bind to different segments of one and the same polypeptide chain forming (ATP.DnaK)m.substrate.DnaJn complexes; in these ternary complexes efficient cis-interaction of the J-domain of DnaJ with DnaK is favored by their propinquity and triggers the hydrolysis of DnaK-bound ATP, converting DnaK to its ADP-liganded high affinity state and thus locking it onto the substrate polypeptide.     

ATPase Domain and Interdomain Linker Play a Key Role in Aggregation of Mitochondrial Hsp70 Chaperone Ssc1

The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Δhep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer.     

Amide hydrogen exchange reveals conformational changes in hsp70 chaperones important for allosteric regulation

Hsp70 chaperones assist protein folding processes by a nucleotide-driven cycle of substrate binding and release. Although structural information is available for the isolated nucleotide-binding (NBD) and substrate-binding domains (SBD) in the high affinity conformation, the low affinity conformations and the conformational changes associated with mutual allosteric regulation remained largely enigmatic. By using amide hydrogen exchange in combination with mass spectrometry, we analyzed the Escherichia coli Hsp70 homologue DnaK as full-length protein and its individual domains in the nucleotide-free and ATP-bound conformation. We found a surprising degree of flexibility in both domains. The comparison of the full-length protein with the isolated domains demonstrates a mutual stabilization of both domains. This protection from solvent was most pronounced and in addition was nucleotide-dependent in the lowerbeta-sheet of the SBD and the loop that connects the last beta-strand with helix alphaA. Interestingly, the linker region, which connects NBD and SBD and which is close to the protected loop in the SBD, is solvent-exposed in the absence of nucleotide and completely protected from hydrogen exchange in the presence of ATP. Peptide binding to DnaK.ATP reverts the ATP-induced conformational changes in the linker and selected parts of the NBD. Our data outline a pathway for allosteric interdomain control and suggest an important role of the linker and the base of helix alphaA.     

Functional defects of the DnaK756 mutant chaperone of Escherichia coli indicate distinct roles for amino- and carboxyl-terminal residues in substrate and co-chaperone interaction and interdomain communication

The first discovery of an Hsp70 chaperone gene was the isolation of an Escherichia coli mutant, dnaK756, which rendered the cells resistant to lytic infection with bacteriophage lambda. The DnaK756 mutant protein has since been used to establish many of the cellular roles and biochemical properties of DnaK. DnaK756 has three glycine-to-aspartate substitutions at residues 32, 455, and 468, which were reported to result in defects in intrinsic and GrpE-stimulated ATPase activities, substrate binding, stability of the substrate-binding domain, interdomain communication, and, consequently, defects in chaperone activity. To dissect the effects of the different amino acid substitutions in DnaK756, we analyzed two DnaK variants carrying only the amino-terminal (residue 32) or the two carboxyl-terminal (residues 455 and 468) substitutions. The amino-terminal substitution interfered with the GrpE-stimulated ATPase activity. The carboxyl-terminal mutations (i) affected stability and function of the substrate-binding domain, (ii) caused a 10-fold elevated ATP hydrolysis rate, but (iii) did not severely affect domain coupling. Surprisingly, DnaK chaperone activity was more severely compromised by the amino-terminal than by the carboxyl-terminal amino acid substitutions both in vivo and in vitro. In the in vitro refolding of denatured firefly luciferase, the defect of the DnaK variant carrying the amino-terminal substitution results from its inability to release, upon GrpE-mediated nucleotide exchange, bound luciferase in a folding competent state. Our results indicate that the DnaK-DnaJ-GrpE chaperone system can tolerate suboptimal substrate binding, whereas the tight kinetic control of substrate dissociation by GrpE is essential.     

The allosteric transition in DnaK probed by infrared difference spectroscopy. Concerted ATP-induced rearrangement of the substrate binding domain

The biological activity of DnaK, the bacterial representative of the Hsp70 protein family, is regulated by the allosteric interaction between its nucleotide and peptide substrate binding domains. Despite the importance of the nucleotide-induced cycling of DnaK between substrate-accepting and releasing states, the heterotropic allosteric mechanism remains as yet undefined. To further characterize this mechanism, the nucleotide-induced absorbance changes in the vibrational spectrum of wild-type DnaK was characterized. To assign the conformation sensitive absorption bands, two deletion mutants (one lacking the C-terminal alpha-helical subdomain and another comprising only the N-terminal ATPase domain), and a single-point DnaK mutant (T199A) with strongly reduced ATPase activity, were investigated by time-resolved infrared difference spectroscopy combined with the use of caged-nucleotides. The results indicate that (1) ATP, but not ADP, binding promotes a conformational change in both subdomains of the peptide binding domain that can be individually resolved; (2) these conformational changes are kinetically coupled, most likely to ensure a decrease in the affinity of DnaK for peptide substrates and a concomitant displacement of the lid away from the peptide binding site that would promote efficient diffusion of the released peptide to the medium; and (3) the alpha-helical subdomain contributes to stabilize the interdomain interface against the thermal challenge and allows bidirectional transmission of the allosteric signal between the ATPase and substrate binding domains at stress temperatures (42 degrees C).     

Binding specificity of an alpha-helical protein sequence to a full-length Hsp70 chaperone and its minimal substrate-binding domain

Hsp70 chaperones are involved in the prevention of misfolding, and possibly the folding, of newly synthesized proteins. The members of this chaperone family are capable of interacting with polypeptide chains both co- and posttranslationally, but it is currently not clear how different structural domains of the chaperone affect binding specificity. We explored the interactions between the bacterial Hsp70, DnaK, and the sequence of a model all-alpha-helical globin (apoMb) by cellulose-bound peptide scanning. The binding specificity of the full-length chaperone was compared with that of its minimal substrate-binding domain, DnaK-beta. Six specific chaperone binding sites evenly distributed along the apoMb sequence were identified. Binding site locations are identical for the full-length chaperone and its substrate-binding domain, but relative affinities differ. The binding specificity of DnaK-beta is only slightly decreased relative to that of full-length DnaK. DnaK's binding motif is known to comprise hydrophobic regions flanked by positively charged residues. We found that the simple fractional mean buried area correlates well with Hsp70's binding site locations along the apoMb sequence. In order to further characterize the properties of the minimal binding host, the stability of DnaK-beta upon chemical denaturation by urea and protons was investigated. Urea unfolding titrations yielded an apparent folding DeltaG degrees of 3.1 +/- 0.9 kcal mol-1 and an m value of 1.7 +/- 0.4 kcal mol-1 M-1.     

Direct comparison of a stable isolated Hsp70 substrate-binding domain in the empty and substrate-bound states

The Hsp70 family of molecular chaperones acts to prevent protein misfolding, import proteins into organelles, unravel protein aggregates, and enhance cell survival under stress conditions. These activities are all mediated by recognition of diverse hydrophobic sequences via a C-terminal substrate-binding domain. ATP-binding/hydrolysis by the N-terminal ATPase domain regulates the interconversion of the substrate-binding domain between low and high affinity conformations. The empty state of the substrate-binding domain has been difficult to study because of its propensity to bind nearly any available protein chain, even if only modestly hydrophobic. We have generated a new stable construct of the substrate-binding domain from the Escherichia coli Hsp70, DnaK, which has enabled us to compare the empty and peptide-bound conformations using NMR chemical shift analysis and hydrogen-deuterium exchange. We have determined that the empty state is, overall, quite similar to the peptide-bound state, contrary to a previous report. Peptide binding leads to a subtle alteration in the packing of the alpha-helical lid relative to the beta-subdomain. Significantly, we have shown that the chemical shifts of the substrate-binding domain and the ATPase domain do not change when they are placed together in a two-domain construct, whether or not peptide is bound, suggesting that, in the absence of nucleotide, the two domains of E. coli DnaK do not interact. We conclude that the isolated substrate-binding domain exists in a stable high affinity state in the absence of influence from a nucleotide-bound ATPase domain.     

Investigation of the interaction between DnaK and DnaJ by surface plasmon resonance spectroscopy.

Hsp70 chaperones assist protein folding through ATP-regulated transient association with substrates. Substrate binding by Hsp70 is controlled by DnaJ co-chaperones which stimulate Hsp70 to hydrolyze ATP and, consequently, to close its substrate binding cavity allowing trapping of substrates. We analyzed the interaction of the Escherichia coli Hsp70 homologue, DnaK, with DnaJ using surface plasmon resonance (SPR) spectroscopy. Resonance signals of complex kinetic characteristics were detected when DnaK was passed over a sensor chip with coupled DnaJ. This interaction was specific as it was not detected with a functionally defective DnaJ mutant protein, DnaJ259, that carries a mutation in the HPD signature motif of the conserved J-domain. Detectable DnaK-DnaJ interaction required ATP hydrolysis by DnaK and was competitively inhibited by chaperone substrates of DnaK. For DnaK mutant proteins with amino acid substitutions in the substrate binding cavity that affect substrate binding, the strength of detected interaction with DnaJ decreased proportionally with increased strength of the substrate binding defects. These findings indicate that the detected response signals resulted from DnaJ and ATP hydrolysis-dependent association of DnaJ as substrate for DnaK. Although not considered as physiologically relevant, this association allowed us to experimentally unravel the mechanism of DnaJ action. Accordingly, DnaJ stimulates ATP hydrolysis only after association of a substrate with the substrate binding cavity of DnaK. Further analysis revealed that this coupling mechanism required the J-domain of DnaJ and was also functional for natural DnaK substrates, and thus is central to the mechanism of action of the DnaK chaperone system.     

Structural features required for the interaction of the Hsp70 molecular chaperone DnaK with its cochaperone DnaJ.

Hsp70 family members together with their Hsp40 cochaperones function as molecular chaperones, using an ATP-controlled cycle of polypeptide binding and release to mediate protein folding. Hsp40 plays a key role in the chaperone reaction by stimulating the ATPase activity and activating the substrate binding of Hsp70. We have explored the interaction between the Escherichia coli Hsp70 family member, DnaK, and its cochaperone partner DnaJ. Our data show that the binding of ATP, subsequent conformational changes in DnaK, and DnaJ-stimulated ATP hydrolysis are all required for the formation of a DnaK-DnaJ complex as monitored by Biacore analysis. In addition, our data imply that the interaction of the J-domain with DnaK depends on the substrate binding state of DnaK.     

Structural basis of interdomain communication in the Hsc70 chaperone

Hsp70 family proteins are highly conserved chaperones involved in protein folding, degradation, targeting and translocation, and protein complex remodeling. They are comprised of an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate binding domain (SBD). ATP binding to the NBD alters SBD conformation and substrate binding kinetics, but an understanding of the mechanism of interdomain communication has been hampered by the lack of a crystal structure of an intact chaperone. We report here the 2.6 angstroms structure of a functionally intact bovine Hsc70 (bHsc70) and a mutational analysis of the observed interdomain interface and the immediately adjacent interdomain linker. This analysis identifies interdomain interactions critical for chaperone function and supports an allosteric mechanism in which the interdomain linker invades and disrupts the interdomain interface when ATP binds.     

The nucleotide‐bound/substrate‐bound conformation of the Mycoplasma genitalium DnaK chaperone

Hsp70 chaperones keep protein homeostasis facilitating the response of organisms to changes in external and internal conditions. Hsp70s have two domains—nucleotide binding domain (NBD) and substrate binding domain (SBD)—connected by a conserved hydrophobic linker. Functioning of Hsp70s depend on tightly regulated cycles of ATP hydrolysis allosterically coupled, often together with cochaperones, to the binding/release of peptide substrates. Here we describe the crystal structure of the Mycoplasma genitalium DnaK (MgDnaK) protein, an Hsp70 homolog, in the noncompact, nucleotide‐bound/substrate‐bound conformation. The MgDnaK structure resembles the one from the thermophilic eubacteria DnaK trapped in the same state. However, in MgDnaK the NBD and SBD domains remain close to each other despite the lack of direct interaction between them and with the linker contacting the two subdomains of SBD. These observations suggest that the structures might represent an intermediate of the protein where the conserved linker binds to the SBD to favor the noncompact state of the protein by stabilizing the SBDβ‐SBDα subdomains interaction, promoting the capacity of the protein to sample different conformations, which is critical for proper functioning of the molecular chaperone allosteric mechanism. Comparison of the solved structures indicates that the NBD remains essentially invariant in presence or absence of nucleotide.     

Modulation of the Chaperone DnaK Allosterism by the Nucleotide Exchange Factor GrpE

Hsp70 chaperones comprise two domains, the nucleotide-binding domain (Hsp70_NBD), responsible for structural and functional changes in the chaperone, and the substrate-binding domain (Hsp70_SBD), involved in substrate interaction. Substrate binding and release in Hsp70 is controlled by the nucleotide state of DnaK_NBD, with ATP inducing the open, substrate-receptive DnaK_SBD conformation, whereas ADP forces its closure. DnaK cycles between the two conformations through interaction with two cofactors, the Hsp40 co-chaperones (DnaJ in Escherichia coli) induce the ADP state, and the nucleotide exchange factors (GrpE in E. coli) induce the ATP state. X-ray crystallography showed that the GrpE dimer is a nucleotide exchange factor that works by interaction of one of its monomers with DnaK_NBD. DnaK_SBD location in this complex is debated; there is evidence that it interacts with the GrpE N-terminal disordered region, far from DnaK_NBD. Although we confirmed this interaction using biochemical and biophysical techniques, our EM-based three-dimensional reconstruction of the DnaK-GrpE complex located DnaK_SBD near DnaK_NBD. 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 DnaK_SBD, whereas the DnaK_NBD-DnaK_SBD linker contacts the GrpE helical region. We suggest that these interactions define a more complex role for GrpE in the control of DnaK function.     

The solution structure of the bacterial HSP70 chaperone protein domain DnaK(393-507) in complex with the peptide NRLLLTG

The Hsp70 family of molecular chaperones participates in a number of cellular processes, including binding to nascent polypeptide chains and assistance in protein (re)folding and degradation. We present the solution structure of the substrate binding domain (residues 393-507) of the Escherichia coli Hsp70, DnaK, that is bound to the peptide NRLLLTG and compare it to the crystal structure of DnaK(389-607) bound to the same peptide. The construct discussed here does not contain the alpha-helical domain that characterizes earlier published peptide-bound structures of the Hsp70s. It is established that removing the alpha-helical domain in its entirety does not affect the primary interactions or structure of the DnaK(393-507) in complex with the peptide NRLLLTG. In particular, the arch that protects the substrate-binding cleft is also formed in the absence of the helical lid. 15N-relaxation measurements show that the peptide-bound form of DnaK(393-507) is relatively rigid. As compared to the peptide-free state, the peptide-bound state of the domain shows distinct, widespread, and contiguous differences in structure extending toward areas previously defined as important to the allosteric regulation of the Hsp70 chaperones.     

Kinetic analysis of interdomain coupling in a lidless variant of the molecular chaperone DnaK: DnaK's lid inhibits transition to the low affinity state

DnaK, the Escherichia coli Hsp70, possesses two functional domains, the N- and C-terminal ATPase and peptide-binding domains, respectively. Elucidation of the mechanism of allosteric coupling between the two domains is key to understanding how Hsp70 chaperones interact with their substrates. We previously reported that ATP reacts with wild-type DnaK-peptide complexes according to the two-step reaction, ATP + DnaK-P if ATP-DnaK-P if ATP-DnaK + P, where ATP binds in the first step, and a conformational change that quenches DnaK's tryptophan fluorescence (denoted by the asterisk) and expels bound peptide occurs in the second step. Here we report that DnaK(2-517), a lidless variant, also reacts with ATP and peptide by this two-step mechanism. Compared to wild-type DnaK, we found that, depending on the sequence of the bound peptide and the temperature, deletion of the lid produces a 27- to 66-fold increase in the rate constant (k(2)) for the ATP-triggered conformational change (ATP-DnaK-P --> ATP-DnaK+P) but only a approximately 2-fold increase in the rate constant (k(-)(2)) for the reverse reaction (ATP-DnaK+P --> ATP-DnaK-P). A model is proposed in which the lid regulates the rate of interdomain communication by retarding motions within the beta-sandwich that occur as a consequence of ATP binding. New evidence in support of the reversible, two-step conformational switch mechanism is also presented.