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.     

GrpE, a nucleotide exchange factor for DnaK

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 N-terminal domain contributes to the interaction with Dnak and modulates the dynamics of the chaperone substrate binding domain

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 β-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 β-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.     

Importance of the D and E helices of the molecular chaperone DnaK for ATP binding and substrate release

The C-terminal domain of the molecular chaperone DnaK is a compact lid-like structure made up of five alpha-helices (alphaA-alphaE) (residues 508-608) that is followed by a 30-residue disordered, flexible region (609-638). The lid encapsulates the peptide molecule bound in the substrate-binding domain, whereas the function of the 30-residue disordered region is not known. By sequentially deleting the flexible subdomain and the individual lid helices, we deduced the importance of each structural unit to creating long-lived DnaK-peptide complexes. Here we report that (i) the alphaD helix is essential for long-lived DnaK-peptide complexes. For example, ATP triggers the dissociation of a acrylodan-labeled p5 peptide (ap5, a-CLLLSAPRR) from wtDnaK and DnaK595(A-D) with k(off) equal to 7.6 and 8.9 s(-1), respectively, whereas when the D-helix is deleted, creating DnaK578(A-C), k(off) jumps to 207 s(-1). (ii) The presence of the alphaB helix impacts the rate of the ATP-induced high-to-low affinity conformational change. For example, ATP induces this conformational change in a lidless variant, DnaK517(1/2A), with a rate constant of 442 s(-1), whereas, after adding back the B-helix (residues 518-554), ATP induces this conformational change in DnaK554(A-B) with a rate constant of 2.5 s(-1). Our interpretation is that this large decrease occurs because the B-helix of the DnaK554(A-B) is bound in the substrate-binding site. (iii) The deletion analysis also revealed that residues 596-638, which comprise the alphaE helix and the flexible subdomain, affect ATP binding. Our results are consistent with this part of the lid producing conformational heterogeneity, perhaps by binding to the ATPase domain.     

The heat-sensitive Escherichia coli grpE280 phenotype: impaired interaction of GrpE(G122D) with DnaK

GrpE is the nucleotide-exchange factor of the DnaK chaperone system. Escherichia coli cells with the classical temperature-sensitive grpE280 phenotype do not grow under heat-shock conditions and have been found to carry the G122D point mutation in GrpE. To date, the molecular mechanism of this defect has not been investigated in detail. Here, we examined the structural and functional properties of isolated GrpE(G122D) in vitro. Similar to wild-type GrpE, GrpE(G122D) is an elongated dimer in solution. Compared to wild-type GrpE, GrpE(G122D) catalyzed the ADP/ATP exchange in DnaK only marginally and did not compete with wild-type GrpE in interacting with DnaK. In the presence of ADP, GrpE(G122D) in contrast to wild-type GrpE, did not form a complex with DnaK detectable by size-exclusion chromatography with on-line static light-scattering and differential refractometry. Apparently, GrpE(G122D) in the presence of ADP binds to DnaK only with much lower affinity than wild-type GrpE. GrpE(G122D) could not substitute for wild-type GrpE in the refolding of denatured proteins by the DnaK/DnaJ/GrpE chaperone system. In the crystal structure of a (Delta1-33)GrpE(G122D).DnaK-ATPase complex, which as yet is the only available structure of a GrpE variant, Asp122 does not interact directly with neighboring residues of GrpE or DnaK. The far-UV circular dichroism spectra of mutant and wild-type GrpE proved slightly different. Possibly, a discrete change in conformation impairs the formation of the complex with DnaK and renders GrpE(G122D) virtually inactive as a nucleotide exchange factor. In view of the drastically reduced ADP/ATP-exchange activity of GrpE(G122D), the heat sensitivity of grpE280 cells might be explained by the ensuing slowing of the chaperone cycle and the increased sequestering of target proteins by high-affinity, ADP-liganded DnaK, both effects being incompatible with efficient chaperone action required for cell growth.     

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.     

A GrpE mutant containing the NH(2)-terminal "tail" region is able to displace bound polypeptide substrate from DnaK

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.     

Formation in vitro of complexes between an abnormal fusion protein and the heat shock proteins from Escherichia coli and yeast mitochondria.

Heat shock proteins (HSPs) of the Hsp70 and GroEL families associate with a variety of cell proteins in vivo. However, the formation of such complexes has not been systematically studied. A 31-kDa fusion protein (CRAG), which contains 12 residues of cro repressor, truncated protein A, and 14 residues of beta-galactosidase, when expressed in Escherichia coli, was found in complexes with DnaK, GrpE, protease La, and GroEL. When an E. coli extract not containing CRAG was applied to an affinity column containing CRAG, DnaK, GroEL, and GrpE were selectively bound. These HSPs did not bind to a normal protein A column. DnaK, GrpE, and the fraction of GroEL could be eluted from the CRAG column with ATP but not with a nonhydrolyzable ATP analog. The ATP-dependent release of DnaK and GroEL also required Mg2+, but GrpE dissociated with ATP alone. The binding and release of DnaK and GroEL were independent events, but the binding of GrpE required DnaK. Inactivation of DnaJ, GrpE, and GroES did not affect the association or dissociation of DnaK or GroEL from CRAG. The DnaK and GrpE proteins could be eluted with 10(-6) M ATP, but 10(-4) M was required for GroEL release. This approach allows a one-step purification of these proteins from E. coli and also the isolation of the DnaK and GroEL homologs from yeast mitochondria. Competition experiments with oligopeptide fragments of CRAG showed that DnaK and GroEL interact with different sites on CRAG and that the cro-derived domain of CRAG contains the DnaK-binding site.     

Physical interactions between members of the DnaK chaperone machinery: characterization of the DnaK.GrpE complex

Many of the functions of the Escherichia coli Hsp 70, DnaK, require two cofactors, DnaJ and GrpE. GrpE acts as a nucleotide exchange factor in the DnaK reaction cycle but the details of its mechanism remain unclear. GrpE has high affinity for monomeric native DnaK, with a K_d estimated at ≤50 nM. GrpE is a very asymmetric molecule and exists as either a dimer or trimer in its native state. The stoichiometry of GrpE to DnaK in the isolated complex was 3:1, suggesting a trimer. Formation of the complex is quite fast (k_on >1 S⁻¹, whereas the off-rate is very slow on the HPLC timescale (k_off ≤ 10⁻⁴ S⁻¹). GrpE has no affinity for ATP or ADP, nor the oligomeric and moltn globule states of DnaK. The complex is much more thermally stable than either GrpE or DnaK alone, and prevents the formation of the molten globule-like state of DnaK at physiologically relevant temperatures. Formation of the complex does not cause any change in secondary structure, as determined by the lack of change in the circular dichroism spectrum. However, binding of GrpE induces a similar tertiary strcutral change in DnaK to that induced by binding of ATP₁ based on the blue shift in λ_max from the fluroscence of the single tryptophan in DnaK. The nucleotide exchange properties of GrpE can be explained by the conformational change which may represent the opening of the nucleotide cleft on DnaK, subsequently inducing a low affinity state for ADP.     

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

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

Structure-function analyses of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli.

The DnaK, DnaJ, and GrpE proteins of Escherichia coli have been universally conserved across the biological kingdoms and work together to constitute a highly efficient molecular chaperone machine. We have examined the extent of functional conservation of Saccharomyces cerevisiae Ssc1p, Mdj1p, and Mge1p by analyzing their ability to substitute for their corresponding E. coli homologs in vivo. We found that the expression of yeast Mge1p, the GrpE homolog, allowed for the deletion of the otherwise essential grpE gene of E. coli, albeit only up to 40 degrees C. The inability of Mge1p to substitute for GrpE at very high temperatures is consistent with our previous finding that it specifically failed to stimulate DnaK's ATPase at such extreme conditions. In contrast to Mge1p, overexpression of Mdj1p, the DnaJ homolog, was lethal in E. coli. This toxicity was specifically relieved by mutations which affected the putative zinc binding region of Mdj1p. Overexpression of a truncated version of Mdj1p, containing the J- and Gly/Phe-rich domains, partially substituted for DnaJ function at high temperature. A chimeric protein, consisting of the J domain of Mdj1p coupled to the rest of DnaJ, acted as a super-DnaJ protein, functioning even more efficiently than wild-type DnaJ. In contrast to the results with Mge1p and Mdj1p, both the expression and function of Ssc1p, the DnaK homolog, were severely compromised in E. coli. We were unable to demonstrate any functional complementation by Ssc1p, even when coexpressed with its Mdj1p cochaperone in E. coli.     

Balance of ATPase stimulation and nucleotide exchange is not required for efficient refolding activity of the DnaK chaperone

The DnaK system from Thermus thermophilus (DnaK(Tth)) exhibits pronounced differences in organisation and regulation to its mesophile counterpart from Escherichia coli (DnaK(Eco)). While the ATPase cycle of DnaK(Eco) is tightly regulated by the concerted action of the two cofactors DnaJ(Eco) and GrpE(Eco), the DnaK(Tth) system features an imbalance in this cochaperone mediated regulation. GrpE(Tth) considerably accelerates the ATP/ADP exchange, but DnaJ(Tth) only slightly stimulates ATPase activity, believed to be a key step for chaperone activity of DnaK(Eco). By in vitro complementation assays, we could not detect significant ATPase-stimulation of orthologous DnaJ(Tth) . DnaKEco or DnaJ(Eco). DnaK(Tth)-complexes as compared to the DnaK(Eco) system, although they were nevertheless active in luciferase refolding experiments. Assistance of protein recovery by DnaK thus seems to be uncoupled of the magnitude of DnaJ mediated ATPase-stimulation.     

Mutational analysis of the energetics of the GrpE.DnaK binding interface: equilibrium association constants by sedimentation velocity analytical ultracentrifugation

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.     

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 insect antimicrobial peptide, L-pyrrhocoricin, binds to and stimulates the ATPase activity of both wild-type and lidless DnaK

Recent reports have indicated that insect antimicrobial peptides kill bacteria by inhibiting the molecular chaperone DnaK. It was proposed that the antimicrobial peptide, all-L-pyrrhocoricin (L-PYR), binds to two sites on DnaK, the conventional substrate-binding site and the multi-helical C-terminal lid, and that inhibition of DnaK comes about from the lid mode of binding. In this report, we show using two different assays that L-PYR binds to and stimulates the ATPase activity of both wild-type and a lidless variant of DnaK. Our study shows that L-PYR interacts with DnaK much like the all-L NR (NRLLLTG) peptide, which is known to bind in the conventional substrate-binding site of DnaK. L-PYR antimicrobial activity is thus a consequence of the competitive inhibition of bacterial DnaK.     

Characterization of a lidless form of the molecular chaperone DnaK: deletion of the lid increases peptide on- and off-rate constants

The C-terminal, polypeptide binding domain of the 70-kDa molecular chaperone DnaK is composed of a unique lidlike subdomain that appears to hinder steric access to the peptide binding site. We have expressed, purified, and characterized a lidless form of DnaK to test the influence of the lid on the ATPase activity, on interdomain communication, and on the kinetics of peptide binding. The principal findings are that loss of the lid creates an activated form of DnaK which is not equivalent to ATP-bound DnaK. For example, at 25 degrees C the NR peptide (NRLLLTG) dissociates from the ADP and ATP states of DnaK with observed off-rate constants of 0.001 and 4.8 s(-1), respectively. In contrast, for DnaK that lacks most of the helical lid, residues 518-638, the NR peptide dissociates with observed off-rate constants of 0.1 and 188 s(-1). These results show that the loss of the lid does not interfere with interdomain communication, that the beta-sandwich peptide binding domain can exist in two discrete conformations, and that the lid functions to increase the lifetime of a DnaK.peptide complex. We discuss several mechanisms to explain how the lid affects the lifetime of a DnaK.peptide complex.     

Interdomain interaction through helices A and B of DnaK peptide binding domain

In order to better define the structural elements involved in allosteric signalling, wild-type DnaK and three deletion mutants of the peptide binding domain have been characterized by biophysical (steady-state and time-resolved fluorescence) and biochemical methods. In the presence of ATP the chemical environment of the single tryptophan residue of DnaK, located in the ATPase domain, becomes less polar, as seen by a blue shift of the emission maximum and a shortening of the fluorescence lifetime, and its accessibility to polar quenchers is drastically reduced. These nucleotide-dependent modifications are also observed for the deletion mutant DnaK1-537, but not for DnaK1-507 or DnaK1-385, and thus rely on the presence of residues 507–537 (helices A and the N-terminal half of B) of the peptide binding domain. These data indicate that αA and half αB contribute to the allosteric communication of DnaK. In the presence of ATP, they promote a conformational change that displaces a residue(s) of the peptide binding domain towards a region of the ATPase domain where the tryptophan residue (W102) is located. A putative role for these helical segments as regulators of the position of the lid is discussed.     

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.     

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.