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: ATP⅐DnaK and DnaJ with its substrate-binding domain bind to different segments of one and the same polypeptide chain forming (ATP⅐DnaK) m ⅐substrate⅐DnaJ n 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. Hsp70 1 chaperones assist a variety of protein folding processes in the cell, including folding of nascent polypeptide chains, rescue of misfolded proteins, translocation of polypeptide chains through membranes, assembly and disassembly of protein complexes, and control of the biological activity of folded regulatory proteins (for a review, see Ref. 1). The chaperone action of DnaK, an Hsp70 homolog in Escherichia coli, is driven by the hydrolysis of ATP. DnaJ, an Hsp40 homolog, triggers the hydrolysis of DnaK-bound ATP and thus converts DnaK from the ATP-liganded low affinity T state to its ADPliganded high affinity R state (2-6). The stimulation of the ATPase activity requires the conserved J-domain of DnaJ (residues 2-78 in E. coli DnaJ; Refs. 2, 4, and 7) and the adjacent G/F region (2). DnaJ itself is also capable of associating with unfolded substrates and preventing aggregation (8 -11), qualifying DnaJ as a chaperone on its own. Binding of substrates requires the zinc finger-like region and the COOH-terminal region of DnaJ (12). Only full-length DnaJ, comprising both the J-domain and the substrate-binding domain, is effective, together with DnaK and GrpE, in refolding denatured firefly luciferase (12, 13). Based on these findings, it has been proposed that substrates first associate with DnaJ, which then transfers them to the substrate-binding site of Hsp70 (6,10,11,14). The function of DnaJ has also been assumed to include a "catalytic activation" of DnaK for trapping target sequences (4, 15). A recent study has shown that DnaJ shares most binding motifs with DnaK, which also seems to qualify DnaJ as a targeting partner for the chaperone DnaK (16).However, no direct experimental evidence has been reported for substrate transfer from DnaJ to DnaK, and the concept of a targeting action of DnaJ has as yet remained obscure (16). The catalytic activation of DnaK by DnaJ (4, 15) has not remained undisputed (17). Recent work (6) has shown that the stimulatory effect of Dn...

Hsp70s assist the folding of proteins in an ATPdependent manner. DnaK, the Hsp70 of Escherichia coli, acts in concert with its co-chaperones DnaJ and GrpE. Amino acid substitutions (D388R and L391S/L392G) in the linker region between the ATPase and substrate-binding domain did not affect the functional domain coupling and oligomerization of DnaK. The intrinsic ATPase activity was enhanced up to 10-fold. However, the ATPase activity of DnaK L391S/L392G, if stimulated by DnaJ plus protein substrate, was five times lower than that of wild-type DnaK and DnaK D388R. This defect correlated with the complete loss of chaperone action in luciferase refolding. Apparently, the conserved leucine residues in the linker mediate the synergistic effects of DnaJ and protein substrate on ATPase activity, a function which might be essential for chaperone action. ß

Chimeric peptides, comprising a DnaK-binding sequence of L-amino acid residues (motif k) and an exclusive DnaJ-binding sequence of D-amino acid residues (motif j) connected through a 22-residue linker, were examined as minisubstrates for the DnaK chaperone system. The DnaJ-stimulated ATPase activity of DnaK was three times higher in the presence of the chimeric peptides pjk or pkj than in the simultaneous presence of the corresponding single-motif peptides ala-p5 (k motif) plus D-p5 (j motif). Apparently, pjk and pkj mimic unfolded proteins by forming ternary (ATPW WDnaK)W WpeptideW WDnaJ complexes which favor cis-interaction of DnaJ with DnaK. Consistent with this interpretation, the speci¢c stimulatory e¡ect of the chimeric peptides was abolished by either single-motif peptide in excess. ß 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

DnaK, a heat-shock protein 70 (Hsp70) homologue in Escherichia coli, possesses a single tryptophan residue in its ATPase domain. Changes in the intrinsic fluorescence of DnaK offer a simple means not only to follow the binding of ATP and of ADP plus the co-chaperone GrpE to the ATPase domain, but also to investigate the kinetics of peptide binding to the substrate-binding domain of ATP.DnaK and GrpE-liganded ADP.DnaK. Addition of ATP or of ADP plus GrpE to nucleotide-free DnaK resulted in a similar decrease in intrinsic fluorescence, indicating similar open conformations of the ATPase domain under these two conditions. Binding of peptide increased the intrinsic fluorescence of both ATP.DnaK and ADP.DnaK.GrpE and rendered their spectra similar to the spectrum of ADP.DnaK with closed conformation of the ATPase domain. These results, together with the differential kinetics of peptide binding to ADP.DnaK on the one hand, and to ATP.DnaK or ADP.DnaK.GrpE on the other, suggest that ligands for either domain, i.e. ATP or ADP plus GrpE for the ATPase domain and peptides for the substrate-binding domain, shift the conformational equilibrium of both domains of DnaK towards the open and closed forms, respectively, in a concerted and parallel manner.

DnaK, a Hsp70 homolog of Escherichia coli, together with its co-chaperones DnaJ and GrpE protects denatured proteins from aggregation and promotes their refolding by an ATP-consuming mechanism. DnaJ not only stimulates the ␥-phosphate cleavage of DnaKbound ATP but also binds polypeptide substrates on its own. Unfolded polypeptides, such as denatured luciferase, thus form ternary complexes with DnaJ and DnaK. A previous study has shown that D-peptides compete with L-peptides for the same binding site in DnaJ but do not bind to DnaK (Feifel, B., Schö nfeld, H.-J., and Christen, P. (1998) J. Biol. Chem. 273, 11999 -12002). Here we report that D-peptides efficiently inhibit the refolding of denatured luciferase by the DnaK/DnaJ/GrpE chaperone system (EC 50 ‫؍‬ 1-2 M). The inhibition of the chaperone action is due to the binding of D-peptide to DnaJ (K d ‫؍‬ 1-2 M), which seems to preclude DnaJ from forming ternary (ATP⅐DnaK) m ⅐substrate⅐DnaJ n complexes. Apparently, simultaneous binding of DnaJ and DnaK to one and the same target polypeptide is essential for effective chaperone action. The Hsp701 chaperone system of Escherichia coli includes DnaK and the two cohort proteins: DnaJ, a Hsp40 homolog, and GrpE. The chaperones assist protein folding by preventing and reversing off-pathway interactions that lead to aggregation (1). The key features of the Hsp70 chaperone system are the binding of unfolded hydrophobic segments of the target polypeptides to the ATP-liganded form of DnaK, the stabilization of the complex upon ATP hydrolysis, and the release of the bound ligands upon ADP/ATP exchange (1-3). This binding/ release cycle is controlled by DnaJ and the nucleotide exchange factor GrpE (4, 5). DnaJ interacts with DnaK through its highly conserved NH 2 -terminal J-domain and stimulates the hydrolysis of DnaK-bound ATP (2, 6). DnaJ also exerts a chaperone action on its own; upon association with denatured polypeptides, such as luciferase or rhodanese, it may prevent their aggregation (3, 6). Recently, it has been shown that D-peptides bind to DnaJ but not to DnaK (7,8). D-Peptides bind to the same site of DnaJ as L-peptides (7). Here we report that two retro-all D-peptides derived from the NH 2 -terminal segment of rhodanese inhibit the DnaK/DnaJ/GrpE chaperone system in refolding denatured firefly luciferase. EXPERIMENTAL PROCEDURESProteins-DnaK was isolated from an overproducing strain of E. coli (JM 83) bearing the plasmid pTPG9 (3). The stock solution of the protein in assay buffer (25 mM Hepes/NaOH, 100 mM KCl, pH 7.0) was stored at Ϫ80°C and contained less than 0.1 mol of ADP/mol of DnaK (9). The concentration of DnaK was determined photometrically with ⑀ 280 ϭ 14.6 mM Ϫ1 cm Ϫ1 . DnaJ and GrpE were prepared as described (10); stock solutions were stored at Ϫ80°C in 50 mM Tris/HCl, 100 mM NaCl at pH 7.7.Peptides-The peptide ala-p5 (ALLLSAPRR) was purchased with a purity of Ͼ90% from Chiron. The peptide was dissolved in 0.1% (v/v) acetic acid, 10% (v/v) acetonitrile and stored at Ϫ20°C. The two Dpeptides RI1-17 (...