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.

The proteins DnaK (hsp70) and GroEL (cpn60) from Escherichia coli are prototypes of two classes of molecular chaperones conserved throughout evolution. The analysis of transferred nuclear Overhauser effects in two-dimensional NMR spectra is ideally suited to determine chaperone-bound conformations of peptides. The peptide vsv-C (amino-acid sequence KLIGVLSSLFRPK) stimulates the ATPase of BiP and Hsc70 (ref. 3) and the intrinsic ATPase of DnaK. The affinity of the vsv-C peptide for DnaK is greatly reduced in the presence of ATP. Here we analyse transferred nuclear Overhauser effects and show that the peptide is in an extended conformation while bound to DnaK but is helical when bound to GroEL. NMR also indicates that the mobility of the peptide backbone is reduced more by binding to DnaK than by binding to GroEL, whereas the side chains are less mobile when bound to GroEL.

The Escherichia coli dnaK gene product, originally defined by mutations that blocked A phage DNA replication, is known to be necessary for E. coli viability. We have purified dnaK protein to homogeneity and have demonstrated that it possesses a weak DNA-independent ATPase activity, which results in the production of ADP and Pi. The proof that this ATPase activity is encoded by the dnaK+ gene relies primarily on the fact that the dnaK756 mutation results in the production of an ATPase activity with altered physical properties. The dnaK protein is phosphorylated in vitro and in vivo, probably as a result of an autophosphorylation reaction. The A 0 and P replication proteins were shown to interact in vitro with the dnaK protein. The ATPase activity of the dnaK protein was inhibited by purified A P protein and stimulated by purified A 0 protein. Moreover, the dnaK protein participates in the initiation of DNA synthesis in an in vitro DNA replication system that is dependent on the 0 and P proteins. Anti-dnaK protein immunoglobulin specifically inhibited DNA synthesis in this system.The study of host-virus interactions has been greatly facilitated through the isolation of host mutants that block viral growth. Through the use of indirect or direct selections, various investigators have uncovered five Escherichia coli genes whose products are necessary for bacteriophage A DNA replication: dnaB, dnaK, dnaJ, grpD, and grpE (1-4). Of these genes, only dnaB has been convincingly shown to be essential for E. coli DNA replication (5). Both the dnaK+ and dnaJ+ gene products are essential for bacterial growth as well, because some mutations in these genes block colony formation at 42°C. Both DNA and RNA bacterial metabolism are affected in dnaK-(4, 6) and dnaJ-mutants (7,8). The effect on RNA metabolism is unique and not shared by mutations in the rest of the dna genes of E. coli (5). Mutations in A phage that enable it to grow on dnaKhosts map in the P gene, which suggests an interaction between the host dnaK protein and the phage P protein (1, 2). The dnaK gene has been cloned into A phage (2), and its gene product has been identified by NaDodSO4/polyacrylamide gel electrophoresis (9) and shown to be identical to protein B66.0 (10), one of the heat shock proteins of E. coli (11)(12)(13). In this communication, we report that the dnaK+ protein (i) possesses a weak DNA-independent ATPase activity that is modulated in vitro by the A 0 and P proteins; (ii) is phosphorylated both in vitro and in vivo, probably as a result of an autophosphorylation reaction; and (iii) is active in an in vitro replication system (unpublished results) that is dependent on the A 0 and P proteins. METHODS AND MATERIALSStrains. The bacteria, bacteriophage, and plasmid strains used in this work have been described (2,9,10,14,15).Purification of dnaK' Protein. The details of the purification will be published elsewhere. Briefly, 12 g of wet paste of B178(pMOB45dnaK+) bacteria was lysed according to Shlomai and Kornberg (16). The supernatant was ...

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Central to the chaperone function of Hsp70 stress proteins including Escherichia coli DnaK is the ability of Hsp70 to bind unfolded protein substrates in an ATP‐dependent manner. Mg2+/ATP dissociates bound substrates and, furthermore, substrate binding stimulates the ATPase of Hsp70. This coupling is proposed to require a glutamate residue, E175 of bovine Hsc70, that is entirely conserved within the Hsp70 family, as it contacts bound Mg2+/ATP and is part of a hinge required for a postulated ATP‐dependent opening/closing movement of the nucleotide binding cleft which then triggers substrate release. We analyzed the effects of dnaK mutations which alter the corresponding glutamate‐171 of DnaK to alanine, leucine or lysine. In vivo, the mutated dnaK alleles failed to complement the delta dnaK52 mutation and were dominant negative in dnaK+ cells. In vitro, all three mutant DnaK proteins were inactive in known DnaK‐dependent reactions, including refolding of denatured luciferase and initiation of lambda DNA replication. The mutant proteins retained ATPase activity, as well as the capacity to bind peptide substrates. The intrinsic ATPase activities of the mutant proteins, however, did exhibit increased Km and Vmax values. More importantly, these mutant proteins showed no stimulation of ATPase activity by substrates and no substrate dissociation by Mg2+/ATP. Thus, glutamate‐171 is required for coupling of ATPase activity with substrate binding, and this coupling is essential for the chaperone function of DnaK.