A Bipartite Signaling Mechanism Involved in DnaJ-mediated Activation of the Escherichia coli DnaK Protein

Abstract

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 Roles of the Two Zinc Binding Sites in DnaJ

“…Adjacent to the J-domain resides the glycine-and phenylalanine-rich G/F domain (residues 77-107). Together with the J-domain, the G/F domain is essential for maximal stimulation of the ATPase activity of DnaK (7). The central domain of DnaJ is the zinc-binding cysteine-rich CR-domain (residues 144 -200), which is followed by a poorly conserved C-terminal CTDdomain (8).…”

“…Interaction of the DnaJsubstrate protein complex with the DnaK-ATP-substrate protein complex maximally stimulates ATP hydrolysis. This is thought to cause conformational changes in DnaK that increase DnaK affinity for unfolded substrate proteins and that causes the lock-in of substrate proteins to the substrate binding site of DnaK (7,(12)(13)(14)(15).…”

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

“…The single turnover assay used in this study was conducted in a HEPES buffer with 150 mM potassium salt; [a-32 P] ATP was separated from [a-32 P] ADP via PEI-cellulose thin layer chromatography. This assay is widely used to measure the rate of ATP hydrolysis by DnaK [14,25,26]. In contrast, the spectrophotometric assay used in a previous study, which detected the release of inorganic phosphate, was conducted in a TRIS buffer without an essential potassium salt [20].…”

“…The single turnover ATPase assay was performed as described by Karzai and McMacken [14]. Briefly, the reaction mixture (30 ll) contained 2.5 lM DnaK or DnaK(2-517), 51 nM ATP (0.05 lCi of [a-32 P]-ATP; 3000 Ci/mmol) in a HEPES buffer (25 mM HEPES/KOH, 10 mM MgCl 2 , and 150 mM KCl, pH 7.6).…”

The Hsp70 chaperone system maintains high concentrations of active proteins and suppresses ATP consumption during heat shock

“…1A) (Schmid et al 1994;Gisler et al 1998;Mayer et al 2000b). Spontaneous transition between the two states is extremely slow but is stimulated synergistically by substrates and DnaJ (Karzai and McMacken 1996;Laufen et al 1999). Under physiological conditions of high ATP concentration, nucleotide exchange is rate limiting for substrate release.…”