Modulation of the ATPase Activity of the Molecular Chaperone DnaK by Peptides and the DnaJ and GrpE Heat Shock Proteins

Abstract

Previous studies have demonstrated that the Escherichia coli DnaK, DnaJ, and GrpE heat shock proteins participate in the initiation of bacteriophage lambda DNA replication by mediating the required disassembly of a preinitiation nucleoprotein structure that is formed at the phage replication origin. To gain some understanding in a simpler system of how the DnaJ and GrpE cochaperonins influence the activity of DnaK, we have examined the effect of the cochaperonins on the weak intrinsic ATPase activity of the molecular chaperone DnaK in the presence and absence of peptide effectors. We have found that random sequence peptide chains of 8 or 9 amino acid residues in length yield optimal (10-fold) activation of the DnaK ATPase, whereas peptides with 5 or fewer residues fail to stimulate the ATPase of this bacterial hsp70 homologue. Furthermore, we have discovered that those peptides that interact best with DnaK, as judged by their KA as activators of ATP hydrolysis by DnaK, also act as strong inhibitors of lambda DNA replication in vitro. The inhibitory effect of peptides on lambda DNA replication was overcome by increasing the concentration of DnaK in the replication system. Diminished inhibition was also found when the replication system was supplemented with GrpE cochaperonin, a protein known to increase the effectiveness of DnaK action in lambda DNA replication. These and other results suggest that the peptide-binding site of DnaK is required for its function in lambda DNA replication. Apparently, peptides sequester free DnaK protein and block lambda DNA replication by reducing the amount of DnaK that is free to mediate disassembly of nucleoprotein preinitiation structures. In related studies, we have found that DnaJ, like short peptides, activates the intrinsic ATPase activity of DnaK. DnaJ, however, is substantially more potent in this regard, since it activates DnaK at concentrations 1000-fold below those required for a peptide of random sequence. By itself, the GrpE cochaperonin has no effect on the peptide-independent ATPase activity of DnaK, but GrpE does vigorously stimulate the peptide-dependent ATPase of the DnaK chaperone. Under steady-state conditions, the Vmax of ATP hydrolysis by DnaK was elevated approximately 40-fold by the presence of GrpE and saturating levels of peptides.

Hsc66 and Hsc20, a new heat shock cognate molecular chaperone system from Escherichia coli

“…The maximal increase observed, -4-fold at 25"C, is less than the maximal stimulation of DnaK intrinsic ATPase activity by DnaJ which is -10-fold (Jordan & McMacken, 1995;McCarty et al, 1995). Because of the higher intrinsic activity of Hsc66 compared to DnaK, however, the maximal Hsc20-stimulated ATPase activity of Hsc66 (-0.5 min-I at 25°C) is in the same range as the maximal Dnd-stimulated ATPase activity of DnaK.…”

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

“…The ATPase activity is regulated by the co-chaperone protein DnaJ [7,8]. The DnaJ proteins stimulate the low basal ATP hydrolysis activity of partner Hsp70s/DnaKs [6,9] and in doing so enhance the Hsp70s/Dnaks substrate-binding activity and hence their chaperone activity.…”

Nucleotide utilization requirements that render ClpB active as a chaperone

“…DnaJ, GrpE were obtained as previously reported [21,22]. Protein concentration was determined by the colorimetric Bradford assay (Bio-Rad), except for DnaK, that was determined spectrophotometrically using e 280 = 15.8 Â 10 3 M À1 cm À1 [23]. Malate dehydrogenase (MDH) was purchased from Sigma and lactate dehydrogenase (LDH) and pyruvate kinase (PK) from Roche.…”