DnaJ Dramatically Stimulates ATP Hydrolysis by DnaK: Insight into Targeting of Hsp70 Proteins to Polypeptide Substrates
- 1 March 1999
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 38 (13), 4165-4176
- https://doi.org/10.1021/bi9824036
Abstract
Most, if not all, of the cellular functions of Hsp70 proteins require the assistance of a DnaJ homologue, which accelerates the weak intrinsic ATPase activity of Hsp70 and serves as a specificity factor by binding and targeting specific polypeptide substrates for Hsp70 action. We have used pre-steady-state kinetics to investigate the interaction of the Escherichia coli DnaJ and DnaK proteins, and the effects of DnaJ on the ATPase reaction of DnaK. DnaJ accelerates hydrolysis of ATP by DnaK to such an extent that ATP binding by DnaK becomes rate-limiting for hydrolysis. At high concentrations of DnaK under single-turnover conditions, the rate-limiting step is a first-order process, apparently a change of DnaK conformation, that accompanies ATP binding and proceeds at 12−15 min-1 at 25 °C and 1−1.5 min-1 at 5 °C. By prebinding ATP to DnaK and subsequently adding DnaJ, the effects of this slow step may be bypassed, and the maximal rate-enhancement of DnaJ on the hydrolysis step is ∼15 000-fold at 5 °C. The interaction of DnaJ with DnaK·ATP is likely a rapid equilibrium relative to ATP hydrolysis, and is relatively weak, with a KD of ∼20 μM at 5 °C, and weaker still at 25 °C. In the presence of saturating DnaJ, the maximal rate of ATP hydrolysis by DnaK is similar to previously reported rates for peptide release from DnaK·ATP. This suggests that when DnaK encounters a DnaJ-bound polypeptide or protein complex, a significant fraction of such events result in ATP hydrolysis by DnaK and concomitant capture of the polypeptide substrate in a tight complex with DnaK·ADP. Furthermore, a broadly applicable kinetic mechanism for DnaJ-mediated specificity of Hsp70 action arises from these observations, in which the specificity arises largely from the acceleration of the hydrolysis step itself, rather than by DnaJ-dependent modulation of the affinity of Hsp70 for substrate polypeptides.Keywords
This publication has 20 references indexed in Scilit:
- Catapult mechanism renders the chaperone action of hsp70 unidirectionalJournal of Molecular Biology, 1998
- Control of the DnaK Chaperone Cycle by Substoichiometric Concentrations of the Co-chaperones DnaJ and GrpEPublished by Elsevier BV ,1998
- The power stroke of the DnaK/DnaJ/GrpE molecular chaperone system 1 1Edited by J.KarnJournal of Molecular Biology, 1997
- The Second Step of ATP Binding to DnaK Induces Peptide ReleaseJournal of Molecular Biology, 1996
- A Bipartite Signaling Mechanism Involved in DnaJ-mediated Activation of the Escherichia coli DnaK ProteinPublished by Elsevier BV ,1996
- The Role of ATP in the Functional Cycle of the DnaK Chaperone SystemJournal of Molecular Biology, 1995
- Regulation of 70-kDa Heat-Shock-Protein ATPase Activity and Substrate Binding by Human DnaJ-Like Proteins, HSJ1a and HSJ1bEuropean Journal of Biochemistry, 1994
- Thermodynamic and Structural Analysis of the Folding/Unfolding Transitions of the Escherichia coli Molecular Chaperone DnaKJournal of Molecular Biology, 1993
- The translation machinery and 70 kd heat shock protein cooperate in protein synthesisCell, 1992
- Uncoating ATPase is a member of the 70 kilodalton family of stress proteinsCell, 1986