Control of the DnaK chaperone cycle by substoichiometric concentrations of the co-chaperones DnaJ and GrpE

Abstract

The polypeptide binding and release cycle of the molecular chaperone DnaK (Hsp70) of Escherichia coli is regulated by the two co-chaperones DnaJ and GrpE. Here, we show that the DnaJ-triggered conversion of DnaK·ATP (T state) to DnaK·ADP·P(i) (R state), as monitored by intrinsic protein fluorescence, is monophasic and occurs simultaneously with ATP hydrolysis. This is in contrast with the T→R conversion in the absence of DnaJ which is biphasic, the first phase occurring simultaneously with the hydrolysis of ATP (Theyssen, H., Schuster, H.-P., Packschies, L., Bukau, B., and Reinstein, J. (1996) J. Mol. Biol. 263, 657-670). Apparently, DnaJ not only stimulates ATP hydrolysis but also couples it with conformational changes of DnaK. In the absence of GrpE, DnaJ forms a tight ternary complex with peptide·DnaK·ADP·P(i) (K(d) = 0.14 μM). However, by monitoring complex formation between DnaK (1 μM) and a fluorophore-labeled peptide in the presence of ATP (1 mM), DnaJ (1 μM), and varying concentrations of the ADP/ATP exchange factor GrpE (0.1-3 μM), substoichiometric concentrations of GrpE were found to shift the equilibrium from the slowly binding and releasing, high-affinity R state of DnaK completely to the fast binding and releasing, low-affinity T state and thus to prevent the formation of a long lived ternary DnaJ-substrate·DnaK·ADP·P(i) complex. Under in vivo conditions with an estimated chaperone ratio of DnaK:DnaJ: GrpE = 10:1:3, beth DnaJ and GrpE appear to control the chaperone cycle by transient interactions with DnaK.