Regulation of ATPase and chaperone cycle of DnaK from Thermus thermophilusby the nucleotide exchange factor GrpE

The nucleotide binding and release cycle of the molecular chaperone DnaK is regulated by the accessory proteins GrpE and DnaJ, also called co-chaperon es. The concerted action of the nucleotide exchange factor GrpE and the ATP ase-stimulating factor DnaJ determines the ratio of the two nucleotide stat es of DnaK, which differ in their mode of interaction with unfolded protein s. In the Escherichia coli system, the stimulation by these two antagonists is comparable in magnitude, resulting in a balance of the two nucleotide s tates of DnaK(Eco) in the absence and the presence of co-chaperones. The regulation of the DnaK chaperone system from Thermus thermophilus is ap parently substantially different. Here, DnaJ does not stimulate the DnaK-me diated ATP hydrolysis and thus does not appear to act as an antagonist of t he nucleotide exchange factor GrpE(Tth). This raises the question of whethe r T. thermophilus GrpE stimulates nucleotide exchange to a smaller degree a s compared to the E. coli system and how the corresponding rates relate to intrinsic ATPase and ATP binding as well as luciferase refolding kinetics o f T. thermophilus DnaK. We determined dissociation constants as well as kinetic constants that desc ribe the interactions between the T. thermophilus molecular chaperone DnaK, its nucleotide exchange factor GrpE and the fluorescent ADP analogue N-8-( 4-N-methylanthraniloylaminobutyl)-8-aminoadenosine-5'-diphosphate by isothe rmal equilibrium titration calorimetry and stopped-flow kinetic experiments and investigated the influence of T. thermophilus DnaJ on the DnaK nucleot ide cycle. The interaction of GrpE with the DnaK ADP complex versus nucleotide-free Dn aK can be described by a simple equilibrium system, where GrpE reduces the affinity of DnaK for ADP by a factor of about 10. Kinetic experiments indic ate that the maximal acceleration of nucleotide release by GrpE is 80,000-f old at a saturating GrpE concentration. Our experiments show that in T. thermophilus, although the thermophilic Dna K system displays no stimulation of the DnaK-ATPase activity by DnaJ, nucle otide exchange is still efficiently stimulated by GrpE. This indicates that two counteracting factors are not absolutely necessary to maintain a funct ional and regulated chaperone cycle. This conclusion is corroborated by dat a that show that the slower ATPase cycle of the DnaK system as well as of h eterologous T. thermophilus DnaK/E. coli DnaK systems is directly reflected in altered refolding kinetics of firefly luciferase but not necessarily in refolding yields. (C) 2001 Academic Press.