The functional cycle and regulation of the Thermus thermophilus DnaK chaperone system

The Escherichia coli DnaK (DnaK(Eco)) chaperone cycle is tightly regulated by the cochaperones DnaJ, which stimulates ATP hydrolysis, and GrpE, which acts as a nucleotide exchange factor. The Thermus thermophilus DnaK (DnaK(T th)) system additionally comprises the DnaK-DnaJ assembly factor (DafA(Tth) ) that is mediating formation of a 300 kDa DnaK(Tth).DnaJ(Tth).DafA(Tth) co mplex. A model peptide derived from the tumor suppressor protein p53 was used to d issect the regulation of the individual kinetic key steps of the DnaK(Tth) nucleotide/chaperone cycle. As with DnaK(Eco) the DnaK(Tth). ATP complex bi nds substrates with reduced affinity and large exchange rates compared to t he DnaK(Tth).ADP.P-i state. In contrast to DnaK(Eco), ADP-P-i release is sl ow compared to the rate of hydrolysis, reversing the balance of the two fun ctional nucleotide states. Whereas GrpE(Tth) stimulates nucleotide release from DnaK(Tth), DnaJ(Tth) does not accelerate ATP hydrolysis under various experimental conditions. However, it exerts influence on the interaction of DnaK(Tth) with substrates: in the presence of DafA(Tth), DnaJ(Tth) inhibit s substrate binding, and substrate already bound to DnaK(Tth) is displaced by DnaJ(Tth) and DafA(Tth), indicating competitive binding of DnaJ(Tth)/Daf A(Tth) and substrate. It thus appears that the DnaK(Tth).DnaJ(Tth).DafA(Tth ) complex as isolated from T. thermophilus does not represent the active sp ecies in the DnaK(Tth) chaperone cycle. Isothermal titration calorimetry sh owed that the ternary complex of DnaK(Tth), DnaJ(Tth) and DafA(Tth) is asse mbling with high affinity, whereas binary complexes of DnaK(Tth) and DnaJ(T th) or DafA(Tth) were not detectable, indicating highly synergistic formati on of the 300 kDa DnaK(Tth). DnaJ(Tth).DafA(Tth) complex. Based on these results, a model describing the DnaK(Tth) chaperone cycle an d its regulation by cochaperones is proposed where DnaK(Tth).DnaJ(Tth).DafA (Tth) constitutes the resting state, and a DnaK(Tth).substrate.DnaJ(Tth) co mplex is the active chaperone species. The novel factor DafA,, that mediate s interaction of DnaK(Tth) with DnaJ(Tth) would thus serve as a "template" to stabilise the ternary DnaK(Tth).DafA(Tth).DnaJ(Tth) complex until it is replaced by substrate proteins under heat shock conditions. (C) 1999 Academ ic Press.