Functional properties of the molecular chaperone DnaK from Thermus thermophilus

Klostermeier, D., Seidel, R., Reinstein, J.

Zusammenfassung

The genes coding for the Thermus thermophilus (Tth) homologues of the molecular chaperones DnaK and GrpE (DnaK(Tth) and GrpE(Tth)) were cloned and expressed in Escherichia coli. The proteins were purified and their functional properties were assessed by equilibrium and transient kinetic methods. DnaK(Tth) has an intrinsic ATPase activity of 3 x 10-4 s-1 at 25°C and 10 x 10-4 s-1 at 75°C under single turnover conditions. It binds the fluorescent nucleotide analogue N8-(4-N'-methylanthraniloylaminobutyl)-8-aminoadenosine 5'-diphosphate (MABA-ADP) with a dissociation constant (K(d)) of 3 nM and ADP with a K(d) of 47 nM at 25°C. At 75°C the affinities are decreased fivefold to 15 nM (MABA-ADP) and 280 nM (ADP). The kinetic constants for two-step binding of MABA-ADP and of ADP to DnaK(Tth) were determined at 25°C and 75°C, respectively. GrpE(Tth) acts as a nucleotide-exchange factor on DnaK(Tth) and accelerates the release of bound MABA-ADP significantly. This shows that the nucleotide-binding domain is functionally intact, and that the specific interaction of DnaK(Tth) and GrpE(Tth) is mediating nucleotide exchange. A fluorescently labelled peptide that comprises a subsequence of the E. coli transcription factor σ32 binds to nucleotide-free DnaK(Tth) with a K(d) of 4.9 μM. Displacement with unlabelled peptide yields a K(d) of 5.0 μM for the unlabelled peptide. Thus the peptide-binding domain also appears to be functional. For the cellular chaperone function of DnaK, a coupling between nucleotide and peptide-binding domains is required. However, with DnaK(Tth) in the ATP as well as in the ADP.P(i)-state, peptide is bound and released within seconds. No correlation between ATP-binding or hydrolysis by DnaK(Tth) and changes in the σ32 peptide exchange rates could be detected. It thus appears that the DnaK system from Th. thermophilus has a different mechanism of coupling the nucleotide state to the fast and slow peptide exchange properties.