Functional properties of the molecular chaperone DnaK from Thermus thermophilus

Abstract

The genes coding for the Thermus thermophilus (Tth) homologues of the molecular chaperones DnaK and GrpE (DnaK Tthand 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 Tthhas an intrinsic ATPase activity of 3×10 −4s −1at 25°C and 10×10 −4 s −1at 75°C under single turnover conditions. It binds the fluorescent nucleotide analogue N 8-(4- N′-methylanthraniloylaminobutyl)-8-aminoadenosine 5′-diphosphate (MABA-ADP) with a dissociation constant ( K d) of 3 nM and ADP with a K dof 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 Tthwere determined at 25°C and 75°C, respectively. GrpE Tthacts as a nucleotide-exchange factor on DnaK Tthand 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 Tthand GrpE Tthis mediating nucleotide exchange. A fluorescently labelled peptide that comprises a subsequence of the E. coli transcription factor σ 32binds to nucleotide-free DnaK Tthwith a K dof 4.9 μM. Displacement with unlabelled peptide yields a K dof 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 Tthin 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 Tthand changes in the σ 32peptide 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.