Folding properties of the nucleotide exchange factor GrpE from Thermus thermophilus: GrpE is a thermosensor that mediates heat shock response.

Hsp70 proteins like DnaK bind unfolded polypeptides in a nucleotide-dependent manner. The switch from high-affinity ADP-state to low- affinity ATP-state with concomitant substrate release is accelerated significantly by GrpE proteins. GrpE thus fulfils an important role in regulation of the chaperone cycle. Here, we analysed the thermal stability of GrpE from Thermus thermophilus using differential scanning calorimetry and CD-spectroscopy. The protein exhibits unusual unfolding characteristics with two observable thermal transitions. The first transition is CD-spectroscopically silent with a transition midpoint at 90 degrees C. The second transition, mainly constituting the CD-signal, ranges between 100 and 105 degrees C depending on the GrpE(Tth) concentration, according to the model N(2) <==> I(2) <==> 2U. Using a C-terminally truncated version of GrpE(Tth) it was possible to assign the second thermal transition to the dimerisation of GrpE(Tth), while the first transition represents the completely reversible unfolding of the globular C-terminal domain. The unfolding of this domain is accompanied by a distinct decrease in nucleotide exchange rates and impaired binding to DnaK(Tth). Under heat shock conditions, the DnaK-ADP-protein-substrate complex is thus stabilised by a reversibly inactivated GrpE-protein that refolds under permissive conditions. In combination with studies on GrpE from Escherichia coli presented recently by Christen and co-workers, it thus appears that the general role of GrpE is to function as a thermosensor that modulates nucleotide exchange rates in a temperature-dependent manner to prevent substrate dissociation at non-permissive conditions.

The chaperone function of ClpB from Thermus thermophilus depends on allosteric interactions of its two ATP-binding sites.

ClpB belongs to the Hsp100 family and assists de-aggregation of protein aggregates by DnaK chaperone systems. It contains two Walker consensus sequences (or P-Loops) that indicate potential nucleotide binding domains (NBD). Both domains appear to be essential for chaperoning function, since mutation of the conserved lysine residue of the GX(4)GKT consensus sequences to glutamine (K204Q and K601Q) abolishes its properties to accelerate renaturation of aggregated firefly luciferase. The underlying biochemical reason for this malfunction appears not to be a dramatically reduced ATPase activity of either P-loop per se but rather changed properties of co-operativity of ATPase activity connected to oligomerization properties to form productive oligomers. This view is corroborated by data that show that structural stability (as judged by CD spectroscopy) or ATPase activity at single turnover conditions (at low ATP concentrations) are not significantly affected by these mutations. In addition nucleotide binding properties of wild-type protein and mutants (as judged by binding studies with fluorescent nucleotide analogues and competitive displacement titrations) do not differ dramatically. However, the general pattern of formation of stable, defined oligomers formed as a function of salt concentration and nucleotides and more importantly, cooperativity of ATPase activity at high ATP concentrations is dramatically changed with the two P-loop mutants described.

Regulation of ATPase and chaperone cycle of DnaK from Thermus thermophilus by 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-chaperones. The concerted action of the nucleotide exchange factor GrpE and the ATPase-stimulating factor DnaJ determines the ratio of the two nucleotide states of DnaK, which differ in their mode of interaction with unfolded proteins. In the Escherichia coli system, the stimulation by these two antagonists is comparable in magnitude, resulting in a balance of the two nucleotide states of DnaK(Eco) in the absence and the presence of co-chaperones. The regulation of the DnaK chaperone system from Thermus thermophilus is apparently substantially different. Here, DnaJ does not stimulate the DnaK-mediated ATP hydrolysis and thus does not appear to act as an antagonist of the nucleotide exchange factor GrpE(Tth). This raises the question of whether T. thermophilus GrpE stimulates nucleotide exchange to a smaller degree as compared to the E. coli system and how the corresponding rates relate to intrinsic ATPase and ATP binding as well as luciferase refolding kinetics of T. thermophilus DnaK. We determined dissociation constants as well as kinetic constants that describe the interactions between the T. thermophilus molecular chaperone DnaK, its nucleotide exchange factor GrpE and the fluorescent ADP analogue N8-(4-N'-methylanthraniloylaminobutyl)-8-aminoadenosine-5'-diphosphate by isothermal equilibrium titration calorimetry and stopped-flow kinetic experiments and investigated the influence of T. thermophilus DnaJ on the DnaK nucleotide cycle. The interaction of GrpE with the DnaK.ADP complex versus nucleotide-free DnaK 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 indicate that the maximal acceleration of nucleotide release by GrpE is 80,000-fold at a saturating GrpE concentration. Our experiments show that in T. thermophilus, although the thermophilic DnaK system displays no stimulation of the DnaK-ATPase activity by DnaJ, nucleotide exchange is still efficiently stimulated by GrpE. This indicates that two counteracting factors are not absolutely necessary to maintain a functional and regulated chaperone cycle. This conclusion is corroborated by data that show that the slower ATPase cycle of the DnaK system as well as of heterologous T. thermophilus DnaK/E. coli DnaK systems is directly reflected in altered refolding kinetics of firefly luciferase but not necessarily in refolding yields.