Physical interactions between members of the DnaK chaperone machinery: characterization of the DnaK.GrpE complex

Many of the functions of the Escherichia coli Hsp 70, DnaK, require two cofactors, DnaJ and GrpE. GrpE acts as a nucleotide exchange factor in the DnaK reaction cycle but the details of its mechanism remain unclear. GrpE has high affinity for monomeric native DnaK, with a K_d estimated at ≤50 nM. GrpE is a very asymmetric molecule and exists as either a dimer or trimer in its native state. The stoichiometry of GrpE to DnaK in the isolated complex was 3:1, suggesting a trimer. Formation of the complex is quite fast (k_on >1 S⁻¹, whereas the off-rate is very slow on the HPLC timescale (k_off ≤ 10⁻⁴ S⁻¹). GrpE has no affinity for ATP or ADP, nor the oligomeric and moltn globule states of DnaK. The complex is much more thermally stable than either GrpE or DnaK alone, and prevents the formation of the molten globule-like state of DnaK at physiologically relevant temperatures. Formation of the complex does not cause any change in secondary structure, as determined by the lack of change in the circular dichroism spectrum. However, binding of GrpE induces a similar tertiary strcutral change in DnaK to that induced by binding of ATP₁ based on the blue shift in λ_max from the fluroscence of the single tryptophan in DnaK. The nucleotide exchange properties of GrpE can be explained by the conformational change which may represent the opening of the nucleotide cleft on DnaK, subsequently inducing a low affinity state for ADP.