The heat-sensitive Escherichia coli grpE280 phenotype: impaired interaction of GrpE(G122D) with DnaK.

Abstract

GrpE is the nucleotide-exchange factor of the DnaK chaperone system. Escherichia coli cells with the classical temperature-sensitive grpE280 phenotype do not grow under heat-shock conditions and have been found to carry the G122D point mutation in GrpE. To date, the molecular mechanism of this defect has not been investigated in detail. Here, we examined the structural and functional properties of isolated GrpE(G122D) in vitro. Similar to wild-type GrpE, GrpE(G122D) is an elongated dimer in solution. Compared to wild-type GrpE, GrpE(G122D) catalyzed the ADP/ATP exchange in DnaK only marginally and did not compete with wild-type GrpE in interacting with DnaK. In the presence of ADP, GrpE(G122D) in contrast to wild-type GrpE, did not form a complex with DnaK detectable by size-exclusion chromatography with on-line static light-scattering and differential refractometry. Apparently, GrpE(G122D) in the presence of ADP binds to DnaK only with much lower affinity than wild-type GrpE. GrpE(G122D) could not substitute for wild-type GrpE in the refolding of denatured proteins by the DnaK/DnaJ/GrpE chaperone system. In the crystal structure of a (Delta1-33)GrpE(G122D).DnaK-ATPase complex, which as yet is the only available structure of a GrpE variant, Asp122 does not interact directly with neighboring residues of GrpE or DnaK. The far-UV circular dichroism spectra of mutant and wild-type GrpE proved slightly different. Possibly, a discrete change in conformation impairs the formation of the complex with DnaK and renders GrpE(G122D) virtually inactive as a nucleotide exchange factor. In view of the drastically reduced ADP/ATP-exchange activity of GrpE(G122D), the heat sensitivity of grpE280 cells might be explained by the ensuing slowing of the chaperone cycle and the increased sequestering of target proteins by high-affinity, ADP-liganded DnaK, both effects being incompatible with efficient chaperone action required for cell growth.