Structure-function analysis of the Escherichia coli GrpE heat shock protein.

Abstract

We have isolated various missense mutations in the essential grpE gene of Escherichia coli based on the inability to propagate bacteriophage lambda. To better understand the biochemical mechanisms of GrpE action in various biological processes, six mutant proteins were overexpressed and purified. All of them, GrpE103, GrpE66, GrpE2/280, GrpE17, GrpE13a and GrpE25, have single amino acid substitutions located in highly conserved regions throughout the GrpE sequence. The biochemical defects of each mutant GrpE protein were identified by examining their abilities to: (i) support in vitro lambda DNA replication; (ii) stimulate the weak ATPase activity of DnaK; (iii) dimerize and oligomerize, as judged by glutaraldehyde crosslinking and HPLC size chromatography; (iv) interact with wild‐type DnaK protein using either an ELISA assay, glutaraldehyde crosslinking or HPLC size chromatography. Our results suggest that GrpE can exist in a dimeric or oligomeric form, depending on its relative concentration, and that it dimerizes/oligomerizes through its N‐terminal region, most likely through a computer predicted coiled‐coil region. Analysis of several mutant GrpE proteins indicates that an oligomer of GrpE is the most active form that interacts stably with DnaK and that the interaction is vital for GrpE biological function. Our results also demonstrate that both the N‐terminal and C‐terminal regions are important for GrpE function in lambda DNA replication and its co‐chaperone activity with DnaK.

Physiology and transcriptome of the polycyclic aromatic hydrocarbon-degrading Sphingomonas sp. LH128 after long-term starvation

“…Reduced expression of many of the genes encoding heat-shock proteins and chaperones was observed during long-term starvation, which was also the case during solute stress of LH128 in biofilms (Fida et al, 2012) and after inoculation in soil (T. Fida, unpublished results). No clear explanation can be given for the reduced expressions since heat-shock proteins prevent the aggregation of stress-denatured proteins in response to temperature stress (Wu et al, 1996). However, this observation is in agreement with gene expression analysis of E. coli during growth arrest, where decreased expression of several heatshock proteins was observed (Chang et al, 2002).…”

Evidence for the Existence of Distinct Mammalian Cytosolic, Microsomal, and Two Mitochondrial GrpE-like Proteins, the Co-chaperones of Specific Hsp70 Members

“…Recently, the crystal structure of E. coli GrpE bound to the ATPase domain of DnaK was determined (10). This confirmed earlier studies that a dimer of GrpE binds asymmetrically to a single DnaK molecule in the absence of ATP (11)(12)(13). GrpE is proposed to facilitate release of DnaK-bound ADP by essentially wedging apart the DnaK ATPase domain, thus weakening the grasp of DnaK on ADP.…”

Structure-function analyses of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli

“…In this study, we have demonstrated that Mge1p⌬43 can compensate for the total lack of GrpE in E. coli cell growth, at temperatures up to 40°C, as well as for DNA replication, as judged by the ability to form plaques. In addition, a recent mutational analysis showed that three conserved residues located in the C-terminal domain of GrpE were involved in the modulation of DnaK's function (51,52). Here, we showed that when the corresponding mutations were introduced in the MGE1⌬43 gene, they resulted in the total loss of Mge1p⌬43 activity in an E. coli background, indicating that both GrpE and Mge1p⌬43 have similar general structures for interacting with and modulating DnaK's activities.…”