The DnaK Chaperone System of Escherichia coli: Quaternary Structures and Interactions of the DnaK and GrpE Components

Abstract

The DnaK (Hsp70), DnaJ, and GrpE heat shock proteins of Escherichia coli constitute a cellular chaperone system for protein folding. Substrate interactions are controlled by the ATPase activity of DnaK which itself is regulated by the nucleotide exchange factor GrpE. To understand the structure-function relationship of this chaperone system, the quaternary structures of DnaK, GrpE, and DnaK-GrpE complexes were analyzed by gel filtration chromatography, dynamic light scattering, analytical ultracentrifugation, and native gel electrophoresis. GrpE formed dimers in solution. DnaK formed monomers, dimers, and higher mole mass oligomers, the equilibrium between these forms being dependent on the DnaK concentration. The behavior of DnaK and GrpE in gel filtration and dynamic light scattering suggested elongated shapes of both molecules. In the absence of added nucleotides, DnaK and GrpE formed stable complexes containing one molecule of DnaK and two molecules of GrpE. A 44-kDa N-terminal ATPase fragment of DnaK also formed complexes with GrpE with the same 1:2 stoichiometry. DnaK-GrpE complex formation was unaffected by elimination of DnaK-bound nucleotides or addition of saturating concentrations of a DnaK peptide substrate. These findings allow the correlation of DnaK-GrpE interactions with a role for GrpE in the functional cycle of the DnaK chaperone system.

Free Human Mitochondrial GrpE Is a Symmetric Dimer in Solution

“…This molecular mass is about twice the molecular mass of the human GrpE monomer. This result is in good agreement with the analytical ultracentrifugation data generated for E. coli GrpE (16). The analysis of the crystal structure of E. coli GrpE confirms that this protein is a dimer with an elongated shape.…”

“…The elongated shape of GrpE is the most likely reason for the apparently much too high molecular mass calculated from molecular exclusion chromatography experiments (16). Several works have indicated that the elongated shape of GrpE serves the purpose of allowing its N terminus to also interact with DnaK-SBD.…”

“…The first indication that GrpE was a homodimer came from cross-linking studies with glutaraldehyde (15). Subsequently, analytical ultracentrifugation experiments (16) showed that GrpE has a dimeric structure with an elongated shape that binds DnaK with 2:1 stoichiometry.…”

Visualization and Mechanism of Assembly of a Glucocorticoid Receptor·Hsp70 Complex That Is Primed for Subsequent Hsp90-dependent Opening of the Steroid Binding Cleft

“…Thus, it seems inappropriate at this time to form rigid models of the priming step based on stoichiometry data that require the action of specifically a monomer or a dimer of hsp70 on the receptor. It is possible that only one molecule of hsp70 is bound directly to and interacting productively with the receptor, and that under these conditions of assembly with purified proteins the receptor-bound hsp70 can associate with other molecules of hsp70 to form dimers and trimers, much as purified hsp70 and its homologs have been shown to do in solution (32)(33)(34). It is perhaps important to note that analysis of both the native and assembled hsp90⅐Hop⅐hsp70 machinery has revealed a molar ratio of 1 hsp70 per hsp90 dimer (6,18).…”

“…Bacterial DnaK and its mammalian mitochondrial and cytoplasmic homologs self-associate in solution into dimers, trimers, and probably higher oligomers, with the equilibrium between these forms being dependent upon the concentration of the chaperone and the equilibrium being shifted toward the monomer by ATP or binding of peptide substrate (32)(33)(34). Under concentrated conditions, our purified hsp70 is predominantly dimeric on native gel electrophoresis with some trimers being detectable, yet under dilute conditions in reticulocyte lysate, the free hsp70 behaves as a monomer on molecular sieve chromatography (6), as did our purified hsp70 (data not shown).…”