The DnaK chaperone system of Escherichia coli: quaternary structures and interactions of the DnaK and GrpE components.
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.
Hoffmann-La Roche Limited, Pharmaceutical Research-New Technologies, Basel, Switzerland.
H J
HJ
Schönfeld
D
D
Schmidt
H
H
Schröder
B
B
Bukau
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000502:physiology*; D002850:Chromatography, Gel; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000502:physiology*; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D018832:Molecular Chaperones / Q000737:chemistry*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins; D012542:Scattering, Radiation; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D018832:Molecular Chaperones; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli