Influence of GrpE on DnaK-substrate interactions.
The DnaK chaperone of Escherichia coli assists protein folding by an ATP-dependent interaction with short peptide stretches within substrate polypeptides. This interaction is regulated by the DnaJ and GrpE co-chaperones, which stimulate ATP hydrolysis and nucleotide exchange by DnaK, respectively. Furthermore, GrpE has been claimed to trigger substrate release independent of its role as a nucleotide exchange factor. However, we show here that GrpE can accelerate substrate release from DnaK exclusively in the presence of ATP. In addition, GrpE prevented the association of peptide substrates with DnaK through an activity of its N-terminal 33 amino acids. A ternary complex of GrpE, DnaK, and a peptide substrate could be observed only when the peptide binding to DnaK precedes GrpE binding. Furthermore, we demonstrate that GrpE slows down the release of a protein substrate, sigma(32), from DnaK in the absence of ATP. These findings suggest that the ATP-triggered dissociation of GrpE and substrates from DnaK occurs in a concerted fashion.
Brehmer
Dirk
D
Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.
Gässler
Claudia
C
Rist
Wolfgang
W
Mayer
Matthias P
MP
Bukau
Bernd
B
D000255:Adenosine Triphosphate / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012808:Sigma Factor / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
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; D010455:Peptides; D012808:Sigma Factor; C069774:heat-shock sigma factor 32; D000255:Adenosine Triphosphate; D008156:Luciferases; C063842:dnaK protein, E coli