GrpE alters the affinity of DnaK for ATP and Mg2+. Implications for the mechanism of nucleotide exchange.
DnaK, DnaJ, and GrpE heat shock proteins of Escherichia coli activate site-specific DNA binding by the RepA replication initiator protein of plasmid P1 in a reaction dependent on ATP and Mg2+. We previously showed that GrpE is essential for in vitro RepA activation specifically at about 1 microM free Mg2+. In this paper, we demonstrate that GrpE lowers the requirement of DnaK ATPase for Mg2+, resulting in a large stimulation of ATP hydrolysis at about 1 microM Mg2+ with and without DnaJ and RepA. In contrast to its effect on the Mg2+ requirement, GrpE increases the ATP requirement for DnaK ATPase and dramatically lowers the affinity of DnaK for ATP in the absence of Mg2+. We propose that GrpE not only lowers the affinity of DnaK for nucleotide but, by increasing affinity of DnaK for Mg2+, also weakens the interactions of Mg2+ with nucleotide prior to its release.
Skowyra
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Laboratory of Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Wickner
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D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism* / Q000494:pharmacology; D004265:DNA Helicases; D004261:DNA Replication; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D011506:Proteins; D015534:Trans-Activators
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli; D004265:DNA Helicases; D008274:Magnesium