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.