Glutathionylation of the bacterial Hsp70 chaperone dnak provides a link between oxidative stress and the heat shock response

Abstract

DnaK is the major bacterial Hsp70, participating in DNA replication, protein folding, and the stress response. DnaK cooperates with the Hsp40 co-chaperone DnaJ and the nucleotide exchange factor GrpE. Under non-stress conditions, DnaK binds to the heat shock transcription factor 32 and facilitates its degradation. Oxidative stress results in temporary inactivation of DnaK due to depletion of cellular ATP and thiol modifications such as glutathionylation until normal cellular ATP levels and a reducing environment are restored. However, the biological significance of DnaK glutathionylation remains unknown, and the mechanisms by which glutathionylation may regulate the activity of DnaK are also unclear. We investigated the conditions under which Escherichia coli DnaK undergoes S-glutathionylation. We observed glutathionylation of DnaK in lysates of E. coli cells that had been subjected to oxidative stress. We also obtained homogeneously glutathionylated DnaK using purified DnaK in the apo state.Wefound that glutathionylation of DnaK reversibly changes the secondary structure and tertiary conformation, leading to reduced nucleotide and peptide binding ability. The chaperone activity ofDnaKwas reversibly downregulated by glutathionylation, accompanying the structural changes.Wefound that interaction of DnaK with DnaJ, GrpE, or σ32 becomes weaker when DnaK is glutathionylated, and the interaction is restored upon deglutathionylation. This study confirms that glutathionylation down-regulates the functions of DnaK under oxidizing conditions, and this down-regulation may facilitate release ofσ32 from its interaction with DnaK, thus triggering the heat shock response. Such a mechanism provides a link between oxidative stress and the heat shock response in bacteria.