Glutathionylation of the Bacterial Hsp70 Chaperone DnaK Provides a Link between Oxidative Stress and the Heat Shock Response

Hong Zhang,Jie Yang,Si Wu,Weibin Gong,Chang Chen,Sarah Perrett

doi:10.1074/jbc.m115.673608

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 σ32 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. We found that glutathionylation of DnaK reversibly changes the secondary structure and tertiary conformation, leading to reduced nucleotide and peptide binding ability. The chaperone activity of DnaK was reversibly down-regulated by glutathionylation, accompanying the structural changes. We found 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 of σ32 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.

Highlights

Glutathionylation of the Bacterial Hsp70 Chaperone DnaK Provides a Link between Oxidative Stress and the Heat Shock Response*
We investigated the conditions under which Escherichia coli DnaK undergoes S-glutathionylation
The Single Conserved Cysteine Residue of E. coli DnaK Can Be Glutathionylated under Oxidative Stress Conditions in Vivo and in Vitro—E. coli DnaK has only one cysteine residue, Cys15, which lies on the ␤-sheet surface of the IA subdomain of the ATPase domain (Fig. 1A)

Summary

Introduction

Glutathionylation of the Bacterial Hsp Chaperone DnaK Provides a Link between Oxidative Stress and the Heat Shock Response*. This study confirms that glutathionylation down-regulates the functions of DnaK under oxidizing conditions, and this down-regulation may facilitate release of ␴32 from its interaction with DnaK, triggering the heat shock response. Such a mechanism provides a link between oxidative stress and the heat shock response in bacteria. The DnaK-DnaJ-GrpE complex works as a chaperone machine to mitigate the effects of heat shock stress, prevent protein aggregation, and facilitate protein folding [20] This machinery controls the level of the heat shock response by interaction with the ␴32 subunit of RNA polymerase, which is MARCH 25, 2016 VOLUME 291 NUMBER 13

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