Allosteric Coupling between the Lid and Interdomain Linker in DnaK Revealed by Inhibitor Binding Studies

ABSTRACT The molecular chaperone DnaK assists protein folding and refolding, translocation across membranes, and regulation of the heat shock response. In Escherichia coli, the protein is a target for insect-derived antimicrobial peptides, pyrrhocoricins. We present here the X-ray crystallographic analysis of the E. coli DnaK substrate-binding domain in complex with pyrrhocoricin-derived peptide inhibitors. The structures show that pyrrhocoricins act as site-specific, dual-mode (competitive and allosteric) inhibitors, occupying the substrate-binding tunnel and disrupting the latch between the lid and the β-sandwich. Our structural analysis revealed an allosteric coupling between the movements of the lid and the interdomain linker, identifying a previously unknown mechanism of the lid-mediated regulation of the chaperone cycle.

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Construction and Analysis of HybridEscherichia coli-Bacillus subtilis dnaK Genes

Journal of Bacteriology ◽

10.1128/jb.181.6.1971-1974.1999 ◽

1999 ◽

Vol 181 (6) ◽

pp. 1971-1974 ◽

Cited By ~ 24

Author(s):

Axel Mogk ◽

Bernd Bukau ◽

Rolf Lutz ◽

Wolfgang Schumann

Keyword(s):

Bacillus Subtilis ◽

Functional Unit ◽

Substrate Binding ◽

Binding Domain ◽

Atpase Domain ◽

E Coli ◽

Terminal Domain ◽

Species Specific ◽

Dnak Protein ◽

Substrate Binding Domain

ABSTRACT The highly conserved DnaK chaperones consist of an N-terminal ATPase domain, a central substrate-binding domain, and a C-terminal domain whose function is not known. Since Bacillus subtilis dnaK was not able to complement an Escherichia coli dnaK null mutant, we performed domain element swap experiments to identify the regions responsible for this finding. It turned out that the B. subtilis DnaK protein needed approximately normal amounts of the cochaperone DnaJ to be functional in E. coli. The ATPase domain and the substrate-binding domain form a species-specific functional unit, while the C-terminal domains, although less conserved, are exchangeable. Deletion of the C-terminal domain in E. coli DnaK affected neither complementation of growth at high temperatures nor propagation of phage λ but abolished degradation of ς32.

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A novel virus-like particle based on hepatitis B core antigen and substrate-binding domain of bacterial molecular chaperone DnaK

Vaccine ◽

10.1016/j.vaccine.2009.09.022 ◽

2009 ◽

Vol 27 (52) ◽

pp. 7377-7384 ◽

Cited By ~ 9

Author(s):

Xue Jun Wang ◽

Kai Gu ◽

Qi Yan Xiong ◽

Liang Shen ◽

Rong Yue Cao ◽

...

Keyword(s):

Hepatitis B ◽

Molecular Chaperone ◽

Substrate Binding ◽

Binding Domain ◽

Core Antigen ◽

Virus Like Particle ◽

Chaperone Dnak ◽

Novel Virus ◽

Hepatitis B Core Antigen ◽

Substrate Binding Domain

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Interdomain communication in the molecular chaperone DnaK

Biochemical Journal ◽

10.1042/bj20020943 ◽

2003 ◽

Vol 369 (3) ◽

pp. 627-634 ◽

Cited By ~ 22

Author(s):

Wanjiang HAN ◽

Philipp CHRISTEN

Keyword(s):

Substrate Binding ◽

Peptide Binding ◽

Binding Domain ◽

Intrinsic Fluorescence ◽

Tryptophan Residue ◽

Atpase Domain ◽

Chaperone Dnak ◽

The One ◽

Kinetics Of ◽

Substrate Binding Domain

DnaK, a heat-shock protein 70 (Hsp70) homologue in Escherichia coli, possesses a single tryptophan residue in its ATPase domain. Changes in the intrinsic fluorescence of DnaK offer a simple means not only to follow the binding of ATP and of ADP plus the co-chaperone GrpE to the ATPase domain, but also to investigate the kinetics of peptide binding to the substrate-binding domain of ATP·DnaK and GrpE-liganded ADP·DnaK. Addition of ATP or of ADP plus GrpE to nucleotide-free DnaK resulted in a similar decrease in intrinsic fluorescence, indicating similar open conformations of the ATPase domain under these two conditions. Binding of peptide increased the intrinsic fluorescence of both ATP·DnaK and ADP·DnaK·GrpE and rendered their spectra similar to the spectrum of ADP·DnaK with closed conformation of the ATPase domain. These results, together with the differential kinetics of peptide binding to ADP·DnaK on the one hand, and to ATP·DnaK or ADP·DnaK·GrpE on the other, suggest that ligands for either domain, i.e. ATP or ADP plus GrpE for the ATPase domain and peptides for the substrate-binding domain, shift the conformational equilibrium of both domains of DnaK towards the open and closed forms, respectively, in a concerted and parallel manner.

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