Construction and Analysis of HybridEscherichia coli-Bacillus subtilis dnaK Genes

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.

Download Full-text

GrpE N-terminal Domain Contributes to the Interaction with DnaK and Modulates the Dynamics of the Chaperone Substrate Binding Domain

Journal of Molecular Biology ◽

10.1016/j.jmb.2007.10.002 ◽

2007 ◽

Vol 374 (4) ◽

pp. 1054-1064 ◽

Cited By ~ 12

Author(s):

Fernando Moro ◽

Stefka G. Taneva ◽

Adrián Velázquez-Campoy ◽

Arturo Muga

Keyword(s):

Substrate Binding ◽

Binding Domain ◽

Terminal Domain ◽

Substrate Binding Domain

Download Full-text

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.

Download Full-text

Defining the structure of the substrate-free state of the DnaK molecular chaperone

Biochemical Society Symposium ◽

10.1042/bss0680069 ◽

2001 ◽

Vol 68 ◽

pp. 69-82 ◽

Cited By ~ 3

Author(s):

Joanna F. Swain ◽

Renuka Sivendran ◽

Lila M. Gierasch

Keyword(s):

Binding Site ◽

Substrate Binding ◽

Binding Domain ◽

Full Length ◽

Free State ◽

Substrate Binding Site ◽

Terminal Domain ◽

C Terminus ◽

Ph Dependent ◽

Substrate Binding Domain

Members of the Hsp70 (heat-shock protein of 70 kDa) family of molecular chaperones bind to exposed hydrophobic stretches on substrate proteins in order to dissociate molecular complexes and prevent aggregation in the cell. Substrate affinity for the C-terminal domain of the Hsp70 is regulated by ATP binding to the N-terminal domain utilizing an allosteric mechanism. Our multi-dimensional NMR studies of a substrate-binding domain fragment (amino acids 387-552) from an Escherichia coli Hsp70, DnaK(387-552), have uncovered a pH-dependent conformational change, which we propose to be relevant for the full-length protein also. At pH 7, the C-terminus of DnaK(387-552) mimics substrate by binding to its own substrate-binding site, as has been observed previously for truncated Hsp70 constructs. At pH 5, the C-terminus is released from the binding site, such that DnaK is in the substrate-free state 10-20% of the time. We propose that the mechanism for the release of the tail is a loss of affinity for substrate at low pH. The pH-dependent fluorescence changes at a tryptophan residue near the substrate-binding pocket in full-length DnaK lead us to extend these conclusions to the full-length DnaK as well. In the context of the DnaK substrate-binding domain fragment, the release of the C-terminus from the substrate-binding site provides our first glimpse of the empty conformation of an Hsp70 substrate-binding domain containing a portion of the helical subdomain.

Download Full-text