Mutations in the interdomain linker region of DnaK abolish the chaperone action of the DnaK/DnaJ/GrpE system

Abstract

Hsp70s assist the folding of proteins in an ATPdependent manner. DnaK, the Hsp70 of Escherichia coli, acts in concert with its co-chaperones DnaJ and GrpE. Amino acid substitutions (D388R and L391S/L392G) in the linker region between the ATPase and substrate-binding domain did not affect the functional domain coupling and oligomerization of DnaK. The intrinsic ATPase activity was enhanced up to 10-fold. However, the ATPase activity of DnaK L391S/L392G, if stimulated by DnaJ plus protein substrate, was five times lower than that of wild-type DnaK and DnaK D388R. This defect correlated with the complete loss of chaperone action in luciferase refolding. Apparently, the conserved leucine residues in the linker mediate the synergistic effects of DnaJ and protein substrate on ATPase activity, a function which might be essential for chaperone action. ß

Allosteric Regulation of Hsp70 Chaperones Involves a Conserved Interdomain Linker

“…These results are consistent with earlier observations (29). The comparison of the results with DnaK-D388R and DnaK-D393R demonstrates that charge reversal in this region of the protein is not sufficient for interruption of interdomain communication but that position 393 is very specific in this respect.…”

“…Mutant proteins with alanine replacements of all four hydrophobic residues or aspartic acid replacements of two of the four residues (DnaK-L390D, L391D) lack all of the three above-mentioned signs of interdomain communication. The contribution of the hydrophobicity of the linker residues was contested by a more recent publication (29) reporting that the substitution of two of the four hydrophobic residues for glycine and serine (DnaK-L391G, L392S) did not obliterate interdomain communication completely. This suggests that not all four hydrophobic residues are essential for domain-domain coupling and that the results of the DnaK-L390D, L391D variant is more likely due to the surplus of two negative charges than to the loss of hydrophobicity within the linker.…”

Crystal Structure of DnaK Protein Complexed with Nucleotide Exchange Factor GrpE in DnaK Chaperone System

“…The GkGrpE dimer and the chain A GkDnaK_SBD interact with the neighboring GkDnaK linker, thereby mimicking a ternary complex configuration (data not shown). This result agrees with previous findings that substrate recognition by DnaK increases when the GrpE dimer binds and subsequently stabilizes the linker (12,17). The spatial relationship between GkDnaK A and the GkGrpE dimer suggests that the allosteric communication between DnaK SBD and NBD occurs via the linker which is co-regulated by the long GrpE N-terminal ␣-helices.…”

“…The difference may be caused by structural and functional diversities across different species as the thermosensor mechanisms are known to be different between T. thermophilus and E. coli. (13), help stabilize the DnaK-substrate complex, and facilitate nucleotide exchange (12,17,31). The interaction between the long GrpE N-terminal ␣-helices and DnaK is therefore important to substrate processing.…”

“…Previous biochemical and thermodynamic studies have emphasized the importance of full-length DnaK and GrpE for formation of a ternary complex and for substrate processing (12,13,16,17). The GrpE N-terminal disordered region may accelerate the release of substrate bound to DnaK (12,13).…”