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

Hsp70s assist the folding of proteins in an ATP-dependent 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 regio n between the ATPase and substrate-binding domain did not affect the functi onal domain coupling and oligomerization of DnaK, The intrinsic ATPase acti vity 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 o f DnaJ and protein substrate on ATPase activity, a function which might be essential for chaperone action, (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B,V, All rights reserved.