Hsp70 chaperones assist a large variety of protein folding processes within
the entire lifespan of proteins. Central to these activities is the regula
tion of Hsp70 bg DnaJ cochaperones. DnaJ stimulates Hsp70 to hydrolyze ATP,
a key step that closes its substrate-binding cavity and thus allows stable
binding of substrate. We show that DnaJ stimulates ATP hydrolysis by Esche
richia coli Hsp70, DnaK, very efficiently to >1000-fold, but only if presen
t at high (micromolar) concentration, In contrast, the chaperone activity o
f DnaK in luciferase refolding was maximal at several hundredfold lower con
centration of DnaJ, However, DnaJ was capable of maximally stimulating the
DnaK ATPase even at this low concentration, provided that protein substrate
was present, indicating synergistic action of DnaJ and substrate. Peptide
substrates were poorly effective in this synergistic action. DnaJ action re
quired binding of protein substrates to the central hydrophobic pocket of t
he substrate-binding cavity of DnaK as evidenced by the reduced ability of
DnaJ to stimulate ATP hydrolysis by a DnaK mutant with defects in substrate
binding. At high concentrations, DnaJ itself served as substrate for DnaK
in a process considered to be unphysiological, Mutant analysis furthermore
revealed that DnaJ-mediated stimulation of ATP hydrolysis requires communic
ation between the ATPase and substrate-binding domains of DnaK. This mechan
ism thus allows DnaJ to tightly couple ATP hydrolysis by DnaK with substrat
e binding and to avoid jamming of the DnaK chaperone with peptides. It prob
ably is conserved among Hsp70 family members and is proposed to account for
their functional diversity.