Mechanism of regulation of hsp70 chaperones by DnaJ cochaperones

Abstract

Hsp70 chaperones assist a large variety of protein folding processes within the entire lifespan of proteins. Central to these activities is the regulation of Hsp70 by 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 Escherichia coli Hsp70, DnaK, very efficiently to >1000-fold, but only if present at high (micromolar) concentration. In contrast, the chaperone activity of DnaK in luciferase refolding was maximal at several hundredfold lower concentration 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 required binding of protein substrates to the central hydrophobic pocket of the 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 communication between the ATPase and substrate-binding domains of DnaK. This mechanism thus allows DnaJ to tightly couple ATP hydrolysis by DnaK with substrate binding and to avoid jamming of the DnaK chaperone with peptides. It probably is conserved among Hsp70 family members and is proposed to account for their functional diversity.

Figure 1

Differences in DnaJ requirements for stimulation of ATP hydrolysis by DnaK and luciferase refolding. (A) Effect of DnaJ on stimulation of ATP hydrolysis. Hydrolysis rates dependent on DnaJ (filled diamonds) were determined by quenched flow experiments and yielded a fitted maximal value of 0.79 s⁻¹. The open diamond indicates the additional effect of σ³² (final concentration 2 μM) at the highest DnaJ concentration. (B) Efficiency of luciferase refolding by the DnaK system dependent on DnaJ. Chemically denatured luciferase was refolded in buffer containing constant concentrations of DnaK, GrpE, and ATP, and various concentrations of DnaJ. The lines between A and B indicate the differences in the DnaJ concentration used in the two experiments.

Figure 2

Stimulation of DnaK ATPase by DnaJ requires protein substrate. (A) Effects of DnaJ, DnaJ259, σ³², and unfolded luciferase (final concentrations indicated) on ATP hydrolysis rates of DnaK as determined by single-turnover assays. Luciferase was unfolded in 3 M Gdn⋅HCl and diluted into refolding buffer. The final Gdn⋅HCl concentration (180 mM) had no effect on unstimulated and DnaJ-stimulated hydrolysis rates (data not shown). (B) Effects of increasing σ³² and peptide substrate [σ³²-Q132-Q144 (28)] on ATP hydrolysis rates of DnaK in the presence of DnaJ. The ATP hydrolysis rates were determined in standard single-turnover assays.

Figure 3

Defects of the DnaK-V436F mutant in substrate binding and DnaJ-dependent stimulation of ATP hydrolysis. (A) Efficiency of binding of σ³² to wild-type DnaK and DnaK-V436F as determined by gel filtration according to Gamer et al. (29). [³H]σ³² (1 μM) was incubated with the indicated amount of DnaK at 30°C for 2 h prior to separation of the σ³²⋅DnaK complex and free σ³² on a Superdex 200 FPLC column (Amersham Pharmacia Biotech). The dissociation constant (K_d) of the DnaK⋅[³H]σ³² complex was calculated from the ratio of DnaK-bound and free [³H]σ³² to be 1.4 and 24 μM for wild-type DnaK and DnaK-V436F, respectively. (B and C) Single-turnover ATP hydrolysis rates of wild-type DnaK and DnaK-V436F in the presence of σ³² without (B) or with 50 nM DnaJ (C). (D) Effects of increasing DnaJ concentrations on single-turnover ATP hydrolysis rates of wild-type DnaK and DnaK-V436F. Open bars, wild-type DnaK; filled bars, DnaK-V436F.

Figure 4

Mutations in the putative linker connecting ATPase and substrate-binding domains of DnaK abolish interdomain communication and stimulation by DnaJ. (A) Schematic representation of the DnaK linker mutants. (B) Effects of σ³² and DnaJ on ATP hydrolysis of wild type and DnaK mutants. The rates of ATP hydrolysis in the presence or absence of σ³² (2 μM) and DnaJ (50 nM) as indicated were determined by single-turnover assays. (C) Effects of ATP on dissociation rates of fluorescent labeled peptide from complex with wild-type DnaK and DnaK mutants. DnaK was prebound to fluorescent labeled peptide (σ³²-Q132-Q144-C-IAANS) (28) in a 1:1 ratio. At time zero, a 100-fold excess of unlabeled peptide in buffer with or without ATP was added and the decrease in fluorescence was monitored.

Figure 5

Cysteine-specific crosslinking of DnaJ and substrate into the substrate-binding cavity of DnaK. DnaK-Q424C, coupled to the heterobifunctional crosslinker BPIA by means of Cys-424, and similarly treated wild-type DnaK (WT) were incubated in the dark at 30°C with combinations of proteins [σ³², 5 μM; trigger factor (TF), 5 μM; DnaJ, 0.5, 1, or 5 μM], peptides (σ³²-H107-N120; σ³²-Q132-Q144, 25 μM), and ATP (5 mM) as indicated by + and − above the lanes. Crosslinking was induced by UV and products were analyzed on SDS/10% polyacrylamide gels (A and B). (Upper) Silver-stained SDS/polyacrylamide gels; (Lower) immunoblots developed with DnaK-, DnaJ-, and σ³²-specific antisera (α-DnaK, etc.). Indicated are positions of molecular weight markers (left) and DnaK, trigger factor (TF), DnaJ, and σ³² (right). Asterisks and open and filled arrowheads denote DnaK-DnaK, DnaK-σ³², and DnaK-DnaJ crosslinks, respectively.

Figure 6

Model for the mechanism of regulation of DnaK by DnaJ. At least four steps (A–D), of unclear order with respect to steps A and B, build up the basic mechanism allowing DnaJ (J) to couple ATP hydrolysis by DnaK (K) with binding of protein substrates (S). See text for details.