A disulfide-bonded DnaK dimer is maintained in an ATP-bound state

Abstract

DnaK, a major Hsp70 molecular chaperones in Escherichia coli, is a widely used model for studying Hsp70s. We recently solved a crystal structure of DnaK in complex with ATP and showed that DnaK was packed as a dimer in the crystal structure. Our previous biochemical studies supported the formation of a specific DnaK dimer as observed in the crystal structure in solution in the presence of ATP and suggested an important role of this dimer in efficient interaction with Hsp40 co-chaperones. In this study, we dissected the biochemical properties of this DnaK dimer. To restrict DnaK in this dimer form, we mutated two residues on the dimer interface to cysteine, A303C, and H541C. Upon oxidation, this DnaK-A303C-H541C protein formed a specific dimer linked by disulfide bonds formed between A303C and H541C only in the presence of ATP, consistent with the crystal structure. Intriguingly, this disulfide-bond-linked dimer of DnaK-A303C-H541C has reduced ATPase activity and decreased affinity for peptide substrate. More interestingly, unlike wild-type DnaK, the peptide substrate-binding kinetics of this dimer is drastically accelerated even in the absence of ATP, suggesting this dimer is restricted in an ATP-bound conformation regardless of nucleotide bound, which was further supported by our analysis using tryptophan fluorescence and ATP-induced peptide release. Thus, formation of the dimer restricted DnaK in an ATP-bound state and blocked the progression through the chaperone cycle. Productive progression through the chaperone cycle requires the dissociation of this transient dimer. Surprisingly, a significantly compromised interaction with Hsp40 co-chaperone was observed for this disulfide-bond-linked dimer. Thus, dissociation of this DnaK dimer is equally crucial for efficient Hsp40 interaction. An initial interaction between Hsp70 and Hsp40 requires the formation of DnaK dimer; but a stable Hsp70-Hsp40 interaction may follow the dissociation of the dimer.

Keywords: ATPase; HSP70; Hsp40; Molecular chaperone; Peptide substrate binding; Protein folding.

Fig. 1

Ala 303 and His541 are on the dimer interfaces of the DnaK-ATP structure. Ribbon diagram of the DnaK-ATP dimer structure. The two protomers are labeled as A and B. The domain coloring is NBD (blue for A and light blue for B), inter-domain linker (purple for A and light purple for B), SBDβ (green for A and light green for B), and SBDα (red for A and pink for B). Ala303 and His 541 are highlighted in space-fill presentation

Fig. 2

The DnaK-C15A-A303C-H541C protein forms specific dimer only in the presence of ATP. a Purified DnaK proteins. WT wild-type DnaK. C15A DnaK-C15A, 303/541(red) DnaK-C15A-A303C-H541C purified under reducing condition, 3030/541(dimer) + DTT purified DnaK-C15A-A303C-H541C protein was oxidized to form dimer in the presence of ATP; after the dimer was purified, DTT was added to reduce the disulfide bonds back to monomer to show that the dimer was formed from disulfide bonds, 303/541(dimer) after purified, DnaK-C15A-A303C-H541C was oxidized to dimer in the presence of ATP, the dimer was purified and loaded on to SDS-PAGE. b Oxidation of DnaK-C15A-A303C-H541C protein. Purified DnaK-C15A-A303C-H541C was oxidized with copper-phenanthroline in the presence of ATP or ADP. The position of the specific dimer formed in the presence of ATP was indicated by an arrow

Fig. 3

Both the intrinsic ATPase activity and peptide substrate-binding affinity were compromised for the dimer of DnaK-C15A-A303C-H541C. a The intrinsic ATPase activity. A single-turnover ATPase assay was used to determine the intrinsic ATPase activity of DnaK proteins. b Fluorescence polarization assay for determination of peptide substrate-binding affinity in the presence of ADP. Model peptide NR was labeled with fluorescein (F-NR) and incubated with serial dilutions of DnaK proteins in the presence of 100 μM ADP. c Summary of the ATPase rate (k _cat) and affinity of peptide NR binding (K _d). k _cat and K _d values were calculated from (a) and (b), respectively. WT wild-type DnaK, C15A DnaK-C15A, 303/541(red) DnaK-C15A-A303C-H541C purified under reducing condition, 303/541(dimer) purified specific dimer of DnaK-C15A-A303C-H541C formed in the presence of ATP

Fig. 4

Kinetics of peptide NR binding to DnaK proteins in the presence of ADP (a) and ATP (b), respectively. DnaK proteins (15 μM) was first incubated with ADP or ATP for 2 min. Then, F-NR was added and the binding was recorded over time. The names of DnaK proteins are the same as in Fig. 3

Fig. 5

The ATP-induced allosteric coupling in DnaK proteins. a The tryptophan fluorescence of DnaK proteins. Tryptophan fluorescence spectra of DnaK proteins (1 μM) were collected with excitation at 295 nm in absence of nucleotide (Apo) and in the presence of ATP and ADP. The peak value for each spectrum is the wavelength of the maximal emission. b The ATP-induced release of bound NR peptide from DnaK proteins. F-NR peptide was incubated with DnaK proteins (15 μM) for more than 3 h to allow binding to reach equilibrium. Then, ATP was added (indicated by an arrow at time zero), and the release of bound NR peptide was recorded over time. The names of DnaK proteins are the same as in Fig. 3

Fig. 6

The dimer of DnaK-C15A-A303C-H541C has a drastically reduced interaction with DnaJ. The DnaK-DnaJ interaction was assayed using surface plasmon Resonance under reducing condition (in the presence of DTT (a)) and oxidizing condition (in absence of DTT (b)); ~200 response units of DnaJ was immobilized on a sensor chip. Then, DnaK proteins at 2 μM were run through the sensor chip. Resonance signals were recorded over time after subtracting the background signals from a control channel. The names of DnaK proteins are the same as in Fig. 3

Fig. 7

Formation of the disulfide-bonded dimer of DnaK-C15A-A303C-H541C abolished chaperone activity. a Refolding of heat-denatured luciferase by DnaK and its co-chaperones DnaJ and GrpE. The activity of unheated luciferase was set as 100%. The names of DnaK proteins are the same as in Fig. 3. b Growth test of DnaK mutants. Serial dilutions of fresh cultures carrying indicated DnaK mutants were spotted on LB agar plates and incubated for 1 overnight at 30 and 35 °C