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. 2015 Apr 3;290(14):8849-62.
doi: 10.1074/jbc.M114.596288. Epub 2015 Jan 28.

A functional DnaK dimer is essential for the efficient interaction with Hsp40 heat shock protein

Evans Boateng Sarbeng  1 , Qingdai Liu  1 , Xueli Tian  1 , Jiao Yang  1 , Hongtao Li  1 , Jennifer Li Wong  1 , Lei Zhou  1 , Qinglian Liu  2
Affiliations

A functional DnaK dimer is essential for the efficient interaction with Hsp40 heat shock protein

Evans Boateng Sarbeng et al. J Biol Chem. .

Abstract

Highly conserved molecular chaperone Hsp70 heat shock proteins play a key role in maintaining protein homeostasis (proteostasis). DnaK, a major Hsp70 in Escherichia coli, has been widely used as a paradigm for studying Hsp70s. In the absence of ATP, purified DnaK forms low-ordered oligomer, whereas ATP binding shifts the equilibrium toward the monomer. Recently, we solved the crystal structure of DnaK in complex with ATP. There are two molecules of DnaK-ATP in the asymmetric unit. Interestingly, the interfaces between the two molecules of DnaK are large with good surface complementarity, suggesting functional importance of this crystallographic dimer. Biochemical analyses of DnaK protein supported the formation of dimer in solution. Furthermore, our cross-linking experiment based on the DnaK-ATP structure confirmed that DnaK forms specific dimer in an ATP-dependent manner. To understand the physiological function of the dimer, we mutated five residues on the dimer interface. Four mutations, R56A, T301A, N537A, and D540A, resulted in loss of chaperone activity and compromised the formation of dimer, indicating the functional importance of the dimer. Surprisingly, neither the intrinsic biochemical activities, the ATP-induced allosteric coupling, nor GrpE co-chaperone interaction is affected appreciably in all of the mutations except for R56A. Unexpectedly, the interaction with co-chaperone Hsp40 is significantly compromised. In summary, this study suggests that DnaK forms a transient dimer upon ATP binding, and this dimer is essential for the efficient interaction of DnaK with Hsp40.

Keywords: 70-Kilodalton Heat Shock Protein (Hsp70); Chaperone DnaJ (DnaJ); Chaperone DnaK (DnaK); Heat Shock Protein (HSP); Hsp40; Protein Folding; Protein-Protein Interaction.

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Figures

FIGURE 1.
FIGURE 1.
Structure of the DnaK-ATP dimer. A and B, worm and ribbon diagrams of the DnaK-ATP homodimer, respectively. A, view has the 2-fold axis vertical and the NBD-NBD′ interface along the line of sight. The two DnaK protomers A and B are colored in red and blue in A, respectively. B, view is rotated 90° from A about a horizontal axis to view the DnaK dimer along the 2-fold axis from below. The DnaK protomers are colored based on domain as follows: NBD (blue for protomer A and marine for protomer B), inter-domain linker (purple), SBDβ (green for protomer A and lemon green for protomer B), and SBDα (red for protomer A and salmon for protomer B). NBD-NBD′ contacts are highlighted with orange circles, and the SBDa-NBD′ contacts are highlighted with green circles. C and D, worm and ribbon diagrams of the Sse1-hHsp70(NBD) heterodimer (Protein Data Bank code 3D2F), respectively. C, Sse1 is colored green and hHsp70(NBD) colored orange. The NBD of Sse1 is superimposed onto the NBD of DnaK protomer A in A, thereby providing an (A)-equivalent view of this complex. D, Sse1 is colored based on domain as in A, and hHsp70(NBD) is colored orange. The view is rotated relative to that in C just as B is rotated relative to A. E, superposition of A and C. F, superposition of B and D in worm diagrams. G, worm diagram of the Sse1 dimer (Protein Data Bank code 2QXL). The two protomers are colored in green and orange, respectively. The NBD of Sse1 protomer A is superimposed onto the NBD of DnaK protomer A in A, thereby providing an (A)-equivalent view of this complex.
FIGURE 2.
FIGURE 2.
DnaK forms specific dimer in solution in the presence of ATP. A, native PAGE analysis of DnaK-T199A/L′3,4. 2 μl of DnaK-T199A/L′3,4 with indicated concentrations were loaded onto an 8–25% gradient PhastGel (GE Healthcare). The positions of the monomer (1×) and dimer (2×) are indicated by arrows. B, DnaK-T199A/L′3,4 was purified to high purity. Equal amounts of DnaK-T199A/L′3,4 for each lane from A were loaded onto SDS-PAGE. There are no apparent contaminants in the purified DnaK-T199A/L′3,4 protein. C, mutant DnaK proteins were purified to a similar purity as that of the WT DnaK. Each mutant protein was loaded and separated on SDS-polyacrylamide gel. WT DnaK protein purified to high purity was used for comparison. D, AUC analysis of the WT DnaK protein in the presence of ATP. Sedimentation velocity experiments were carried out with WT DnaK protein at 0.25 (top panel) and 1 mg/ml (bottom panel). The positions of the monomer and dimer are labeled as 1× and 2×, respectively. E, AUC analysis of the WT DnaK protein in the presence of different nucleotides. Sedimentation velocity experiments were performed on WT DnaK protein at 1 mg/ml in the presence of ATP, ADP, or in the absence of nucleotide (apo-form). F, glutaraldehyde cross-linking. WT DnaK protein was treated with 0.00625, 0.0125, and 0.025% glutaraldehyde and separated on SDS-PAGE. The concentrations of glutaraldehyde were labeled on the top of the gel. The positions of the monomer (1×) and dimer (2×) are labeled on the right. G, disulfide bond formation between A303C and H541C. A303C, DnaK-C15A/A303C; H541C, DnaK-C15A/H541C; A303C+H541C, DnaK-C15A/A303C and DnaK-C15A/H541C were mixed in 1:1 ratio. Oxidation with copper-phenanthroline was carried out in the presence of ATP (+ATP) or ADP (+ADP). Samples in the presence of ATP were treated with DTT as loading controls (+DTT). The positions of the monomer (1×) and dimer (2×) are labeled on the right.
FIGURE 3.
FIGURE 3.
DnaK dimer is essential for in vivo chaperone activity. A, contacts between the NBDs in the DnaK dimer. The NBDs of protomer A and B are colored blue and cyan, respectively. Viewpoint is as in Fig. 1B. B, contacts between NBD′ (cyan) and the partner SBDα (red) in the DnaK dimer. Viewpoint is as in Fig. 1B. C, growth test of the dimer mutants in DnaK. Serial dilutions of fresh cultures carrying indicated dimer mutations were spotted on LB agar plates and grew for 1 overnight at 30 and 37 °C. We used WT DnaK and empty vector as positive and negative controls, respectively. D, protein expression levels of the dimer mutants. Equal amounts of E. coli cultures carrying each dimer mutant or the WT DnaK were loaded onto SDS-PAGE. The empty vector was used as negative control. The top panel is the Western blot analysis with an anti-DnaK antibody. The bottom panel is a SDS-polyacrylamide gel stained with Coomassie Blue.
FIGURE 4.
FIGURE 4.
Dimer mutations R56A, T301A, N537A, and D540A compromise the formation of DnaK dimer. A, native gel analysis of the dimer mutations in the DnaK-T199A/L′3,4 construct. 2 μl of each protein at 12 mg/ml was loaded onto an 8–25% gradient PhastGel (GE Healthcare). The positions of the monomer (1×) and dimer (2×) are indicated by arrows. The DnaK-T199A/L′3,4 protein (WT) was used as control. B, all the dimer mutants in the DnaK-T199A/L′3,4 construct were purified to a similar purity as that of the DnaK-T199A/L′3,4 (WT). 24 μg of each protein were loaded onto SDS-PAGE. C and D, AUC analysis of the dimer mutants in the presence of ATP (C) and ADP (D), respectively. Each mutant protein (at 1 mg/ml) was subjected to sedimentation velocity experiment as in Fig. 2D. The WT DnaK was used as control. The positions of the monomer and dimer are labeled as 1× and 2×, respectively. E, glutaraldehyde cross-linking. The mutant DnaK proteins were treated with 0.00625, 0.0125, and 0.025% glutaraldehyde and separated on SDS-PAGE as in Fig. 2F. The concentrations of glutaraldehyde were labeled at the top of the gel. The positions of the monomer (1×) and dimer (2×) are labeled on the right. F, dimer mutants T301A and N537A/D540A compromise the formation of the disulfide bond between A303C and H541C. T301A and N537A/D540A were introduced into the cysteine mutants A303C and H541C. Purified proteins were oxidized with increasing concentrations of copper-phenanthroline (50, 100, and 200 μm; labeled at the top of the gel) in the presence of ATP as described in Fig. 2G. The positions of the monomer (1×) and dimer (2×) are labeled on the right. G, cysteine mutant DnaK proteins used in F were purified to the similar purity as that of the WT DnaK. Each DnaK protein was loaded and separated on an SDS-polyacrylamide gel.
FIGURE 5.
FIGURE 5.
Hydrogen bonds formed between Asn-537 and Glu-467 are not essential for DnaK's chaperone activity. A, ribbon diagram of the isolated DnaK SBD structure. SBDβ and SBDα are colored in green and red, respectively. The bound NR peptide is shown in cyan. Asn-537, Asp-540, Glu-467, and Met-404 are highlighted in stick representation. B, close-up view of the hydrogen bonds formed between Asn-537 and Glu-467 and between Asp-540 and the main chain of Met-404. Coloring is the same as in A. C, E467C mutation has little effect on DnaK's in vivo chaperone activity. The growth test was done the same way as that of Fig. 3C.
FIGURE 6.
FIGURE 6.
Tests of the ATP-induced allosteric coupling in the dimer mutant proteins. A, ATP-induced tryptophan fluorescence shift. The blue shift for each protein was calculated as the wavelength difference of the maximal emission between the samples incubated with ATP and ADP. B, ATP-induced bound peptide substrate release. F-NR was incubated with 5 μm each of DnaK protein for more than 3 h to allow binding to reach equilibrium. Fluorescence anisotropy was measured for binding in the absence of ATP (black bars). Then ATP was added to a final concentration of 2 mm and incubated for 2 min. The resulting anisotropy measurements represent the binding in the presence of ATP (red bars). C, NR peptide stimulation of the intrinsic ATPase activity of DnaK. Fold of stimulation in the presence of 40 μm NR peptide was calculated by setting the intrinsic ATPase activity rate (kcat) as 1.
FIGURE 7.
FIGURE 7.
ATP release activity of GrpE in the single-turnover ATPase assay. DnaK-[32P]ATP complexes were incubated with 250 μm unlabeled ATP (purple diamonds) or 3 and 6 μm GrpE together with 250 μm unlabeled ATP (green triangles and blue upside-down triangles). Samples were taken at the indicated times, and the fraction of ATP converted to ADP was calculated after quantification. The samples of the DnaK-[32P]ATP complexes incubated with buffer were used as control (red-filled circles). A, WT DnaK; B, DnaK-R56A; C, DnaK-T301A; D, DnaK-N537A; and E, DnaK-N540A.
FIGURE 8.
FIGURE 8.
DnaJ interaction is compromised in the dimer mutant proteins. A and B, SPR analysis of the DnaK-DnaJ interaction with DnaJ immobilized. 2 μm DnaK proteins were injected over a sensor chip with DnaJ immobilized on the surface. Resonance signals after subtracting the background binding to a control channel were recorded over time. All the analyses in B were carried out in the presence of ATP. C, DnaJ stimulation of DnaK's ATPase activity. Various concentrations of DnaJ were incubated with DnaK-[32P]ATP complexes in the single-turnover ATPase assay. The hydrolysis rate, kcat, for each concentration of DnaJ was determined, and the fold of stimulation was calculated by setting the intrinsic ATPase rate as 1. D, SPR analysis with DnaK immobilized on a sensor chip. DnaJ protein was injected as analyte. The concentrations of DnaJ were labeled on the right. E, DnaJ dependence in refolding the heat-denatured firefly luciferase. Various concentrations of DnaJ were included in the refolding reactions of denatured firefly luciferase. The refolding activity was calculated by setting the undenatured luciferase activity as 100%.
FIGURE 9.
FIGURE 9.
Proposed model of the DnaK chaperone cycle. DnaK domain coloring is the same as protomer A in Fig. 1B. The domains of DnaJ are as follows: J-domain (yellow), substrate binding domain (pink), and dimerization domain (cyan). J-domain has been shown to be the site that binds Hsp70s. Polypeptide substrates are highlighted in black.
FIGURE 10.
FIGURE 10.
Sequence alignment of segments containing key dimer contacts. Residues corresponding to Glu-28, Arg-56, Thr-301, Asn-537, and Asp-540 in DnaK are highlighted in red.
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