Two J domains ensure high cochaperone activity of DnaJ, Escherichia coli heat shock protein 40

Two J domains ensure high cochaperone activity of DnaJ, Escherichia coli heat shock protein 40 Abstract Heat shock protein 70 (Hsp70) chaperone systems consist of Hsp70, Hsp40 and a nucleotide-exchange factor and function to help unfolded proteins achieve their native conformations. Typical Hsp40s assume a homodimeric structure and have both chaperone and cochaperone activity. The dimeric structure is critical for chaperone function, whereas the relationship between the dimeric structure and cochaperone function is hardly known. Here, we examined whether two intact protomers are required for cochaperone activity of Hsp40 using an Escherichia coli Hsp70 chaperone system consisting of DnaK, DnaJ and GrpE. The expression systems were generated and two heterodimeric DnaJs that included a mutated protomer lacking cochaperone activity were purified. Normal chaperone activity was demonstrated by assessing aggregation prevention activity using urea-denatured luciferase. The heterodimeric DnaJs were investigated for cochaperone activity by measuring DnaK ATPase activity and the heat-denatured glucose-6-phosphate dehydrogenase refolding activity of the DnaK chaperone system, and they showed reduced cochaperone activity. These results indicate that two intact protomers are required for high cochaperone activity of DnaJ, suggesting that one homodimeric DnaJ molecule promotes the simultaneous binding of multiple DnaK molecules to one substrate molecule, and that this binding mode is required for the efficient folding of denatured proteins. chaperones, DnaJ, DnaK, Hsp40, Hsp70 Newly synthesized polypeptides fold into their specific conformations to fulfil their functions. Although the final conformation of a polypeptide is determined by its primary structure, many polypeptides need help from a set of proteins called molecular chaperones to assume their final conformations. Molecular chaperones are also involved in various cellular functions, such as the refolding and degradation of denatured proteins. Heat shock protein 100 (Hsp100)s, Hsp90s, Hsp70s, chaperonins and small Hsps are known to be major molecular chaperones, and Hsp70s play a central role among them (1–8). Hsp70 is thought to bind the hydrophobic regions exposed on the surface of denatured polypeptides and to prevent aggregation driven by incorrect hydrophobic interactions (9). In addition to the passive function, Hsp70 disentangles protein aggregates and unfolds collapsed proteins (10–12). Hsp70 has ATPase activity, binding to and releasing an oligopeptide or polypeptide substrate using the ATP hydrolytic cycle (13–16). Hsp70 assumes two conformations, an open form and a closed form, and an equilibrium exists between them (17–22). The ATP-bound form shifts the equilibrium toward the open state and has low affinity for substrates, whereas the ADP-bound form shifts the equilibrium toward the closed state and has high affinity for substrates. Two components called cochaperones, Hsp40 and a nucleotide-exchange factor (NEF), are required to drive the Hsp70 reaction cycle (or chaperone cycle) (23–29). Hsp40 stimulates Hsp70 ATPase activity as a cochaperone, and Hsp40 is also a chaperone that prevents aggregation of denatured polypeptides (25, 30), likely by binding to the hydrophobic regions of denatured polypeptides (31). In a case, Hsp40 seems to induce conformational changes of a substrate (32). In the Hsp70 chaperone cycle, when ATP-bound Hsp70 interacts with Hsp40 on a polypeptide substrate, Hsp70 becomes the ADP-bound form through its interaction with Hsp40, and ADP-bound Hsp70 tightly binds to the substrate. NEF promotes ADP release from Hsp70. As a result, Hsp70 returns to the ATP-bound form, leading to dissociation of the substrate from Hsp70. Hsp70 proteins belong to a big family because they are highly conserved through evolution, and one species encodes multiple paralogous Hsp70 genes. The N-terminal of Hsp70 contains the nucleotide-binding domain, and the substrate-binding domain is located at the C-terminal. Hsp40s are also conserved through evolution, and members of the Hsp40 family are divided into three or four subfamilies (3, 33). According to Cheetham and Caplan (33), Hsp40s can be divided into three groups: Types I–III. All types contain a J domain that interacts with Hsp70 and stimulates its ATPase activity (34–37), which is why Hsp40s are called J proteins. Because the His-Pro-Asp (HPD) motif in the J domain is important for Hsp70 interactions, Hsp40 mutants with amino acid substitutions in the HPD motif do not exhibit cochaperone function (34, 37–39). Types I and II Hsp40s have a glycine/phenylalanine rich region (G/F rich region) and a C-terminal domain (CTD) in addition to the N-terminal J domain. The functional form is a homodimer consisting of two protomers bound at the C-termini (40–44). The G/F rich region is a flexible region and modulates Hsp70–substrate interactions (45–47). Type I Hsp40s include a zinc finger-like motif (ZFLM) in the CTD (48). Each CTD contains a substrate-binding site (43, 49–51), and the G/F rich region and ZFLM are also required for binding to some substrates (52–55). Type III Hsp40s contain the J domain and other specific domains. Types I and II Hsp40s bind to denatured proteins as a chaperone and the homodimeric structure is crucial for Hsp40 chaperone activity, because purified monomeric Hsp40 mutant proteins cannot bind to denatured proteins (42, 44) or act with Hsp70 to catalyse the refolding of denatured luciferase (42), although each protomer of Hsp40 contains a substrate-binding site. Two substrate-binding sites seem to be required for stable binding of Hsp40 to a substrate. However, a monomeric mutant of Sis1, a Type II Hsp40 in Saccharomyces cerevisiae, stimulates the ATPase activity of Hsp70 to a similar level as wild-type (WT) Sis1 in the absence of a substrate (42). Furthermore, Sahi and Craig reported that expressing only a J domain derived from various Hsp40s compensates for severe growth defects in the Saccharomyces cerevisiae Δydj1 (a Type I Hsp40) strain (56). Georgopoulos et al. also reported that a purified monomeric polypeptide containing only the J domain and the G/F rich region of DnaJ, an E. coli Type I Hsp40, stimulates the ATPase activity of Hsp70 and supports some Hsp70 chaperone functions, although, in those cases, more truncated DnaJ than intact DnaJ was required for those effects (34, 57). These results seemingly suggest that the dimeric conformation of Hsp40 is important for chaperone activity but is not absolutely required for cochaperone activity. However, because Hsp40 function was not completely complemented by monomeric Hsp40s, the dimeric structure seems required for full cochaperone activity. Is the dimeric structure sufficient for cochaperone activity that stimulates the ATPase activity of Hsp70 that binds near the Hsp40-binding site on the same substrate molecule? Whether the cochaperone function of Hsp40 requires one or two J domains is an important question that will further elucidate the Hsp70–substrate interaction. In this paper, we addressed this question using an E. coli Hsp70 chaperone system [DnaK (Hsp70), DnaJ (Hsp40; Fig. 1A), GrpE (NEF)] as a model. To this end, two heterodimeric DnaJ mutants that maintained chaperone activity and included an inactive J domain in one subunit were constructed. We found that the two heterodimeric DnaJs cannot efficiently support DnaK chaperone function. Fig. 1 View largeDownload slide Confirmation of purified DnaJs. (A) The DnaJ protomer is 376 amino acids with a J domain (1–70), a G/F rich region (71–105) and a CTD. The CTD is divided into two subdomains, CTD I (110–253) and CTD II (254–331). The J domain contains a HPD motif and the CTD I contains a ZFLM. Two protomers associate at the C-termini and form a homodimer. (B) pKKU1029 carries the dnaJ gene fused to a sequence coding for a Strep-tag at the 3’-end, whereas pKKU1125 carries the dnaJ gene fused with a sequence coding for a His-tag at the 5’-end. (C) DnaJ (10 μM) was incubated at 30°C for 30 min. After addition of molecular mass standards (β-amylase, 200 kDa; apo-transferrin, 81 kDa; carbonic anhydrase, 29 kDa), the mixture was loaded onto a Superdex 200 gel-filtration column. Aliquots from eluted fractions were subjected to SDS-PAGE. Proteins were detected by staining with CBB. Gel images were captured using an LAS3000 image analyzer. Fraction numbers are shown above the gel images. Molecular mass markers (in kilodaltons) are indicated to the left, and proteins are indicated to the right: 1, apo-transferrin; 2, β-amylase; 3, DnaJ; 4, carbonic anhydrase. (D) DnaJs (6 μg) were treated with thrombin (0.8 unit) in Buffer J4 (30 μl) at 22°C for 48 h, and 2 μg was electrophoresed (lanes 8–10) with untreated proteins (2 μg; lanes 5–7). Other DnaJs [(WT)-(WT), thrombin-treated (His-WT)-(His-WT), (WT-Strep)-(WT-Strep), (His-WT)-(His-WT)] (1 μg each) were also electrophoresed (lanes 1–4). Proteins were detected by staining with CBB. DnaJ is often observed as two or three bands due to the modification during electrophoresis. The components of each DnaJ are shown above the image: W, WT protomer; W × 2, two WT protomers; 33, H33Q protomer; 35, D35N protomer. The tag added to each protomer is indicated to the left, and ‘−’ means the authentic DnaJ protomer. Tb is an abbreviation for thrombin: +, thrombin treated; −, thrombin untreated. Fig. 1 View largeDownload slide Confirmation of purified DnaJs. (A) The DnaJ protomer is 376 amino acids with a J domain (1–70), a G/F rich region (71–105) and a CTD. The CTD is divided into two subdomains, CTD I (110–253) and CTD II (254–331). The J domain contains a HPD motif and the CTD I contains a ZFLM. Two protomers associate at the C-termini and form a homodimer. (B) pKKU1029 carries the dnaJ gene fused to a sequence coding for a Strep-tag at the 3’-end, whereas pKKU1125 carries the dnaJ gene fused with a sequence coding for a His-tag at the 5’-end. (C) DnaJ (10 μM) was incubated at 30°C for 30 min. After addition of molecular mass standards (β-amylase, 200 kDa; apo-transferrin, 81 kDa; carbonic anhydrase, 29 kDa), the mixture was loaded onto a Superdex 200 gel-filtration column. Aliquots from eluted fractions were subjected to SDS-PAGE. Proteins were detected by staining with CBB. Gel images were captured using an LAS3000 image analyzer. Fraction numbers are shown above the gel images. Molecular mass markers (in kilodaltons) are indicated to the left, and proteins are indicated to the right: 1, apo-transferrin; 2, β-amylase; 3, DnaJ; 4, carbonic anhydrase. (D) DnaJs (6 μg) were treated with thrombin (0.8 unit) in Buffer J4 (30 μl) at 22°C for 48 h, and 2 μg was electrophoresed (lanes 8–10) with untreated proteins (2 μg; lanes 5–7). Other DnaJs [(WT)-(WT), thrombin-treated (His-WT)-(His-WT), (WT-Strep)-(WT-Strep), (His-WT)-(His-WT)] (1 μg each) were also electrophoresed (lanes 1–4). Proteins were detected by staining with CBB. DnaJ is often observed as two or three bands due to the modification during electrophoresis. The components of each DnaJ are shown above the image: W, WT protomer; W × 2, two WT protomers; 33, H33Q protomer; 35, D35N protomer. The tag added to each protomer is indicated to the left, and ‘−’ means the authentic DnaJ protomer. Tb is an abbreviation for thrombin: +, thrombin treated; −, thrombin untreated. Materials and Methods Bacterial strains and plasmids The following Escherichia coli strains were used. HB101 [F-supE Δ(mcrC-mrr) recA ara-14 proA lacY galK rpsL xyl-5 mtl-1 leuB thi-1] was used for plasmid construction. MC4100 [F-araD Δ(argF-lac) U169 rpsL relA flbB deoC ptsF rbsR], KY1456 (MC4100 dnaJ:: Tn10-42) (58), HI1006 [F-araD139 Δ(araABIOC leu)7697 Δ(lacIPOZY)X74 galU galK strA trpA38 recA], and HMS174(DE3) [F-recA1 hsdR19 rpoB331 (DE3)] were used for protein purification. The λDE3 prophage carries a gene cording for T7 RNA polymerase that recognizes the T7 promoter (T7p). T7 RNA polymerase is induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to the culture medium. KY1456 was also used for testing the activity of tagged DnaJs in vivo. pKV1142 is a pTrc99A (ori: pMB1) (Pharmacia) derivative that carries ampicillin resistance gene (bla), the lacIq gene and the IPTG-dependent trc promoter (trcp) (59). pKV1961 and pKV2237 are pKV1142 derivatives carrying the dnaJ and dnaJ-his gene, respectively (60), and based on these plasmids, expression vectors for DnaJ mutants (H33Q and D35N-His) were constructed. pKV1900 is a pKV1142 derivative carrying the grpE gene. pAR3 is a pACYC184 (ori: p15A) derivative that carries the araC gene and the araB promoter (araBp) from Salmonella typhimurium, and chloramphenicol resistance gene (cat) (61). The dnaJ gene fused with a sequence coding for a Strep-tag (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) at the 3’-end was cloned under the araBp, and the resulting plasmid was designated pKKU1029 (Fig. 1B). The strep-tag sequence was derived from pASG-IBA162 (IBA). Three codons for glycine, serine, and alanine were inserted between the last codon of dnaJ and the strep-tag sequence. In the following sections, Strep-tagged DnaJ will be referred to as ‘DnaJ-Strep’ and especially as ‘WT-Strep’ for WT DnaJ. pET15b (ori: pMB1) (Novagen) carries the bla gene, the lacI gene and the T7 promoter. In addition, two sequences coding for a His-tag (six histidine residues) and a thrombin digestion site followed by the dnaJ gene were cloned downstream of the T7 promoter. The resulting plasmid was designated pKKU1125 (Fig. 1B). The DnaJ produced has the His-tag at the N-terminus of DnaJ, and the thrombin digestion site is located between the His-tag and DnaJ. His-tagged DnaJ will be referred to as ‘His-DnaJ’ and especially as ‘His-WT’ for WT DnaJ. Both pKKU1029 and pKKU1125 were constructed using an InFusion HD cloning kit (Clontech) with proper primer sets. Based on pKKU1125, a plasmid carrying the dnaJ mutant coding for H33Q or D35N DnaJ was constructed by site-directed mutagenesis using a PrimeSTAR Max DNA polymerase (Takara) and the appropriate primer sets according to the manufacturer’s instructions. The sequence of each dnaJ gene was confirmed by DNA sequence analysis. Media and chemicals L broth (per 1 litre; 10 g tryptone, 5 g yeast extract, 5 g NaCl, pH 7.4) was used for cell growth. Ampicillin (50 μg/ml) and chloramphenicol (20 μg/ml) were added when necessary. Chemicals were purchased from Nacalai Tesque, Wako Pure Chemicals or Sigma-Aldrich, unless otherwise specified. Protein purification All the protein purification steps were performed at 4°C. DnaK, the authentic DnaJ [(untagged WT)-(untagged WT) homodimeric DnaJ], a DnaJ mutant [(untagged H33Q)-(untagged H33Q) homodimeric DnaJ], and σ32 were purified as described previously in (60, 62). (WT-Strep)-(WT-Strep) was purified from HI1006 cells harbouring pKKU1029. The cells were grown to late log phase in L broth containing 20 μg/ml chloramphenicol at 30°C, and (WT-Strep)-(WT-Strep) synthesis was induced with 1 mg/ml arabinose. The cells were harvested after 3 h, resuspended in Buffer J1 [50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10 mM dithiothreitol, 10% (w/v) sucrose, 1 mg/ml lysozyme, and 0.6% (w/v) Brij58] (47), maintained on ice for 45 min, and disrupted by sonication. The resulting lysate was centrifuged at 75,000 × g for 90 min. The pellet was resuspended in Buffer J3 [50 mM Tris-HCl (pH 7.5), 2 mM β-mercaptoethanol, 10% sucrose, 100 mM KCl, and 0.5% (v/v) TritonX-100] and gently rotated for 60 min. After centrifugation at 75,000 × g for 90 min, the supernatant was loaded onto a Strep-Tactin Sepharose column (IBA) equilibrated with Buffer J3. After washing with Buffer J4 [50 mM Tris-HCl (pH 7.5), 2 mM β-mercaptoethanol, 10% (w/v) sucrose and 500 mM NaCl], (WT-Strep)-(WT-Strep) was eluted with Buffer J4 containing 2.5 mM desthiobiotin (IBA). (WT-Strep)-(WT-Strep) was concentrated, and desthiobiotin was removed, using a centrifugal filter device (Millipore). (His-WT)-(His-WT) was purified from HMS174(DE3) cells harbouring pKKU1125. The cells were grown to late log phase in L broth containing 50 μg/ml ampicillin at 30°C, and (His-WT)-(His-WT) synthesis was induced with 1 mM IPTG. The cells were harvested after 3 h and resuspended in Buffer J1. Cell disruption, the first ultracentrifugation, resuspension and the second ultracentrifugation were performed identically as the purification of (WT-Strep)-(WT-Strep). After the second ultracentrifugation, the supernatant was loaded onto a Ni2+-NTA agarose column (QIAGEN) equilibrated with Buffer J3. After washing with Buffer J4 containing 50 mM imidazole, (His-WT)-(His-WT) was eluted with Buffer J4 containing 250 mM imidazole. (His-WT)-(His-WT) was concentrated, and imidazole was removed, using a centrifugal filter device. (WT-His)-(WT-His) and (D35N-His)-(D35N-His) were purified from KY1456 cells harbouring pKV2237 and the pKV2237 derivative, respectively, in the same manner. GrpE was purified from HI1006 cells harbouring pKV1900. The cells were grown until the late-log phase in L broth containing 50 μg/ml ampicillin at 30°C, and GrpE synthesis was induced with 1 mM IPTG. Cells were harvested after 3 h, resuspended in Buffer A [50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, 10% (v/v) glycerol] containing 0.1% lysozyme, and kept on ice for 30 min. After addition of sodium deoxycholate to 0.05%, cells were disrupted by sonication, and the resulting lysate was centrifuged at 75,000 × g for 90 min. The supernatant was treated with ammonium sulphate. Proteins that were precipitated in a range of ammonium sulphate concentrations between 0.24 and 0.31 g/ml, were dissolved, dialysed against Buffer A and loaded onto a HiTrap heparin column (GE Healthcare). The flowthrough fraction was loaded onto a HiTrap Q-Sepharose column (GE Healthcare). Proteins were eluted with Buffer A containing a linear gradient of NaCl. The fractions containing GrpE were applied to a HiPrep Sephacryl S-100 column (GE Healthcare) equilibrated with Buffer A containing 0.1 M KCl. Fractions containing GrpE were stored. All the purified proteins, except for σ32, were more than 90% pure, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie Brilliant Blue (CBB). The purity of σ32 was ∼80%. The purified proteins were quantified using Bradford protein assay reagent (Bio-Rad). Because the functional forms of DnaJ and GrpE are dimer, their concentrations were calculated based on the molecular masses of their dimeric forms. Gel filtration DnaJ (10 μM) was incubated in Buffer G2 [20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM dithiothreitol, 500 mM NaCl, 5 mM Mg(CH3COO)2, 10% (v/v) glycerol] at 30°C for 30 min (total volume: 120 μl). After addition of molecular mass standards (β-amylase, 200 kD; apo-transferrin, 81 kD; carbonic anhydrase, 29 kD) (total volume: 150 μl), 100 μl of the mixture was loaded on a Superdex 200 gel filtration column (GE Healthcare). Aliquots (15 μl each) from eluted fractions (500 μl each) were subjected to SDS-PAGE. Proteins were detected by staining with CBB. Gel images were captured using an LAS3000 image analyzer (Fuji film). Stability of the DnaJ dimeric conformation DnaJ (1 μM) was incubated in Buffer G3 [20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5 mM Mg(CH3COO)2, and 10% (v/v) glycerol] at 30°C for 2 h (total volume: 100 μl). Next, 1 ml of Buffer G3 containing 30 mM imidazole and 0.1% Tween20 was added. After the addition of 20 μl of a 50% slurry of Ni2+-NTA agarose (QIAGEN), the mixture was rotated at 4°C for 1 h. Agarose beads were washed three times with Buffer G3 containing 30 mM imidazole and 0.1% Tween20, and DnaJ was dissociated from the agarose beads by treating the precipitate with 35 μl of Buffer J4 containing 250 mM imidazole. Half of the eluate was treated with thrombin (0.47 U) at 22°C for 48 h. Proteins were subjected to SDS-PAGE and detected by CBB staining and immunoblotting with an anti-DnaJ antiserum (60, 62) or an anti-His-tag antibody (Wako). Gel and membrane images were captured using an LAS3000 image analyzer, and the protein bands were quantified with Multi Gauge software (Fuji film). Chaperone activity of DnaJs To test chaperone activity of DnaJs, the ability to suppress the aggregation of urea-denatured luciferase was measured. Luciferase (Sigma) from Photinus pyralis was denatured by dissolving (8 μM) and incubating it in Denaturation buffer [30 mM Tris-HCl (pH 7.5), 5 mM dithiothreitol, 8 M urea] at room temperature for 12 h. The denatured luciferase was diluted 100 times in Assay buffer [30 mM HEPES-KOH (pH 7.5), 1 mM dithiothreitol, 40 mM KCl, 50 mM NaCl, 7 mM Mg(CH3COO)2] (total volume: 600 μl) containing BSA (40 or 80 nM) or DnaJ (40 or 80 nM) and incubated at room temperature in the chamber of a RF-5300 fluorescence photometer (Shimadzu). Light scattering was measured at an excitation and emission wavelength of 350 nm. ATPase assay To test cochaperone activity of DnaJs, DnaK ATPase activity was measured in the presence of GrpE and σ32 as well as each DnaJ protein. DnaK (0.46 μM), GrpE (0.25 μM), σ32 (1.84 μM), and each DnaJ (0.2, 0.4, 0.6 or 0.8 μM) were mixed in Buffer M2 [50 mM Tris-HCl (pH 7.5) 100 mM KCl, 25 mM MgCl2] at 30°C. The reactions were initiated by adding ATP (100 μM), and aliquots (80 μl each) were withdrawn at 2.5 min intervals. The concentration of inorganic free phosphate was measured using the Malachite Green Phosphate Assay Kit (Bio Assay Systems). Glucose-6-phosphate dehydrogenase refolding assay Glucose-6-phosphate dehydrogenase (G6PDH) refolding assay was performed as previously described (63). The G6PDH concentration was calculated from the molecular mass of its dimeric form. G6PDH (Wako) from Leuconostoc mesenteroides was dissolved in Buffer A to a concentration of 100–200 μM and diluted in G6PDH refolding buffer [50 mM HEPES-KOH (pH7.5), 150 mM KCl, 20 mM Mg(CH3COO)2, 10 mM DTT] (30 μl) to a concentration of 0.5 μM. After G6PDH was denatured at 52°C for 10 min, the solution was incubated at 25°C for 10 min. An equal volume of G6PDH refolding buffer containing the DnaK chaperone system (12 μM DnaK, 0.2–0.8 μM DnaJ, 0.6 μM GrpE), 8 mM ATP and an ATP regeneration system (200 μg/ml creatine kinase, 40 mM creatine phosphate) was added and the refolding reaction was initiated (final concentrations: 0.25 μM G6PDH, 6 μM DnaK, 0.1–0.4 μM DnaJ, 0.3 μM GrpE, 4 mM ATP). At indicated times, 10 μl aliquots were collected, and G6PDH was diluted in G6PDH reaction buffer [50 mM Tris-HCl (pH7.8), 3 mM MgCl2] to a concentration of 100 nM. Furthermore, 10 μl of 100 nM G6PDH was added to 990 μl of G6PDH reaction buffer containing glucose 6-phosphate and NAD+, and G6PDH reaction was started (final concentrations: 1 nM G6PDH, 3.3 mM glucose 6-phosphate, 2 mM NAD+). After 5 min, the absorbance was measured at 340 nm. The concentration of NADH was calculated based on the molar extinction coefficient (ε) of 6300/M/cm. Results A Strep-tagged protomer and a His-tagged protomer form a stable dimeric DnaJ molecule Expression vectors for WT-Strep (pKKU1029) and His-WT (pKKU1125) were constructed as described in the Materials and Methods (Fig. 1B). pKKU1029 is a derivative of pAR3, and the dnaJ-strep gene is expressed from the araBp by adding arabinose to the culture medium. Repression by AraC was incomplete. Therefore, when pKKU1029 was transformed into a ΔdnaJ strain (KY1456), the temperature sensitivity of the mutant was complemented at 42 and 44°C without arabinose addition, similar to the WT dnaJ expression vector [pKV1961, which also exhibited leaky dnaJ expression without IPTG addition to the culture medium] (data not shown). pKKU1125 is a derivative of pET15b, and the his-dnaJ gene is expressed from the T7p upon IPTG addition because the gene coding for T7 RNA polymerase is under the control of an IPTG-dependent promoter on the λDE3 prophage. When the same his-dnaJ gene was inserted under the trc promoter on pKV1142 and the resulting plasmid was transformed into KY1456, the temperature sensitivity was complemented at both 42 and 44°C (data not shown). Because the two vectors (pKKU1029 and pKKU1125) are compatible, they were co-transformed into HMS174(DE3). To induce the synthesis of both proteins at equal levels, the concentrations of arabinose and IPTG used in the culture medium were 1 mg/ml and 20 μM, respectively. Three hours after induction, cells were harvested, resuspended in Buffer J1, and disrupted by sonication. The lysate was centrifuged at 75,000 × g for 90 min. The DnaJ in the pellet was dissolved in Buffer J4. Because DnaJ tends to aggregate, dimeric DnaJ molecules were separated by gel filtration using a HiPrep Sephacryl S-300 column (GE healthcare) equilibrated with Buffer J4. Next, to obtain (WT-Strep)–(His-WT) dimeric molecules, two kinds of affinity chromatography were performed. Gel-filtration fractions containing dimeric DnaJ were loaded onto a Ni2+-NTA agarose column equilibrated with Buffer J4. After washing with Buffer J4 containing 30 mM imidazole, His-tagged DnaJs [(His-WT)-(His-WT) and (WT-Strep)-(His-WT)] were eluted with Buffer J4 containing 100 mM imidazole. The eluate was loaded onto a Strep-Tactin Sepharose column equilibrated with Buffer J4. After washing with Buffer J4, (WT-Strep)-(His-WT) was eluted with Buffer J4 containing 2.5 mM desthiobiotin. To confirm that the dimeric conformation was maintained during purification, purified DnaJ was separated by gel filtration using a Superdex 200 column (GE healthcare). The DnaJ protomer is 376 amino acids with a molecular mass of 41.1 kDa, whereas the molecular mass of (WT-Strep)-(His-WT) deduced from amino acid sequences is 85.4 kDa [the deduced molecular mass of WT-Strep (387 aa) is 42.4 kDa and that of His-WT (394 aa) is 43.0 kDa]. Purified (WT-Strep)-(His-WT) eluted in the same fractions as apo-transferrin (81 kDa) (Fig. 1C top panel). This result indicates that purified (WT-Strep)-(His-WT) maintains a dimeric conformation at 4°C. Nearly all Hsp40s contain the HPD motif in the J domain, and in DnaJ, this motif contains His33, Pro34 and Asp35. When a mutation substituting H33Q or D35N was introduced into the dnaJ-his gene on pKV2237 (pKV1142 trcp-dnaJ-his) and the resulting plasmid was transformed into KY1456, the mutant genes did not complement the temperature sensitivity of the mutant at 42°C unlike the WT dnaJ-his gene (data not shown). A codon substitution encoding H33Q or D35N was also introduced into the his-dnaJ gene on pKKU1125. The individual expression vectors were transformed into HMS174(DE3) with pKKU1029, and two heterodimeric DnaJs, (WT-Strep)-(His-H33Q) and (WT-Strep)-(His-D35N), were purified in the same manner as (WT-Strep)-(His-WT). When purified heterodimeric DnaJs were analysed by gel filtration, the results were similar to (WT-Strep)-(His-WT) (Fig. 1C middle and bottom panels), indicating that both heterodimers were also stable at 4°C. DnaJ-Strep and His-DnaJ have similar molecular masses, causing these proteins to run close together on a gel. Therefore, to confirm that purified DnaJs contained a Strep-tagged DnaJ protomer and a His-tagged DnaJ protomer, they were treated with thrombin to cleave the His-tag. The deduced molecular mass of thrombin-digested His-DnaJ (377 aa) was 41.1 kDa. Digestion reactions were performed under conditions that completely digested (His-WT)-(His-WT) (Fig. 1D lane 2 compared with lane 4). When purified DnaJs (Fig. 1D lanes 5–7) were treated and the resulting products were electrophoresed, two distinct bands were detected near a molecular mass of 40 kDa (Fig. 1D lanes 8–10). The upper bands corresponded to DnaJ-Strep (Fig. 1D lane 3), and the lower bands corresponded to thrombin-digested His-DnaJ (Fig. 1D lane 2). This result indicates that purified DnaJ molecules assume dimeric conformations consisting of a DnaJ-Strep protomer and one of three His-DnaJ protomers. Heterodimeric DnaJs containing a WT protomer and a mutated protomer retain chaperone activity The above results demonstrated that the (DnaJ-Strep)-(His-DnaJ) dimeric conformation was stable at 4°C during DnaJ purification. To test whether the (DnaJ-Strep)-(His-DnaJ) dimeric conformation is also maintained at high temperatures, the three (DnaJ-Strep)-(His-DnaJ) dimers were incubated at 30°C for 2 h and precipitated with Ni2+-NTA agarose. Then, the precipitated DnaJs were eluted with imidazole and electrophoresed. More than 50% of each DnaJ was recovered (Fig. 2A lanes 7–9). When the eluates were treated with thrombin, two bands from individual DnaJs were detected whose mobilities were similar to those of the DnaJ-Strep and digested His-DnaJ shown in Fig. 1D (Fig. 2A lanes 10–12), and their intensities were similar. Under the same conditions, (His-DnaJ)-(His-DnaJ) was almost completely digested (Fig. 2A lane 13 compared with lane 15), indicating that DnaJs precipitated by Ni2+-NTA agarose contain DnaJ-Strep and His-DnaJ at a molar ratio of 1:1. To confirm this result, DnaJ bands were detected by immunoblotting with an anti-DnaJ antiserum and an anti-His-tag antibody (Fig. 2B). When the anti-DnaJ antiserum was used to detect DnaJ, the results were identical to those obtained by CBB staining (Fig. 2B top panel). In detecting DnaJ with the anti-His-tag antibody, although the band intensity did not exhibit a linear relationship with DnaJ quantity (Fig. 2B middle and bottom panels lanes 1–3), the bands were not detected in the thrombin-treated products (Fig. 2B middle and bottom panels lanes 7–9). When signals were intensified (Fig. 2B bottom panel), 305 ng of DnaJ was detected with the anti-His-tag antibody, but no signals were obtained from the thrombin-treated DnaJs (Fig. 2B bottom panel lanes 7–9), although the band intensity for the thrombin-treated DnaJs was higher than that of 305 ng of DnaJ in the detection with the anti-DnaJ antiserum (Fig. 2B top panel lanes 7–9 compared with lane 2). These results indicate that the (DnaJ-Strep)-(His-DnaJ) dimeric conformation is stable at 30°C for at least 2 h. Fig. 2 View largeDownload slide Stability of the DnaJ dimeric conformation. DnaJ (1 μM) [(WT-Strep)-(WT-Strep) (negative control), (WT-Strep)-(His-WT), (WT-Strep)-(His-H33Q) or (WT-Strep)-(His-D35N)] was incubated at 30°C for 2 h. After precipitation with Ni2+-NTA agarose, DnaJ was eluted with 250 mM imidazole. Half of the eluate was treated with thrombin. (A) Thrombin-treated proteins (lanes 10–12) were subjected to SDS-PAGE with untreated proteins (lanes 6–9) and detected by CBB staining. The image was captured using an LAS3000 image analyzer. Serial dilutions of purified (WT-Strep)-(His-WT) were also electrophoresed to estimate the recovery efficiency by Ni2+-NTA agarose (lane 1–5); 2,440 ng corresponds to 100% recovery. Thrombin-treated (His-WT)-(His-WT) (1 μg; lane 13), (WT-Strep)-(WT-Strep) (1 μg; lane 14) and (His-WT)-(His-WT) (1 μg; lane 15) were also electrophoresed as molecular mass markers. (B) Thrombin-treated proteins shown in (A) (lanes 7–9) were subjected to SDS-PAGE with untreated proteins (lanes 4–6) and detected by immunoblotting with an anti-DnaJ antiserum (top panel) or an anti-His-tag antibody (middle and bottom panels). Images were captured using an LAS3000 image analyzer. The bottom panel is an intensified image of the middle panel. The image was intensified without changing the contrast using Multi Gauge software. Serial dilutions of purified (WT-Strep)-(His-WT) were also electrophoresed (lane 1–3); 610 ng corresponds to 50% recovery. Thrombin-treated (His-WT)-(His-WT) (0.5 μg; lane 10), (WT-Strep)-(WT-Strep) (0.5 μg; lane 11) and (His-WT)-(His-WT) (0.5 μg; lane 12) were also electrophoresed. In panels (A) and (B), molecular mass markers (in kilodaltons) are indicated to the left. The components of each DnaJ are shown above the images: W, WT protomer; W × 2, two WT protomers; 33, H33Q protomer; 35, D35N protomer. The tag added to each protomer is indicated to the left. Tb is an abbreviation for thrombin: +, thrombin treated; −, thrombin untreated. Fig. 2 View largeDownload slide Stability of the DnaJ dimeric conformation. DnaJ (1 μM) [(WT-Strep)-(WT-Strep) (negative control), (WT-Strep)-(His-WT), (WT-Strep)-(His-H33Q) or (WT-Strep)-(His-D35N)] was incubated at 30°C for 2 h. After precipitation with Ni2+-NTA agarose, DnaJ was eluted with 250 mM imidazole. Half of the eluate was treated with thrombin. (A) Thrombin-treated proteins (lanes 10–12) were subjected to SDS-PAGE with untreated proteins (lanes 6–9) and detected by CBB staining. The image was captured using an LAS3000 image analyzer. Serial dilutions of purified (WT-Strep)-(His-WT) were also electrophoresed to estimate the recovery efficiency by Ni2+-NTA agarose (lane 1–5); 2,440 ng corresponds to 100% recovery. Thrombin-treated (His-WT)-(His-WT) (1 μg; lane 13), (WT-Strep)-(WT-Strep) (1 μg; lane 14) and (His-WT)-(His-WT) (1 μg; lane 15) were also electrophoresed as molecular mass markers. (B) Thrombin-treated proteins shown in (A) (lanes 7–9) were subjected to SDS-PAGE with untreated proteins (lanes 4–6) and detected by immunoblotting with an anti-DnaJ antiserum (top panel) or an anti-His-tag antibody (middle and bottom panels). Images were captured using an LAS3000 image analyzer. The bottom panel is an intensified image of the middle panel. The image was intensified without changing the contrast using Multi Gauge software. Serial dilutions of purified (WT-Strep)-(His-WT) were also electrophoresed (lane 1–3); 610 ng corresponds to 50% recovery. Thrombin-treated (His-WT)-(His-WT) (0.5 μg; lane 10), (WT-Strep)-(WT-Strep) (0.5 μg; lane 11) and (His-WT)-(His-WT) (0.5 μg; lane 12) were also electrophoresed. In panels (A) and (B), molecular mass markers (in kilodaltons) are indicated to the left. The components of each DnaJ are shown above the images: W, WT protomer; W × 2, two WT protomers; 33, H33Q protomer; 35, D35N protomer. The tag added to each protomer is indicated to the left. Tb is an abbreviation for thrombin: +, thrombin treated; −, thrombin untreated. Hsp40 has chaperone activity that prevents the aggregation of denatured proteins in addition to cochaperone activity (25, 30). The chaperone activity of heterodimeric DnaJs was investigated by performing an aggregation prevention assay with denatured luciferase (Fig. 3). After urea-denatured luciferase was diluted 100 times to 80 nM, the light scattering intensity gradually increased over a period of 20 min (Fig. 3 cross symbols), indicating luciferase aggregation. However, when the authentic DnaJ [(untagged WT)-(untagged WT)] was added at the same concentration as the luciferase, the light scattering intensity minimally increased, indicating that luciferase aggregation was almost completely inhibited. The same phenomenon was observed with all three (DnaJ-Strep)-(His-DnaJ) DnaJs (Fig. 3 straight lines). When the DnaJ concentration was decreased to half that of luciferase, moderate inhibition of luciferase aggregation was observed for all the DnaJ constructs including the authentic DnaJ (Fig. 3 dotted lines). These results demonstrate that the heterodimeric DnaJs possess adequate substrate-binding activity, or chaperone activity. Fig. 3 View largeDownload slide Chaperone activity of DnaJs. Denatured luciferase (8 μM) was diluted 100 times in Assay buffer containing BSA or DnaJ [40 nM (dotted lines) or 80 nM (straight lines)] and incubated at room temperature. Light scattering was measured at an excitation and emission wavelength of 350 nm. Crosses, BSA; closed circles, authentic DnaJ; open circles, (WT-Strep)-(His-WT); triangles, (WT-Strep)-(His-H33Q); squares, (WT-Strep)-(His-D35N). The mean values from three experiments are shown with SD (error bars). Fig. 3 View largeDownload slide Chaperone activity of DnaJs. Denatured luciferase (8 μM) was diluted 100 times in Assay buffer containing BSA or DnaJ [40 nM (dotted lines) or 80 nM (straight lines)] and incubated at room temperature. Light scattering was measured at an excitation and emission wavelength of 350 nm. Crosses, BSA; closed circles, authentic DnaJ; open circles, (WT-Strep)-(His-WT); triangles, (WT-Strep)-(His-H33Q); squares, (WT-Strep)-(His-D35N). The mean values from three experiments are shown with SD (error bars). Cochaperone activity of the heterodimeric DnaJs is reduced to half of the WT DnaJ activity Hsp70 has weak ATPase activity, and its activity is stimulated >50-fold by cochaperones and substrates (13, 64, 65). To examine whether the heterodimeric DnaJs have cochaperone activity, the ATPase activity of DnaK was measured in the presence of GrpE and a substrate, σ32 as well as individual DnaJs. σ32, the E. coli heat shock factor, is known to bind to DnaK and DnaJ (57, 60, 62, 66–69), and to stimulate DnaK ATPase activity (70). The ATPase activity linearly increased in the presence of WT DnaJs [the authentic DnaJ and (WT-His)-(WT-His)] (Fig. 4A closed circles and closed diamonds, respectively), whereas ATPase activity was not detected in the absence of DnaJ (Fig. 4A open diamonds). In the cases of homodimeric DnaJ mutants, (H33Q)-(H33Q) did not accelerate DnaK ATPase activity (Fig. 4A closed triangles), whereas (D35N-His)-(D35N-His) slightly stimulated DnaK ATPase activity (Fig. 4A closed squares). When the concentration of DnaJ was changed, the ATPase activity linearly increased for 7.5 min at any concentration of the authentic DnaJ (Fig. 4B closed circles). For (WT-Strep)-(His-WT), DnaK ATPase activity was comparable to that for the authentic DnaJ (Fig. 4B open circles). This result indicates that neither the Strep-tag nor the His-tag hinder the cochaperone activity of DnaJ. In the presence of the two heterodimeric DnaJs, DnaK ATPase activity decreased for all ranges of DnaJ concentration used (Fig. 4B). The ATP hydrolysis rates calculated from the slopes of the graphs were reduced to ∼55% at 0.2 μM DnaJ, ∼60% at 0.4 μM DnaJ, ∼65% at 0.6 μM DnaJ, and ∼70% at 0.8 μM DnaJ compared with those of WT DnaJs [the authentic DnaJ and (WT-Strep)-(His-WT)] (Fig. 4C). These results indicate that two intact J domains are required for full DnaJ cochaperone activity. Intriguingly, the two heterodimeric DnaJs similarly stimulated DnaK ATPase activity at twice the concentrations of WT DnaJs, i.e. the ATP hydrolysis rates in the presence of either heterodimeric DnaJ at concentrations of 0.4 and 0.8 μM were similar to those in the presence of WT DnaJs at the concentrations of 0.2 and 0.4 μM, respectively (Fig. 4C). These results indicate that the heterodimeric DnaJs simply lose half of their cochaperone activity. Fig. 4 View largeDownload slide DnaK ATPase activity in the presence of various DnaJs. (A) DnaK (0.46 μM), GrpE (0.25 μM), σ32 (1.84 μM) and each DnaJ (0 or 0.8 μM) were mixed at 30°C. Reactions were initiated by adding ATP (100 μM), and aliquots were withdrawn at 2.5 min intervals. The concentration of inorganic free phosphate was measured. Open diamonds, without DnaJ; closed circles, authentic DnaJ; closed diamonds, (WT-His)-(WT-His); closed triangles, (H33Q)-(H33Q); closed squares, (D35N-His)-(D35N-His). Closed triangles overlap with open diamonds. (B) DnaK (0.46 μM), GrpE (0.25 μM), σ32 (1.84 μM), and each DnaJ (0.2, 0.4, 0.6 or 0.8 μM) were mixed and DnaK ATPase activity was measured shown in (A). Closed circles, authentic DnaJ; open circles, (WT-Strep)-(His-WT); open triangles, (WT-Strep)-(His-H33Q); open squares, (WT-Strep)-(His-D35N). In most cases, open triangles overlap with open squares. (C) The ATP hydrolysis rates were calculated from the slopes of the graphs shown in (B). Black bars, authentic DnaJ; dark grey bars, (WT-Strep)-(His-WT); light grey bars, (WT-Strep)-(His-H33Q); white bars, (WT-Strep)-(His-D35N). In panels (A–C), the mean values from three experiments are shown with SD (error bars). Fig. 4 View largeDownload slide DnaK ATPase activity in the presence of various DnaJs. (A) DnaK (0.46 μM), GrpE (0.25 μM), σ32 (1.84 μM) and each DnaJ (0 or 0.8 μM) were mixed at 30°C. Reactions were initiated by adding ATP (100 μM), and aliquots were withdrawn at 2.5 min intervals. The concentration of inorganic free phosphate was measured. Open diamonds, without DnaJ; closed circles, authentic DnaJ; closed diamonds, (WT-His)-(WT-His); closed triangles, (H33Q)-(H33Q); closed squares, (D35N-His)-(D35N-His). Closed triangles overlap with open diamonds. (B) DnaK (0.46 μM), GrpE (0.25 μM), σ32 (1.84 μM), and each DnaJ (0.2, 0.4, 0.6 or 0.8 μM) were mixed and DnaK ATPase activity was measured shown in (A). Closed circles, authentic DnaJ; open circles, (WT-Strep)-(His-WT); open triangles, (WT-Strep)-(His-H33Q); open squares, (WT-Strep)-(His-D35N). In most cases, open triangles overlap with open squares. (C) The ATP hydrolysis rates were calculated from the slopes of the graphs shown in (B). Black bars, authentic DnaJ; dark grey bars, (WT-Strep)-(His-WT); light grey bars, (WT-Strep)-(His-H33Q); white bars, (WT-Strep)-(His-D35N). In panels (A–C), the mean values from three experiments are shown with SD (error bars). The DnaK chaperone system refolds denatured proteins (10, 14, 25, 28, 29, 71). In this process, DnaJ binds to denatured proteins to prevent their aggregation and accelerates DnaK ATPase activity to promote tight DnaK binding to denatured proteins. To further investigate the cochaperone activity of the heterodimeric DnaJs, a refolding assay using heat-denatured G6PDH was performed. The refolding ratio (%) for denatured G6PDH was calculated by dividing the produced NADH at the indicated times by the amount of produced NADH by an equal amount of native G6PDH. After denaturing G6PDH at 52°C for 10 min, activity was not spontaneously restored at 25°C even after 2 h (data not shown). When heat-denatured G6PDH was incubated at 25°C in the presence of the DnaK chaperone system, activity was gradually restored (Fig. 5A). However, renaturation of G6PDH was not observed either in the absence of DnaJ or in the presence of homodimeric DnaJ mutants (Fig. 5A). When the concentration of DnaJ was changed, the final yields of active G6PDH increased with increasing concentration of DnaJ (Fig. 5B). In the presence of WT DnaJs at 0.4 μM, denatured G6PDH (0.25 μM) recovered up to ∼80% activity after 90 min. To easily compare the refolding activity of DnaJs, refolding rates (%/min) were calculated from the refolding ratios using the steepest slope values from individual graphs in Fig. 5B (Fig. 5C). Whereas (WT-Strep)-(His-WT) showed refolding rates comparable to the authentic DnaJ at all DnaJ concentrations, the refolding rates in the presence of either heterodimeric DnaJ decreased by 50–70% at 0.1 μM DnaJ, ∼40% at 0.2 μM DnaJ, and ∼35% at 0.4 μM DnaJ. Again, these results indicate that two intact J domains per dimeric DnaJ molecule are required for effective cochaperone activity. The reduced DnaK ATPase activity probably decreased the amount of DnaK bound to G6PDH and, in turn, resulted in a low efficiency for G6PDH refolding. Similar to the results for DnaK ATPase activity, the refolding rates for the DnaK chaperone system including either heterodimeric DnaJ at doubled concentrations showed comparable values to WT DnaJs (Fig. 5C). Fig. 5 View largeDownload slide G6PDH refolding activity by a DnaK chaperone system with a heterodimeric DnaJ. (A) G6PDH (0.5 μM) was denatured at 52°C for 10 min. After the solution was incubated at 25°C for 10 min, an equal volume of the G6PDH refolding buffer containing the DnaK chaperone system was added, and the refolding reaction was initiated (final concentrations: 0.25 μM G6PDH, 6 μM DnaK, 0 or 0.4 μM DnaJ, 0.3 μM GrpE, 4 mM ATP). G6PDH activity was measured at the indicated times. Native G6PDH was treated in the same manner, and the refolding ratios were determined by comparing to the values obtained with native G6PDH (100%). Open diamonds, without DnaJ; closed circles, authentic DnaJ; closed diamonds, (WT-His)-(WT-His); closed triangles, (H33Q)-(H33Q); closed squares, (D35N-His)-(D35N-His). Open diamonds, closed triangles, and closed squares overlap each other. (B) G6PDH refolding assays were performed in the same manner as in (A). Final concentration of DnaJ ranged from 0.1 to 0.4 μM. Closed circles, authentic DnaJ; open circles, (WT-Strep)-(His-WT); open triangles, (WT-Strep)-(His-H33Q); open squares, (WT-Strep)-(His-D35N). In the case of 0.4 μM DnaJ, closed circles overlap with open circles. (C) The G6PDH refolding rates were calculated from the steepest slope in each graph shown in (B). Black bars, authentic DnaJ; dark grey bars, (WT-Strep)-(His-WT); light grey bars, (WT-Strep)-(His-H33Q); white bars, (WT-Strep)-(His-D35N). In panels (A–C), the mean values from three experiments are shown with SD (error bars). Fig. 5 View largeDownload slide G6PDH refolding activity by a DnaK chaperone system with a heterodimeric DnaJ. (A) G6PDH (0.5 μM) was denatured at 52°C for 10 min. After the solution was incubated at 25°C for 10 min, an equal volume of the G6PDH refolding buffer containing the DnaK chaperone system was added, and the refolding reaction was initiated (final concentrations: 0.25 μM G6PDH, 6 μM DnaK, 0 or 0.4 μM DnaJ, 0.3 μM GrpE, 4 mM ATP). G6PDH activity was measured at the indicated times. Native G6PDH was treated in the same manner, and the refolding ratios were determined by comparing to the values obtained with native G6PDH (100%). Open diamonds, without DnaJ; closed circles, authentic DnaJ; closed diamonds, (WT-His)-(WT-His); closed triangles, (H33Q)-(H33Q); closed squares, (D35N-His)-(D35N-His). Open diamonds, closed triangles, and closed squares overlap each other. (B) G6PDH refolding assays were performed in the same manner as in (A). Final concentration of DnaJ ranged from 0.1 to 0.4 μM. Closed circles, authentic DnaJ; open circles, (WT-Strep)-(His-WT); open triangles, (WT-Strep)-(His-H33Q); open squares, (WT-Strep)-(His-D35N). In the case of 0.4 μM DnaJ, closed circles overlap with open circles. (C) The G6PDH refolding rates were calculated from the steepest slope in each graph shown in (B). Black bars, authentic DnaJ; dark grey bars, (WT-Strep)-(His-WT); light grey bars, (WT-Strep)-(His-H33Q); white bars, (WT-Strep)-(His-D35N). In panels (A–C), the mean values from three experiments are shown with SD (error bars). Discussion The dimeric conformation of Hsp40 is needed for its chaperone activity (42, 44). It also seems necessary for Hsp40 cochaperone activity, because monomeric Hsp40 mutants compensate for defects caused by Hsp40 insufficiency to some extent but the compensation is not complete (34, 56, 57). However, whether or not the dimeric structure of Hsp40 is sufficient for cochaperone activity is unknown. In this study, to address this question, two heterodimeric DnaJs [(WT-Strep)-(His-H33Q), (WT-Strep)-(His-D35N)] with an amino acid substitution in one protomer were constructed. The H33Q and D35N mutations were located in the HPD motif, which interacts with the DnaK nucleotide-binding domain to stimulate ATPase activity. Although the two heterodimeric DnaJs maintained chaperone activity (Fig. 3), they exhibited reduced cochaperone activity (Figs 4 and 5), indicating that the dimeric structure of Hsp40 is not sufficient for full cochaperone activity and that two intact J domains per homodimeric Hsp40 molecule are needed to efficiently stimulate Hsp70 ATPase activity on substrates. These results suggest that the interaction of DnaJ with DnaK on a substrate does not occur at a molar ratio of 1:1, i.e. one homodimeric DnaJ molecule induces the simultaneous binding of multiple DnaK molecules to the same substrate molecule. Sarbeng et al. (72) reported that DnaK transiently forms a dimeric structure that is responsible for efficient DnaJ interaction. Their model supports the notion that one homodimeric DnaJ molecule simultaneously stimulates multiple DnaK molecules on one substrate molecule. The DnaK ATPase assays shown in Fig. 4 were performed under conditions in which the amount of substrate (σ32; 1.84 μM) was larger than the amounts of DnaK (0.46 μM) and DnaJ (0.2, 0.4, 0.6 and 0.8 μM). Given that the dissociation constant for DnaJ–σ32 interaction is 20 nM (69), most DnaJ molecules would bind to σ32 under the conditions used and DnaK would interact with DnaJ on σ32. For WT DnaJs [the authentic DnaJ and (WT-Strep)-(His-WT)], the ATPase activity of DnaK increased as the DnaJ concentration increased. Similar results were obtained for the two heterodimeric DnaJs, but DnaK ATPase activity was reduced to 55–70% compared with WT DnaJs, probably due to the inactivation of one of two J domains. The difference in stimulation of DnaK ATPase activity between WT DnaJs and the heterodimeric DnaJs became smaller at higher DnaJ concentrations. It is likely that DnaK was the limiting factor at higher DnaJ concentrations. These results indicate that DnaK ATPase activity is stimulated through the interaction between DnaK and the J domain at a molar ratio of 1:1. The G6PDH refolding assays shown in Fig. 5 were performed under conditions of excess DnaK (6 μM) compared with DnaJ (0.1, 0.2 and 0.4 μM) and G6PDH (0.25 μM). Refolding activity for the DnaK chaperone system including the heterodimeric DnaJs was reduced to half activity of the DnaK chaperone system including WT DnaJs. Because heat-denatured G6PDH molecules form aggregates, a G6PDH molecule must be disentangled from an aggregate before refolding (10). Considering that in the disentanglement reaction, several DnaK molecules were needed per G6PDH molecule (10), it seems that multiple DnaK molecules bind to one G6PDH aggregate. Therefore, the present data suggests that one homodimeric WT DnaJ molecule efficiently stimulates multiple DnaK molecules to bind to the same substrate molecule, which leads to efficient refolding of denatured proteins. Although the promotion of G6PDH refolding by the heterodimeric DnaJs was not efficient, the heterodimeric DnaJs significantly promoted G6PDH refolding even at 0.1 μM (Fig. 5A). Cobinding of multiple DnaJs or DnaJ rebinding to one G6PDH aggregate might enable multipe DnaK molecules to bind to the same G6PDH aggregate. The percentage of the refolding rate for the heterodimeric DnaJs compared with that of WT DnaJs was smaller at 0.1 μM DnaJ (30–50%) than that at 0.4 μM DanJ (65%), which might reflect the difficulty of cobinding of multiple DnaJs or DnaJ rebinding to the same G6PDH aggregate at lower DnaJ concentrations. The activity of (WT-Strep)-(His-D35N) was slightly higher than that of (WT-Strep)-(His-H33Q). It might be related to the fact that a J domain fragment containing the D35N mutation shows higher affinity for the DnaK nucleotide-binding domain fragment than the WT J domain fragment (73). In fact, DnaK ATPase activity was slightly stimulated by (D35N-His)-(D35N-His) homodimeric mutant (Fig. 4A). Both heterodimeric DnaJs showed a similar acceleration of ATPase activity and refolding activity of DnaK at twice the concentrations of WT DnaJs (Figs 4C and 5C). These results indicate that two J domains in one homodimeric DnaJ molecule function additively, not synergistically. It means that each J domain independently stimulates a DnaK molecule to bind to a substrate. The idea is consistent with the fact that monomeric Hsp40 mutnats lacking substrate-binding activity have significant cochaperone ativity. 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( 2003) The J-domain of Hsp40 couples ATP hydrolysis to substrate capture in Hsp70. Biochemistry  42, 4937– 4944 Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations araBp araB promoter CBB Coomassie Brilliant Blue CTD C-terminal domain G6PDH glucose-6-phosphate dehydrogenase HPD His-Pro-Asp Hsp heat shock protein IPTG isopropyl-β-D-thiogalactopyranoside NEF nucleotide-exchange factor SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis T7p T7 promoter; trcp, trc promoter ZFLM zinc finger-like motif © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Two J domains ensure high cochaperone activity of DnaJ, Escherichia coli heat shock protein 40

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
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0021-924X
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1756-2651
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10.1093/jb/mvy038
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Abstract

Abstract Heat shock protein 70 (Hsp70) chaperone systems consist of Hsp70, Hsp40 and a nucleotide-exchange factor and function to help unfolded proteins achieve their native conformations. Typical Hsp40s assume a homodimeric structure and have both chaperone and cochaperone activity. The dimeric structure is critical for chaperone function, whereas the relationship between the dimeric structure and cochaperone function is hardly known. Here, we examined whether two intact protomers are required for cochaperone activity of Hsp40 using an Escherichia coli Hsp70 chaperone system consisting of DnaK, DnaJ and GrpE. The expression systems were generated and two heterodimeric DnaJs that included a mutated protomer lacking cochaperone activity were purified. Normal chaperone activity was demonstrated by assessing aggregation prevention activity using urea-denatured luciferase. The heterodimeric DnaJs were investigated for cochaperone activity by measuring DnaK ATPase activity and the heat-denatured glucose-6-phosphate dehydrogenase refolding activity of the DnaK chaperone system, and they showed reduced cochaperone activity. These results indicate that two intact protomers are required for high cochaperone activity of DnaJ, suggesting that one homodimeric DnaJ molecule promotes the simultaneous binding of multiple DnaK molecules to one substrate molecule, and that this binding mode is required for the efficient folding of denatured proteins. chaperones, DnaJ, DnaK, Hsp40, Hsp70 Newly synthesized polypeptides fold into their specific conformations to fulfil their functions. Although the final conformation of a polypeptide is determined by its primary structure, many polypeptides need help from a set of proteins called molecular chaperones to assume their final conformations. Molecular chaperones are also involved in various cellular functions, such as the refolding and degradation of denatured proteins. Heat shock protein 100 (Hsp100)s, Hsp90s, Hsp70s, chaperonins and small Hsps are known to be major molecular chaperones, and Hsp70s play a central role among them (1–8). Hsp70 is thought to bind the hydrophobic regions exposed on the surface of denatured polypeptides and to prevent aggregation driven by incorrect hydrophobic interactions (9). In addition to the passive function, Hsp70 disentangles protein aggregates and unfolds collapsed proteins (10–12). Hsp70 has ATPase activity, binding to and releasing an oligopeptide or polypeptide substrate using the ATP hydrolytic cycle (13–16). Hsp70 assumes two conformations, an open form and a closed form, and an equilibrium exists between them (17–22). The ATP-bound form shifts the equilibrium toward the open state and has low affinity for substrates, whereas the ADP-bound form shifts the equilibrium toward the closed state and has high affinity for substrates. Two components called cochaperones, Hsp40 and a nucleotide-exchange factor (NEF), are required to drive the Hsp70 reaction cycle (or chaperone cycle) (23–29). Hsp40 stimulates Hsp70 ATPase activity as a cochaperone, and Hsp40 is also a chaperone that prevents aggregation of denatured polypeptides (25, 30), likely by binding to the hydrophobic regions of denatured polypeptides (31). In a case, Hsp40 seems to induce conformational changes of a substrate (32). In the Hsp70 chaperone cycle, when ATP-bound Hsp70 interacts with Hsp40 on a polypeptide substrate, Hsp70 becomes the ADP-bound form through its interaction with Hsp40, and ADP-bound Hsp70 tightly binds to the substrate. NEF promotes ADP release from Hsp70. As a result, Hsp70 returns to the ATP-bound form, leading to dissociation of the substrate from Hsp70. Hsp70 proteins belong to a big family because they are highly conserved through evolution, and one species encodes multiple paralogous Hsp70 genes. The N-terminal of Hsp70 contains the nucleotide-binding domain, and the substrate-binding domain is located at the C-terminal. Hsp40s are also conserved through evolution, and members of the Hsp40 family are divided into three or four subfamilies (3, 33). According to Cheetham and Caplan (33), Hsp40s can be divided into three groups: Types I–III. All types contain a J domain that interacts with Hsp70 and stimulates its ATPase activity (34–37), which is why Hsp40s are called J proteins. Because the His-Pro-Asp (HPD) motif in the J domain is important for Hsp70 interactions, Hsp40 mutants with amino acid substitutions in the HPD motif do not exhibit cochaperone function (34, 37–39). Types I and II Hsp40s have a glycine/phenylalanine rich region (G/F rich region) and a C-terminal domain (CTD) in addition to the N-terminal J domain. The functional form is a homodimer consisting of two protomers bound at the C-termini (40–44). The G/F rich region is a flexible region and modulates Hsp70–substrate interactions (45–47). Type I Hsp40s include a zinc finger-like motif (ZFLM) in the CTD (48). Each CTD contains a substrate-binding site (43, 49–51), and the G/F rich region and ZFLM are also required for binding to some substrates (52–55). Type III Hsp40s contain the J domain and other specific domains. Types I and II Hsp40s bind to denatured proteins as a chaperone and the homodimeric structure is crucial for Hsp40 chaperone activity, because purified monomeric Hsp40 mutant proteins cannot bind to denatured proteins (42, 44) or act with Hsp70 to catalyse the refolding of denatured luciferase (42), although each protomer of Hsp40 contains a substrate-binding site. Two substrate-binding sites seem to be required for stable binding of Hsp40 to a substrate. However, a monomeric mutant of Sis1, a Type II Hsp40 in Saccharomyces cerevisiae, stimulates the ATPase activity of Hsp70 to a similar level as wild-type (WT) Sis1 in the absence of a substrate (42). Furthermore, Sahi and Craig reported that expressing only a J domain derived from various Hsp40s compensates for severe growth defects in the Saccharomyces cerevisiae Δydj1 (a Type I Hsp40) strain (56). Georgopoulos et al. also reported that a purified monomeric polypeptide containing only the J domain and the G/F rich region of DnaJ, an E. coli Type I Hsp40, stimulates the ATPase activity of Hsp70 and supports some Hsp70 chaperone functions, although, in those cases, more truncated DnaJ than intact DnaJ was required for those effects (34, 57). These results seemingly suggest that the dimeric conformation of Hsp40 is important for chaperone activity but is not absolutely required for cochaperone activity. However, because Hsp40 function was not completely complemented by monomeric Hsp40s, the dimeric structure seems required for full cochaperone activity. Is the dimeric structure sufficient for cochaperone activity that stimulates the ATPase activity of Hsp70 that binds near the Hsp40-binding site on the same substrate molecule? Whether the cochaperone function of Hsp40 requires one or two J domains is an important question that will further elucidate the Hsp70–substrate interaction. In this paper, we addressed this question using an E. coli Hsp70 chaperone system [DnaK (Hsp70), DnaJ (Hsp40; Fig. 1A), GrpE (NEF)] as a model. To this end, two heterodimeric DnaJ mutants that maintained chaperone activity and included an inactive J domain in one subunit were constructed. We found that the two heterodimeric DnaJs cannot efficiently support DnaK chaperone function. Fig. 1 View largeDownload slide Confirmation of purified DnaJs. (A) The DnaJ protomer is 376 amino acids with a J domain (1–70), a G/F rich region (71–105) and a CTD. The CTD is divided into two subdomains, CTD I (110–253) and CTD II (254–331). The J domain contains a HPD motif and the CTD I contains a ZFLM. Two protomers associate at the C-termini and form a homodimer. (B) pKKU1029 carries the dnaJ gene fused to a sequence coding for a Strep-tag at the 3’-end, whereas pKKU1125 carries the dnaJ gene fused with a sequence coding for a His-tag at the 5’-end. (C) DnaJ (10 μM) was incubated at 30°C for 30 min. After addition of molecular mass standards (β-amylase, 200 kDa; apo-transferrin, 81 kDa; carbonic anhydrase, 29 kDa), the mixture was loaded onto a Superdex 200 gel-filtration column. Aliquots from eluted fractions were subjected to SDS-PAGE. Proteins were detected by staining with CBB. Gel images were captured using an LAS3000 image analyzer. Fraction numbers are shown above the gel images. Molecular mass markers (in kilodaltons) are indicated to the left, and proteins are indicated to the right: 1, apo-transferrin; 2, β-amylase; 3, DnaJ; 4, carbonic anhydrase. (D) DnaJs (6 μg) were treated with thrombin (0.8 unit) in Buffer J4 (30 μl) at 22°C for 48 h, and 2 μg was electrophoresed (lanes 8–10) with untreated proteins (2 μg; lanes 5–7). Other DnaJs [(WT)-(WT), thrombin-treated (His-WT)-(His-WT), (WT-Strep)-(WT-Strep), (His-WT)-(His-WT)] (1 μg each) were also electrophoresed (lanes 1–4). Proteins were detected by staining with CBB. DnaJ is often observed as two or three bands due to the modification during electrophoresis. The components of each DnaJ are shown above the image: W, WT protomer; W × 2, two WT protomers; 33, H33Q protomer; 35, D35N protomer. The tag added to each protomer is indicated to the left, and ‘−’ means the authentic DnaJ protomer. Tb is an abbreviation for thrombin: +, thrombin treated; −, thrombin untreated. Fig. 1 View largeDownload slide Confirmation of purified DnaJs. (A) The DnaJ protomer is 376 amino acids with a J domain (1–70), a G/F rich region (71–105) and a CTD. The CTD is divided into two subdomains, CTD I (110–253) and CTD II (254–331). The J domain contains a HPD motif and the CTD I contains a ZFLM. Two protomers associate at the C-termini and form a homodimer. (B) pKKU1029 carries the dnaJ gene fused to a sequence coding for a Strep-tag at the 3’-end, whereas pKKU1125 carries the dnaJ gene fused with a sequence coding for a His-tag at the 5’-end. (C) DnaJ (10 μM) was incubated at 30°C for 30 min. After addition of molecular mass standards (β-amylase, 200 kDa; apo-transferrin, 81 kDa; carbonic anhydrase, 29 kDa), the mixture was loaded onto a Superdex 200 gel-filtration column. Aliquots from eluted fractions were subjected to SDS-PAGE. Proteins were detected by staining with CBB. Gel images were captured using an LAS3000 image analyzer. Fraction numbers are shown above the gel images. Molecular mass markers (in kilodaltons) are indicated to the left, and proteins are indicated to the right: 1, apo-transferrin; 2, β-amylase; 3, DnaJ; 4, carbonic anhydrase. (D) DnaJs (6 μg) were treated with thrombin (0.8 unit) in Buffer J4 (30 μl) at 22°C for 48 h, and 2 μg was electrophoresed (lanes 8–10) with untreated proteins (2 μg; lanes 5–7). Other DnaJs [(WT)-(WT), thrombin-treated (His-WT)-(His-WT), (WT-Strep)-(WT-Strep), (His-WT)-(His-WT)] (1 μg each) were also electrophoresed (lanes 1–4). Proteins were detected by staining with CBB. DnaJ is often observed as two or three bands due to the modification during electrophoresis. The components of each DnaJ are shown above the image: W, WT protomer; W × 2, two WT protomers; 33, H33Q protomer; 35, D35N protomer. The tag added to each protomer is indicated to the left, and ‘−’ means the authentic DnaJ protomer. Tb is an abbreviation for thrombin: +, thrombin treated; −, thrombin untreated. Materials and Methods Bacterial strains and plasmids The following Escherichia coli strains were used. HB101 [F-supE Δ(mcrC-mrr) recA ara-14 proA lacY galK rpsL xyl-5 mtl-1 leuB thi-1] was used for plasmid construction. MC4100 [F-araD Δ(argF-lac) U169 rpsL relA flbB deoC ptsF rbsR], KY1456 (MC4100 dnaJ:: Tn10-42) (58), HI1006 [F-araD139 Δ(araABIOC leu)7697 Δ(lacIPOZY)X74 galU galK strA trpA38 recA], and HMS174(DE3) [F-recA1 hsdR19 rpoB331 (DE3)] were used for protein purification. The λDE3 prophage carries a gene cording for T7 RNA polymerase that recognizes the T7 promoter (T7p). T7 RNA polymerase is induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to the culture medium. KY1456 was also used for testing the activity of tagged DnaJs in vivo. pKV1142 is a pTrc99A (ori: pMB1) (Pharmacia) derivative that carries ampicillin resistance gene (bla), the lacIq gene and the IPTG-dependent trc promoter (trcp) (59). pKV1961 and pKV2237 are pKV1142 derivatives carrying the dnaJ and dnaJ-his gene, respectively (60), and based on these plasmids, expression vectors for DnaJ mutants (H33Q and D35N-His) were constructed. pKV1900 is a pKV1142 derivative carrying the grpE gene. pAR3 is a pACYC184 (ori: p15A) derivative that carries the araC gene and the araB promoter (araBp) from Salmonella typhimurium, and chloramphenicol resistance gene (cat) (61). The dnaJ gene fused with a sequence coding for a Strep-tag (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) at the 3’-end was cloned under the araBp, and the resulting plasmid was designated pKKU1029 (Fig. 1B). The strep-tag sequence was derived from pASG-IBA162 (IBA). Three codons for glycine, serine, and alanine were inserted between the last codon of dnaJ and the strep-tag sequence. In the following sections, Strep-tagged DnaJ will be referred to as ‘DnaJ-Strep’ and especially as ‘WT-Strep’ for WT DnaJ. pET15b (ori: pMB1) (Novagen) carries the bla gene, the lacI gene and the T7 promoter. In addition, two sequences coding for a His-tag (six histidine residues) and a thrombin digestion site followed by the dnaJ gene were cloned downstream of the T7 promoter. The resulting plasmid was designated pKKU1125 (Fig. 1B). The DnaJ produced has the His-tag at the N-terminus of DnaJ, and the thrombin digestion site is located between the His-tag and DnaJ. His-tagged DnaJ will be referred to as ‘His-DnaJ’ and especially as ‘His-WT’ for WT DnaJ. Both pKKU1029 and pKKU1125 were constructed using an InFusion HD cloning kit (Clontech) with proper primer sets. Based on pKKU1125, a plasmid carrying the dnaJ mutant coding for H33Q or D35N DnaJ was constructed by site-directed mutagenesis using a PrimeSTAR Max DNA polymerase (Takara) and the appropriate primer sets according to the manufacturer’s instructions. The sequence of each dnaJ gene was confirmed by DNA sequence analysis. Media and chemicals L broth (per 1 litre; 10 g tryptone, 5 g yeast extract, 5 g NaCl, pH 7.4) was used for cell growth. Ampicillin (50 μg/ml) and chloramphenicol (20 μg/ml) were added when necessary. Chemicals were purchased from Nacalai Tesque, Wako Pure Chemicals or Sigma-Aldrich, unless otherwise specified. Protein purification All the protein purification steps were performed at 4°C. DnaK, the authentic DnaJ [(untagged WT)-(untagged WT) homodimeric DnaJ], a DnaJ mutant [(untagged H33Q)-(untagged H33Q) homodimeric DnaJ], and σ32 were purified as described previously in (60, 62). (WT-Strep)-(WT-Strep) was purified from HI1006 cells harbouring pKKU1029. The cells were grown to late log phase in L broth containing 20 μg/ml chloramphenicol at 30°C, and (WT-Strep)-(WT-Strep) synthesis was induced with 1 mg/ml arabinose. The cells were harvested after 3 h, resuspended in Buffer J1 [50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10 mM dithiothreitol, 10% (w/v) sucrose, 1 mg/ml lysozyme, and 0.6% (w/v) Brij58] (47), maintained on ice for 45 min, and disrupted by sonication. The resulting lysate was centrifuged at 75,000 × g for 90 min. The pellet was resuspended in Buffer J3 [50 mM Tris-HCl (pH 7.5), 2 mM β-mercaptoethanol, 10% sucrose, 100 mM KCl, and 0.5% (v/v) TritonX-100] and gently rotated for 60 min. After centrifugation at 75,000 × g for 90 min, the supernatant was loaded onto a Strep-Tactin Sepharose column (IBA) equilibrated with Buffer J3. After washing with Buffer J4 [50 mM Tris-HCl (pH 7.5), 2 mM β-mercaptoethanol, 10% (w/v) sucrose and 500 mM NaCl], (WT-Strep)-(WT-Strep) was eluted with Buffer J4 containing 2.5 mM desthiobiotin (IBA). (WT-Strep)-(WT-Strep) was concentrated, and desthiobiotin was removed, using a centrifugal filter device (Millipore). (His-WT)-(His-WT) was purified from HMS174(DE3) cells harbouring pKKU1125. The cells were grown to late log phase in L broth containing 50 μg/ml ampicillin at 30°C, and (His-WT)-(His-WT) synthesis was induced with 1 mM IPTG. The cells were harvested after 3 h and resuspended in Buffer J1. Cell disruption, the first ultracentrifugation, resuspension and the second ultracentrifugation were performed identically as the purification of (WT-Strep)-(WT-Strep). After the second ultracentrifugation, the supernatant was loaded onto a Ni2+-NTA agarose column (QIAGEN) equilibrated with Buffer J3. After washing with Buffer J4 containing 50 mM imidazole, (His-WT)-(His-WT) was eluted with Buffer J4 containing 250 mM imidazole. (His-WT)-(His-WT) was concentrated, and imidazole was removed, using a centrifugal filter device. (WT-His)-(WT-His) and (D35N-His)-(D35N-His) were purified from KY1456 cells harbouring pKV2237 and the pKV2237 derivative, respectively, in the same manner. GrpE was purified from HI1006 cells harbouring pKV1900. The cells were grown until the late-log phase in L broth containing 50 μg/ml ampicillin at 30°C, and GrpE synthesis was induced with 1 mM IPTG. Cells were harvested after 3 h, resuspended in Buffer A [50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, 10% (v/v) glycerol] containing 0.1% lysozyme, and kept on ice for 30 min. After addition of sodium deoxycholate to 0.05%, cells were disrupted by sonication, and the resulting lysate was centrifuged at 75,000 × g for 90 min. The supernatant was treated with ammonium sulphate. Proteins that were precipitated in a range of ammonium sulphate concentrations between 0.24 and 0.31 g/ml, were dissolved, dialysed against Buffer A and loaded onto a HiTrap heparin column (GE Healthcare). The flowthrough fraction was loaded onto a HiTrap Q-Sepharose column (GE Healthcare). Proteins were eluted with Buffer A containing a linear gradient of NaCl. The fractions containing GrpE were applied to a HiPrep Sephacryl S-100 column (GE Healthcare) equilibrated with Buffer A containing 0.1 M KCl. Fractions containing GrpE were stored. All the purified proteins, except for σ32, were more than 90% pure, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie Brilliant Blue (CBB). The purity of σ32 was ∼80%. The purified proteins were quantified using Bradford protein assay reagent (Bio-Rad). Because the functional forms of DnaJ and GrpE are dimer, their concentrations were calculated based on the molecular masses of their dimeric forms. Gel filtration DnaJ (10 μM) was incubated in Buffer G2 [20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM dithiothreitol, 500 mM NaCl, 5 mM Mg(CH3COO)2, 10% (v/v) glycerol] at 30°C for 30 min (total volume: 120 μl). After addition of molecular mass standards (β-amylase, 200 kD; apo-transferrin, 81 kD; carbonic anhydrase, 29 kD) (total volume: 150 μl), 100 μl of the mixture was loaded on a Superdex 200 gel filtration column (GE Healthcare). Aliquots (15 μl each) from eluted fractions (500 μl each) were subjected to SDS-PAGE. Proteins were detected by staining with CBB. Gel images were captured using an LAS3000 image analyzer (Fuji film). Stability of the DnaJ dimeric conformation DnaJ (1 μM) was incubated in Buffer G3 [20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5 mM Mg(CH3COO)2, and 10% (v/v) glycerol] at 30°C for 2 h (total volume: 100 μl). Next, 1 ml of Buffer G3 containing 30 mM imidazole and 0.1% Tween20 was added. After the addition of 20 μl of a 50% slurry of Ni2+-NTA agarose (QIAGEN), the mixture was rotated at 4°C for 1 h. Agarose beads were washed three times with Buffer G3 containing 30 mM imidazole and 0.1% Tween20, and DnaJ was dissociated from the agarose beads by treating the precipitate with 35 μl of Buffer J4 containing 250 mM imidazole. Half of the eluate was treated with thrombin (0.47 U) at 22°C for 48 h. Proteins were subjected to SDS-PAGE and detected by CBB staining and immunoblotting with an anti-DnaJ antiserum (60, 62) or an anti-His-tag antibody (Wako). Gel and membrane images were captured using an LAS3000 image analyzer, and the protein bands were quantified with Multi Gauge software (Fuji film). Chaperone activity of DnaJs To test chaperone activity of DnaJs, the ability to suppress the aggregation of urea-denatured luciferase was measured. Luciferase (Sigma) from Photinus pyralis was denatured by dissolving (8 μM) and incubating it in Denaturation buffer [30 mM Tris-HCl (pH 7.5), 5 mM dithiothreitol, 8 M urea] at room temperature for 12 h. The denatured luciferase was diluted 100 times in Assay buffer [30 mM HEPES-KOH (pH 7.5), 1 mM dithiothreitol, 40 mM KCl, 50 mM NaCl, 7 mM Mg(CH3COO)2] (total volume: 600 μl) containing BSA (40 or 80 nM) or DnaJ (40 or 80 nM) and incubated at room temperature in the chamber of a RF-5300 fluorescence photometer (Shimadzu). Light scattering was measured at an excitation and emission wavelength of 350 nm. ATPase assay To test cochaperone activity of DnaJs, DnaK ATPase activity was measured in the presence of GrpE and σ32 as well as each DnaJ protein. DnaK (0.46 μM), GrpE (0.25 μM), σ32 (1.84 μM), and each DnaJ (0.2, 0.4, 0.6 or 0.8 μM) were mixed in Buffer M2 [50 mM Tris-HCl (pH 7.5) 100 mM KCl, 25 mM MgCl2] at 30°C. The reactions were initiated by adding ATP (100 μM), and aliquots (80 μl each) were withdrawn at 2.5 min intervals. The concentration of inorganic free phosphate was measured using the Malachite Green Phosphate Assay Kit (Bio Assay Systems). Glucose-6-phosphate dehydrogenase refolding assay Glucose-6-phosphate dehydrogenase (G6PDH) refolding assay was performed as previously described (63). The G6PDH concentration was calculated from the molecular mass of its dimeric form. G6PDH (Wako) from Leuconostoc mesenteroides was dissolved in Buffer A to a concentration of 100–200 μM and diluted in G6PDH refolding buffer [50 mM HEPES-KOH (pH7.5), 150 mM KCl, 20 mM Mg(CH3COO)2, 10 mM DTT] (30 μl) to a concentration of 0.5 μM. After G6PDH was denatured at 52°C for 10 min, the solution was incubated at 25°C for 10 min. An equal volume of G6PDH refolding buffer containing the DnaK chaperone system (12 μM DnaK, 0.2–0.8 μM DnaJ, 0.6 μM GrpE), 8 mM ATP and an ATP regeneration system (200 μg/ml creatine kinase, 40 mM creatine phosphate) was added and the refolding reaction was initiated (final concentrations: 0.25 μM G6PDH, 6 μM DnaK, 0.1–0.4 μM DnaJ, 0.3 μM GrpE, 4 mM ATP). At indicated times, 10 μl aliquots were collected, and G6PDH was diluted in G6PDH reaction buffer [50 mM Tris-HCl (pH7.8), 3 mM MgCl2] to a concentration of 100 nM. Furthermore, 10 μl of 100 nM G6PDH was added to 990 μl of G6PDH reaction buffer containing glucose 6-phosphate and NAD+, and G6PDH reaction was started (final concentrations: 1 nM G6PDH, 3.3 mM glucose 6-phosphate, 2 mM NAD+). After 5 min, the absorbance was measured at 340 nm. The concentration of NADH was calculated based on the molar extinction coefficient (ε) of 6300/M/cm. Results A Strep-tagged protomer and a His-tagged protomer form a stable dimeric DnaJ molecule Expression vectors for WT-Strep (pKKU1029) and His-WT (pKKU1125) were constructed as described in the Materials and Methods (Fig. 1B). pKKU1029 is a derivative of pAR3, and the dnaJ-strep gene is expressed from the araBp by adding arabinose to the culture medium. Repression by AraC was incomplete. Therefore, when pKKU1029 was transformed into a ΔdnaJ strain (KY1456), the temperature sensitivity of the mutant was complemented at 42 and 44°C without arabinose addition, similar to the WT dnaJ expression vector [pKV1961, which also exhibited leaky dnaJ expression without IPTG addition to the culture medium] (data not shown). pKKU1125 is a derivative of pET15b, and the his-dnaJ gene is expressed from the T7p upon IPTG addition because the gene coding for T7 RNA polymerase is under the control of an IPTG-dependent promoter on the λDE3 prophage. When the same his-dnaJ gene was inserted under the trc promoter on pKV1142 and the resulting plasmid was transformed into KY1456, the temperature sensitivity was complemented at both 42 and 44°C (data not shown). Because the two vectors (pKKU1029 and pKKU1125) are compatible, they were co-transformed into HMS174(DE3). To induce the synthesis of both proteins at equal levels, the concentrations of arabinose and IPTG used in the culture medium were 1 mg/ml and 20 μM, respectively. Three hours after induction, cells were harvested, resuspended in Buffer J1, and disrupted by sonication. The lysate was centrifuged at 75,000 × g for 90 min. The DnaJ in the pellet was dissolved in Buffer J4. Because DnaJ tends to aggregate, dimeric DnaJ molecules were separated by gel filtration using a HiPrep Sephacryl S-300 column (GE healthcare) equilibrated with Buffer J4. Next, to obtain (WT-Strep)–(His-WT) dimeric molecules, two kinds of affinity chromatography were performed. Gel-filtration fractions containing dimeric DnaJ were loaded onto a Ni2+-NTA agarose column equilibrated with Buffer J4. After washing with Buffer J4 containing 30 mM imidazole, His-tagged DnaJs [(His-WT)-(His-WT) and (WT-Strep)-(His-WT)] were eluted with Buffer J4 containing 100 mM imidazole. The eluate was loaded onto a Strep-Tactin Sepharose column equilibrated with Buffer J4. After washing with Buffer J4, (WT-Strep)-(His-WT) was eluted with Buffer J4 containing 2.5 mM desthiobiotin. To confirm that the dimeric conformation was maintained during purification, purified DnaJ was separated by gel filtration using a Superdex 200 column (GE healthcare). The DnaJ protomer is 376 amino acids with a molecular mass of 41.1 kDa, whereas the molecular mass of (WT-Strep)-(His-WT) deduced from amino acid sequences is 85.4 kDa [the deduced molecular mass of WT-Strep (387 aa) is 42.4 kDa and that of His-WT (394 aa) is 43.0 kDa]. Purified (WT-Strep)-(His-WT) eluted in the same fractions as apo-transferrin (81 kDa) (Fig. 1C top panel). This result indicates that purified (WT-Strep)-(His-WT) maintains a dimeric conformation at 4°C. Nearly all Hsp40s contain the HPD motif in the J domain, and in DnaJ, this motif contains His33, Pro34 and Asp35. When a mutation substituting H33Q or D35N was introduced into the dnaJ-his gene on pKV2237 (pKV1142 trcp-dnaJ-his) and the resulting plasmid was transformed into KY1456, the mutant genes did not complement the temperature sensitivity of the mutant at 42°C unlike the WT dnaJ-his gene (data not shown). A codon substitution encoding H33Q or D35N was also introduced into the his-dnaJ gene on pKKU1125. The individual expression vectors were transformed into HMS174(DE3) with pKKU1029, and two heterodimeric DnaJs, (WT-Strep)-(His-H33Q) and (WT-Strep)-(His-D35N), were purified in the same manner as (WT-Strep)-(His-WT). When purified heterodimeric DnaJs were analysed by gel filtration, the results were similar to (WT-Strep)-(His-WT) (Fig. 1C middle and bottom panels), indicating that both heterodimers were also stable at 4°C. DnaJ-Strep and His-DnaJ have similar molecular masses, causing these proteins to run close together on a gel. Therefore, to confirm that purified DnaJs contained a Strep-tagged DnaJ protomer and a His-tagged DnaJ protomer, they were treated with thrombin to cleave the His-tag. The deduced molecular mass of thrombin-digested His-DnaJ (377 aa) was 41.1 kDa. Digestion reactions were performed under conditions that completely digested (His-WT)-(His-WT) (Fig. 1D lane 2 compared with lane 4). When purified DnaJs (Fig. 1D lanes 5–7) were treated and the resulting products were electrophoresed, two distinct bands were detected near a molecular mass of 40 kDa (Fig. 1D lanes 8–10). The upper bands corresponded to DnaJ-Strep (Fig. 1D lane 3), and the lower bands corresponded to thrombin-digested His-DnaJ (Fig. 1D lane 2). This result indicates that purified DnaJ molecules assume dimeric conformations consisting of a DnaJ-Strep protomer and one of three His-DnaJ protomers. Heterodimeric DnaJs containing a WT protomer and a mutated protomer retain chaperone activity The above results demonstrated that the (DnaJ-Strep)-(His-DnaJ) dimeric conformation was stable at 4°C during DnaJ purification. To test whether the (DnaJ-Strep)-(His-DnaJ) dimeric conformation is also maintained at high temperatures, the three (DnaJ-Strep)-(His-DnaJ) dimers were incubated at 30°C for 2 h and precipitated with Ni2+-NTA agarose. Then, the precipitated DnaJs were eluted with imidazole and electrophoresed. More than 50% of each DnaJ was recovered (Fig. 2A lanes 7–9). When the eluates were treated with thrombin, two bands from individual DnaJs were detected whose mobilities were similar to those of the DnaJ-Strep and digested His-DnaJ shown in Fig. 1D (Fig. 2A lanes 10–12), and their intensities were similar. Under the same conditions, (His-DnaJ)-(His-DnaJ) was almost completely digested (Fig. 2A lane 13 compared with lane 15), indicating that DnaJs precipitated by Ni2+-NTA agarose contain DnaJ-Strep and His-DnaJ at a molar ratio of 1:1. To confirm this result, DnaJ bands were detected by immunoblotting with an anti-DnaJ antiserum and an anti-His-tag antibody (Fig. 2B). When the anti-DnaJ antiserum was used to detect DnaJ, the results were identical to those obtained by CBB staining (Fig. 2B top panel). In detecting DnaJ with the anti-His-tag antibody, although the band intensity did not exhibit a linear relationship with DnaJ quantity (Fig. 2B middle and bottom panels lanes 1–3), the bands were not detected in the thrombin-treated products (Fig. 2B middle and bottom panels lanes 7–9). When signals were intensified (Fig. 2B bottom panel), 305 ng of DnaJ was detected with the anti-His-tag antibody, but no signals were obtained from the thrombin-treated DnaJs (Fig. 2B bottom panel lanes 7–9), although the band intensity for the thrombin-treated DnaJs was higher than that of 305 ng of DnaJ in the detection with the anti-DnaJ antiserum (Fig. 2B top panel lanes 7–9 compared with lane 2). These results indicate that the (DnaJ-Strep)-(His-DnaJ) dimeric conformation is stable at 30°C for at least 2 h. Fig. 2 View largeDownload slide Stability of the DnaJ dimeric conformation. DnaJ (1 μM) [(WT-Strep)-(WT-Strep) (negative control), (WT-Strep)-(His-WT), (WT-Strep)-(His-H33Q) or (WT-Strep)-(His-D35N)] was incubated at 30°C for 2 h. After precipitation with Ni2+-NTA agarose, DnaJ was eluted with 250 mM imidazole. Half of the eluate was treated with thrombin. (A) Thrombin-treated proteins (lanes 10–12) were subjected to SDS-PAGE with untreated proteins (lanes 6–9) and detected by CBB staining. The image was captured using an LAS3000 image analyzer. Serial dilutions of purified (WT-Strep)-(His-WT) were also electrophoresed to estimate the recovery efficiency by Ni2+-NTA agarose (lane 1–5); 2,440 ng corresponds to 100% recovery. Thrombin-treated (His-WT)-(His-WT) (1 μg; lane 13), (WT-Strep)-(WT-Strep) (1 μg; lane 14) and (His-WT)-(His-WT) (1 μg; lane 15) were also electrophoresed as molecular mass markers. (B) Thrombin-treated proteins shown in (A) (lanes 7–9) were subjected to SDS-PAGE with untreated proteins (lanes 4–6) and detected by immunoblotting with an anti-DnaJ antiserum (top panel) or an anti-His-tag antibody (middle and bottom panels). Images were captured using an LAS3000 image analyzer. The bottom panel is an intensified image of the middle panel. The image was intensified without changing the contrast using Multi Gauge software. Serial dilutions of purified (WT-Strep)-(His-WT) were also electrophoresed (lane 1–3); 610 ng corresponds to 50% recovery. Thrombin-treated (His-WT)-(His-WT) (0.5 μg; lane 10), (WT-Strep)-(WT-Strep) (0.5 μg; lane 11) and (His-WT)-(His-WT) (0.5 μg; lane 12) were also electrophoresed. In panels (A) and (B), molecular mass markers (in kilodaltons) are indicated to the left. The components of each DnaJ are shown above the images: W, WT protomer; W × 2, two WT protomers; 33, H33Q protomer; 35, D35N protomer. The tag added to each protomer is indicated to the left. Tb is an abbreviation for thrombin: +, thrombin treated; −, thrombin untreated. Fig. 2 View largeDownload slide Stability of the DnaJ dimeric conformation. DnaJ (1 μM) [(WT-Strep)-(WT-Strep) (negative control), (WT-Strep)-(His-WT), (WT-Strep)-(His-H33Q) or (WT-Strep)-(His-D35N)] was incubated at 30°C for 2 h. After precipitation with Ni2+-NTA agarose, DnaJ was eluted with 250 mM imidazole. Half of the eluate was treated with thrombin. (A) Thrombin-treated proteins (lanes 10–12) were subjected to SDS-PAGE with untreated proteins (lanes 6–9) and detected by CBB staining. The image was captured using an LAS3000 image analyzer. Serial dilutions of purified (WT-Strep)-(His-WT) were also electrophoresed to estimate the recovery efficiency by Ni2+-NTA agarose (lane 1–5); 2,440 ng corresponds to 100% recovery. Thrombin-treated (His-WT)-(His-WT) (1 μg; lane 13), (WT-Strep)-(WT-Strep) (1 μg; lane 14) and (His-WT)-(His-WT) (1 μg; lane 15) were also electrophoresed as molecular mass markers. (B) Thrombin-treated proteins shown in (A) (lanes 7–9) were subjected to SDS-PAGE with untreated proteins (lanes 4–6) and detected by immunoblotting with an anti-DnaJ antiserum (top panel) or an anti-His-tag antibody (middle and bottom panels). Images were captured using an LAS3000 image analyzer. The bottom panel is an intensified image of the middle panel. The image was intensified without changing the contrast using Multi Gauge software. Serial dilutions of purified (WT-Strep)-(His-WT) were also electrophoresed (lane 1–3); 610 ng corresponds to 50% recovery. Thrombin-treated (His-WT)-(His-WT) (0.5 μg; lane 10), (WT-Strep)-(WT-Strep) (0.5 μg; lane 11) and (His-WT)-(His-WT) (0.5 μg; lane 12) were also electrophoresed. In panels (A) and (B), molecular mass markers (in kilodaltons) are indicated to the left. The components of each DnaJ are shown above the images: W, WT protomer; W × 2, two WT protomers; 33, H33Q protomer; 35, D35N protomer. The tag added to each protomer is indicated to the left. Tb is an abbreviation for thrombin: +, thrombin treated; −, thrombin untreated. Hsp40 has chaperone activity that prevents the aggregation of denatured proteins in addition to cochaperone activity (25, 30). The chaperone activity of heterodimeric DnaJs was investigated by performing an aggregation prevention assay with denatured luciferase (Fig. 3). After urea-denatured luciferase was diluted 100 times to 80 nM, the light scattering intensity gradually increased over a period of 20 min (Fig. 3 cross symbols), indicating luciferase aggregation. However, when the authentic DnaJ [(untagged WT)-(untagged WT)] was added at the same concentration as the luciferase, the light scattering intensity minimally increased, indicating that luciferase aggregation was almost completely inhibited. The same phenomenon was observed with all three (DnaJ-Strep)-(His-DnaJ) DnaJs (Fig. 3 straight lines). When the DnaJ concentration was decreased to half that of luciferase, moderate inhibition of luciferase aggregation was observed for all the DnaJ constructs including the authentic DnaJ (Fig. 3 dotted lines). These results demonstrate that the heterodimeric DnaJs possess adequate substrate-binding activity, or chaperone activity. Fig. 3 View largeDownload slide Chaperone activity of DnaJs. Denatured luciferase (8 μM) was diluted 100 times in Assay buffer containing BSA or DnaJ [40 nM (dotted lines) or 80 nM (straight lines)] and incubated at room temperature. Light scattering was measured at an excitation and emission wavelength of 350 nm. Crosses, BSA; closed circles, authentic DnaJ; open circles, (WT-Strep)-(His-WT); triangles, (WT-Strep)-(His-H33Q); squares, (WT-Strep)-(His-D35N). The mean values from three experiments are shown with SD (error bars). Fig. 3 View largeDownload slide Chaperone activity of DnaJs. Denatured luciferase (8 μM) was diluted 100 times in Assay buffer containing BSA or DnaJ [40 nM (dotted lines) or 80 nM (straight lines)] and incubated at room temperature. Light scattering was measured at an excitation and emission wavelength of 350 nm. Crosses, BSA; closed circles, authentic DnaJ; open circles, (WT-Strep)-(His-WT); triangles, (WT-Strep)-(His-H33Q); squares, (WT-Strep)-(His-D35N). The mean values from three experiments are shown with SD (error bars). Cochaperone activity of the heterodimeric DnaJs is reduced to half of the WT DnaJ activity Hsp70 has weak ATPase activity, and its activity is stimulated >50-fold by cochaperones and substrates (13, 64, 65). To examine whether the heterodimeric DnaJs have cochaperone activity, the ATPase activity of DnaK was measured in the presence of GrpE and a substrate, σ32 as well as individual DnaJs. σ32, the E. coli heat shock factor, is known to bind to DnaK and DnaJ (57, 60, 62, 66–69), and to stimulate DnaK ATPase activity (70). The ATPase activity linearly increased in the presence of WT DnaJs [the authentic DnaJ and (WT-His)-(WT-His)] (Fig. 4A closed circles and closed diamonds, respectively), whereas ATPase activity was not detected in the absence of DnaJ (Fig. 4A open diamonds). In the cases of homodimeric DnaJ mutants, (H33Q)-(H33Q) did not accelerate DnaK ATPase activity (Fig. 4A closed triangles), whereas (D35N-His)-(D35N-His) slightly stimulated DnaK ATPase activity (Fig. 4A closed squares). When the concentration of DnaJ was changed, the ATPase activity linearly increased for 7.5 min at any concentration of the authentic DnaJ (Fig. 4B closed circles). For (WT-Strep)-(His-WT), DnaK ATPase activity was comparable to that for the authentic DnaJ (Fig. 4B open circles). This result indicates that neither the Strep-tag nor the His-tag hinder the cochaperone activity of DnaJ. In the presence of the two heterodimeric DnaJs, DnaK ATPase activity decreased for all ranges of DnaJ concentration used (Fig. 4B). The ATP hydrolysis rates calculated from the slopes of the graphs were reduced to ∼55% at 0.2 μM DnaJ, ∼60% at 0.4 μM DnaJ, ∼65% at 0.6 μM DnaJ, and ∼70% at 0.8 μM DnaJ compared with those of WT DnaJs [the authentic DnaJ and (WT-Strep)-(His-WT)] (Fig. 4C). These results indicate that two intact J domains are required for full DnaJ cochaperone activity. Intriguingly, the two heterodimeric DnaJs similarly stimulated DnaK ATPase activity at twice the concentrations of WT DnaJs, i.e. the ATP hydrolysis rates in the presence of either heterodimeric DnaJ at concentrations of 0.4 and 0.8 μM were similar to those in the presence of WT DnaJs at the concentrations of 0.2 and 0.4 μM, respectively (Fig. 4C). These results indicate that the heterodimeric DnaJs simply lose half of their cochaperone activity. Fig. 4 View largeDownload slide DnaK ATPase activity in the presence of various DnaJs. (A) DnaK (0.46 μM), GrpE (0.25 μM), σ32 (1.84 μM) and each DnaJ (0 or 0.8 μM) were mixed at 30°C. Reactions were initiated by adding ATP (100 μM), and aliquots were withdrawn at 2.5 min intervals. The concentration of inorganic free phosphate was measured. Open diamonds, without DnaJ; closed circles, authentic DnaJ; closed diamonds, (WT-His)-(WT-His); closed triangles, (H33Q)-(H33Q); closed squares, (D35N-His)-(D35N-His). Closed triangles overlap with open diamonds. (B) DnaK (0.46 μM), GrpE (0.25 μM), σ32 (1.84 μM), and each DnaJ (0.2, 0.4, 0.6 or 0.8 μM) were mixed and DnaK ATPase activity was measured shown in (A). Closed circles, authentic DnaJ; open circles, (WT-Strep)-(His-WT); open triangles, (WT-Strep)-(His-H33Q); open squares, (WT-Strep)-(His-D35N). In most cases, open triangles overlap with open squares. (C) The ATP hydrolysis rates were calculated from the slopes of the graphs shown in (B). Black bars, authentic DnaJ; dark grey bars, (WT-Strep)-(His-WT); light grey bars, (WT-Strep)-(His-H33Q); white bars, (WT-Strep)-(His-D35N). In panels (A–C), the mean values from three experiments are shown with SD (error bars). Fig. 4 View largeDownload slide DnaK ATPase activity in the presence of various DnaJs. (A) DnaK (0.46 μM), GrpE (0.25 μM), σ32 (1.84 μM) and each DnaJ (0 or 0.8 μM) were mixed at 30°C. Reactions were initiated by adding ATP (100 μM), and aliquots were withdrawn at 2.5 min intervals. The concentration of inorganic free phosphate was measured. Open diamonds, without DnaJ; closed circles, authentic DnaJ; closed diamonds, (WT-His)-(WT-His); closed triangles, (H33Q)-(H33Q); closed squares, (D35N-His)-(D35N-His). Closed triangles overlap with open diamonds. (B) DnaK (0.46 μM), GrpE (0.25 μM), σ32 (1.84 μM), and each DnaJ (0.2, 0.4, 0.6 or 0.8 μM) were mixed and DnaK ATPase activity was measured shown in (A). Closed circles, authentic DnaJ; open circles, (WT-Strep)-(His-WT); open triangles, (WT-Strep)-(His-H33Q); open squares, (WT-Strep)-(His-D35N). In most cases, open triangles overlap with open squares. (C) The ATP hydrolysis rates were calculated from the slopes of the graphs shown in (B). Black bars, authentic DnaJ; dark grey bars, (WT-Strep)-(His-WT); light grey bars, (WT-Strep)-(His-H33Q); white bars, (WT-Strep)-(His-D35N). In panels (A–C), the mean values from three experiments are shown with SD (error bars). The DnaK chaperone system refolds denatured proteins (10, 14, 25, 28, 29, 71). In this process, DnaJ binds to denatured proteins to prevent their aggregation and accelerates DnaK ATPase activity to promote tight DnaK binding to denatured proteins. To further investigate the cochaperone activity of the heterodimeric DnaJs, a refolding assay using heat-denatured G6PDH was performed. The refolding ratio (%) for denatured G6PDH was calculated by dividing the produced NADH at the indicated times by the amount of produced NADH by an equal amount of native G6PDH. After denaturing G6PDH at 52°C for 10 min, activity was not spontaneously restored at 25°C even after 2 h (data not shown). When heat-denatured G6PDH was incubated at 25°C in the presence of the DnaK chaperone system, activity was gradually restored (Fig. 5A). However, renaturation of G6PDH was not observed either in the absence of DnaJ or in the presence of homodimeric DnaJ mutants (Fig. 5A). When the concentration of DnaJ was changed, the final yields of active G6PDH increased with increasing concentration of DnaJ (Fig. 5B). In the presence of WT DnaJs at 0.4 μM, denatured G6PDH (0.25 μM) recovered up to ∼80% activity after 90 min. To easily compare the refolding activity of DnaJs, refolding rates (%/min) were calculated from the refolding ratios using the steepest slope values from individual graphs in Fig. 5B (Fig. 5C). Whereas (WT-Strep)-(His-WT) showed refolding rates comparable to the authentic DnaJ at all DnaJ concentrations, the refolding rates in the presence of either heterodimeric DnaJ decreased by 50–70% at 0.1 μM DnaJ, ∼40% at 0.2 μM DnaJ, and ∼35% at 0.4 μM DnaJ. Again, these results indicate that two intact J domains per dimeric DnaJ molecule are required for effective cochaperone activity. The reduced DnaK ATPase activity probably decreased the amount of DnaK bound to G6PDH and, in turn, resulted in a low efficiency for G6PDH refolding. Similar to the results for DnaK ATPase activity, the refolding rates for the DnaK chaperone system including either heterodimeric DnaJ at doubled concentrations showed comparable values to WT DnaJs (Fig. 5C). Fig. 5 View largeDownload slide G6PDH refolding activity by a DnaK chaperone system with a heterodimeric DnaJ. (A) G6PDH (0.5 μM) was denatured at 52°C for 10 min. After the solution was incubated at 25°C for 10 min, an equal volume of the G6PDH refolding buffer containing the DnaK chaperone system was added, and the refolding reaction was initiated (final concentrations: 0.25 μM G6PDH, 6 μM DnaK, 0 or 0.4 μM DnaJ, 0.3 μM GrpE, 4 mM ATP). G6PDH activity was measured at the indicated times. Native G6PDH was treated in the same manner, and the refolding ratios were determined by comparing to the values obtained with native G6PDH (100%). Open diamonds, without DnaJ; closed circles, authentic DnaJ; closed diamonds, (WT-His)-(WT-His); closed triangles, (H33Q)-(H33Q); closed squares, (D35N-His)-(D35N-His). Open diamonds, closed triangles, and closed squares overlap each other. (B) G6PDH refolding assays were performed in the same manner as in (A). Final concentration of DnaJ ranged from 0.1 to 0.4 μM. Closed circles, authentic DnaJ; open circles, (WT-Strep)-(His-WT); open triangles, (WT-Strep)-(His-H33Q); open squares, (WT-Strep)-(His-D35N). In the case of 0.4 μM DnaJ, closed circles overlap with open circles. (C) The G6PDH refolding rates were calculated from the steepest slope in each graph shown in (B). Black bars, authentic DnaJ; dark grey bars, (WT-Strep)-(His-WT); light grey bars, (WT-Strep)-(His-H33Q); white bars, (WT-Strep)-(His-D35N). In panels (A–C), the mean values from three experiments are shown with SD (error bars). Fig. 5 View largeDownload slide G6PDH refolding activity by a DnaK chaperone system with a heterodimeric DnaJ. (A) G6PDH (0.5 μM) was denatured at 52°C for 10 min. After the solution was incubated at 25°C for 10 min, an equal volume of the G6PDH refolding buffer containing the DnaK chaperone system was added, and the refolding reaction was initiated (final concentrations: 0.25 μM G6PDH, 6 μM DnaK, 0 or 0.4 μM DnaJ, 0.3 μM GrpE, 4 mM ATP). G6PDH activity was measured at the indicated times. Native G6PDH was treated in the same manner, and the refolding ratios were determined by comparing to the values obtained with native G6PDH (100%). Open diamonds, without DnaJ; closed circles, authentic DnaJ; closed diamonds, (WT-His)-(WT-His); closed triangles, (H33Q)-(H33Q); closed squares, (D35N-His)-(D35N-His). Open diamonds, closed triangles, and closed squares overlap each other. (B) G6PDH refolding assays were performed in the same manner as in (A). Final concentration of DnaJ ranged from 0.1 to 0.4 μM. Closed circles, authentic DnaJ; open circles, (WT-Strep)-(His-WT); open triangles, (WT-Strep)-(His-H33Q); open squares, (WT-Strep)-(His-D35N). In the case of 0.4 μM DnaJ, closed circles overlap with open circles. (C) The G6PDH refolding rates were calculated from the steepest slope in each graph shown in (B). Black bars, authentic DnaJ; dark grey bars, (WT-Strep)-(His-WT); light grey bars, (WT-Strep)-(His-H33Q); white bars, (WT-Strep)-(His-D35N). In panels (A–C), the mean values from three experiments are shown with SD (error bars). Discussion The dimeric conformation of Hsp40 is needed for its chaperone activity (42, 44). It also seems necessary for Hsp40 cochaperone activity, because monomeric Hsp40 mutants compensate for defects caused by Hsp40 insufficiency to some extent but the compensation is not complete (34, 56, 57). However, whether or not the dimeric structure of Hsp40 is sufficient for cochaperone activity is unknown. In this study, to address this question, two heterodimeric DnaJs [(WT-Strep)-(His-H33Q), (WT-Strep)-(His-D35N)] with an amino acid substitution in one protomer were constructed. The H33Q and D35N mutations were located in the HPD motif, which interacts with the DnaK nucleotide-binding domain to stimulate ATPase activity. Although the two heterodimeric DnaJs maintained chaperone activity (Fig. 3), they exhibited reduced cochaperone activity (Figs 4 and 5), indicating that the dimeric structure of Hsp40 is not sufficient for full cochaperone activity and that two intact J domains per homodimeric Hsp40 molecule are needed to efficiently stimulate Hsp70 ATPase activity on substrates. These results suggest that the interaction of DnaJ with DnaK on a substrate does not occur at a molar ratio of 1:1, i.e. one homodimeric DnaJ molecule induces the simultaneous binding of multiple DnaK molecules to the same substrate molecule. Sarbeng et al. (72) reported that DnaK transiently forms a dimeric structure that is responsible for efficient DnaJ interaction. Their model supports the notion that one homodimeric DnaJ molecule simultaneously stimulates multiple DnaK molecules on one substrate molecule. The DnaK ATPase assays shown in Fig. 4 were performed under conditions in which the amount of substrate (σ32; 1.84 μM) was larger than the amounts of DnaK (0.46 μM) and DnaJ (0.2, 0.4, 0.6 and 0.8 μM). Given that the dissociation constant for DnaJ–σ32 interaction is 20 nM (69), most DnaJ molecules would bind to σ32 under the conditions used and DnaK would interact with DnaJ on σ32. For WT DnaJs [the authentic DnaJ and (WT-Strep)-(His-WT)], the ATPase activity of DnaK increased as the DnaJ concentration increased. Similar results were obtained for the two heterodimeric DnaJs, but DnaK ATPase activity was reduced to 55–70% compared with WT DnaJs, probably due to the inactivation of one of two J domains. The difference in stimulation of DnaK ATPase activity between WT DnaJs and the heterodimeric DnaJs became smaller at higher DnaJ concentrations. It is likely that DnaK was the limiting factor at higher DnaJ concentrations. These results indicate that DnaK ATPase activity is stimulated through the interaction between DnaK and the J domain at a molar ratio of 1:1. The G6PDH refolding assays shown in Fig. 5 were performed under conditions of excess DnaK (6 μM) compared with DnaJ (0.1, 0.2 and 0.4 μM) and G6PDH (0.25 μM). Refolding activity for the DnaK chaperone system including the heterodimeric DnaJs was reduced to half activity of the DnaK chaperone system including WT DnaJs. Because heat-denatured G6PDH molecules form aggregates, a G6PDH molecule must be disentangled from an aggregate before refolding (10). Considering that in the disentanglement reaction, several DnaK molecules were needed per G6PDH molecule (10), it seems that multiple DnaK molecules bind to one G6PDH aggregate. Therefore, the present data suggests that one homodimeric WT DnaJ molecule efficiently stimulates multiple DnaK molecules to bind to the same substrate molecule, which leads to efficient refolding of denatured proteins. Although the promotion of G6PDH refolding by the heterodimeric DnaJs was not efficient, the heterodimeric DnaJs significantly promoted G6PDH refolding even at 0.1 μM (Fig. 5A). Cobinding of multiple DnaJs or DnaJ rebinding to one G6PDH aggregate might enable multipe DnaK molecules to bind to the same G6PDH aggregate. The percentage of the refolding rate for the heterodimeric DnaJs compared with that of WT DnaJs was smaller at 0.1 μM DnaJ (30–50%) than that at 0.4 μM DanJ (65%), which might reflect the difficulty of cobinding of multiple DnaJs or DnaJ rebinding to the same G6PDH aggregate at lower DnaJ concentrations. The activity of (WT-Strep)-(His-D35N) was slightly higher than that of (WT-Strep)-(His-H33Q). It might be related to the fact that a J domain fragment containing the D35N mutation shows higher affinity for the DnaK nucleotide-binding domain fragment than the WT J domain fragment (73). In fact, DnaK ATPase activity was slightly stimulated by (D35N-His)-(D35N-His) homodimeric mutant (Fig. 4A). Both heterodimeric DnaJs showed a similar acceleration of ATPase activity and refolding activity of DnaK at twice the concentrations of WT DnaJs (Figs 4C and 5C). These results indicate that two J domains in one homodimeric DnaJ molecule function additively, not synergistically. It means that each J domain independently stimulates a DnaK molecule to bind to a substrate. The idea is consistent with the fact that monomeric Hsp40 mutnats lacking substrate-binding activity have significant cochaperone ativity. 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( 2003) The J-domain of Hsp40 couples ATP hydrolysis to substrate capture in Hsp70. Biochemistry  42, 4937– 4944 Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations araBp araB promoter CBB Coomassie Brilliant Blue CTD C-terminal domain G6PDH glucose-6-phosphate dehydrogenase HPD His-Pro-Asp Hsp heat shock protein IPTG isopropyl-β-D-thiogalactopyranoside NEF nucleotide-exchange factor SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis T7p T7 promoter; trcp, trc promoter ZFLM zinc finger-like motif © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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The Journal of BiochemistryOxford University Press

Published: Apr 4, 2018

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