Biochemical Coupling of the Two Nucleotide Binding Domains of ClpB
2005; Elsevier BV; Volume: 280; Issue: 45 Linguagem: Inglês
10.1074/jbc.m506672200
ISSN1083-351X
AutoresPhilipp Beinker, Sandra Schlee, Rajeswari Auvula, Jochen Reinstein,
Tópico(s)Protein Structure and Dynamics
ResumoClpB cooperates with the DnaK chaperone system in the reactivation of protein from aggregates and is a member of the ATPases associated with a variety of cellular activities (AAA+) protein family. The underlying disaggregation reaction is dependent on ATP hydrolysis at both AAA cassettes of ClpB but the role of each AAA cassette in the reaction cycle is largely unknown. Here we analyze the activity of the separately expressed and purified nucleotide binding domains of ClpB from Thermus thermophilus. The two fragments show different biochemical properties: the first construct is inactive in ATPase activity assays and binds nucleotides weakly, the second construct has a very high ATPase activity and interacts tightly with nucleotides. Both individual fragments have lost their chaperone function and are not able to form large oligomers. When combined in solution, however, the two fragments form a stable heterodimer with oligomerization capacities equivalent to wild-type ClpB. This non-covalent complex regains activity in reactivating protein aggregates in cooperation with the DnaK chaperone system. Upon complex formation the ATPase activity of fragment 2 is reduced to a level similar to wild-type ClpB. Hence functional ClpB can be reassembled from its isolated AAA cassettes showing that covalent linkage of these domains is not a prerequisite for the chaperone activity. The observation that the intrinsically high ATPase activity of AAA2 is suppressed by AAA1 allows a hypothetical assignment of their mechanistic function. Whereas the energy gained upon ATP hydrolysis at the AAA2 is likely to drive a conformational change of the structure of ClpB, AAA1 might function as a regulator of the chaperone cycle. ClpB cooperates with the DnaK chaperone system in the reactivation of protein from aggregates and is a member of the ATPases associated with a variety of cellular activities (AAA+) protein family. The underlying disaggregation reaction is dependent on ATP hydrolysis at both AAA cassettes of ClpB but the role of each AAA cassette in the reaction cycle is largely unknown. Here we analyze the activity of the separately expressed and purified nucleotide binding domains of ClpB from Thermus thermophilus. The two fragments show different biochemical properties: the first construct is inactive in ATPase activity assays and binds nucleotides weakly, the second construct has a very high ATPase activity and interacts tightly with nucleotides. Both individual fragments have lost their chaperone function and are not able to form large oligomers. When combined in solution, however, the two fragments form a stable heterodimer with oligomerization capacities equivalent to wild-type ClpB. This non-covalent complex regains activity in reactivating protein aggregates in cooperation with the DnaK chaperone system. Upon complex formation the ATPase activity of fragment 2 is reduced to a level similar to wild-type ClpB. Hence functional ClpB can be reassembled from its isolated AAA cassettes showing that covalent linkage of these domains is not a prerequisite for the chaperone activity. The observation that the intrinsically high ATPase activity of AAA2 is suppressed by AAA1 allows a hypothetical assignment of their mechanistic function. Whereas the energy gained upon ATP hydrolysis at the AAA2 is likely to drive a conformational change of the structure of ClpB, AAA1 might function as a regulator of the chaperone cycle. ClpB co-operates with the DnaK chaperone system consisting of the principal component DnaK and the co-chaperones DnaJ and GrpE in the disaggregation and reactivation of protein aggregates. This chaperone network was first identified in Saccharomyces cerevisiae to be essential for the survival of yeast at elevated temperatures (1Sanchez Y. Taulien J. Borkovich K.A. Lindquist S. EMBO J. 1992; 11: 2357-2364Crossref PubMed Scopus (464) Google Scholar). The mechanism of this chaperone-mediated thermotolerance was elucidated and linked to the protein disaggregation activity of the ClpB-DnaK chaperone network (2Parsell D.A. Kowal A.S. Singer M.A. Lindquist S. Nature. 1994; 372: 475-478Crossref PubMed Scopus (726) Google Scholar). This reaction is strictly dependent on the presence and hydrolysis of ATP. In Escherichia coli 25% of the heat-labile proteins were found to interact with these chaperones under heat shock conditions, indicating a broad substrate specificity of the ClpB-DnaK system in vivo and a preference for large multidomain proteins as substrates (3Mogk A. Tomoyasu T. Goloubinoff P. Rudiger S. Roder D. Langen H. Bukau B. EMBO J. 1999; 18: 6934-6949Crossref PubMed Scopus (508) Google Scholar). The chaperone activity of ClpB and the DnaK system was also studied in vitro using a variety of substrate proteins (2Parsell D.A. Kowal A.S. Singer M.A. Lindquist S. Nature. 1994; 372: 475-478Crossref PubMed Scopus (726) Google Scholar, 3Mogk A. Tomoyasu T. Goloubinoff P. Rudiger S. Roder D. Langen H. Bukau B. EMBO J. 1999; 18: 6934-6949Crossref PubMed Scopus (508) Google Scholar, 4Glover J.R. Lindquist S. Cell. 1998; 94: 73-82Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, 5Motohashi K. Watanabe Y. Yohda M. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7184-7189Crossref PubMed Scopus (221) Google Scholar, 6Zolkiewski M. J. Biol. Chem. 1999; 274: 28083-28086Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Model substrates from different organisms, with different sizes and functions have been established leading to the characterization of chaperone function under a variety of experimental conditions (5Motohashi K. Watanabe Y. Yohda M. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7184-7189Crossref PubMed Scopus (221) Google Scholar, 6Zolkiewski M. J. Biol. 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According to all determined crystal structures, the fold of AAA cassettes (ATPases associated with a variety of cellular activities) 4The abbreviations used are: AAA, ATPases associated with a variety of cellular activities; AAA cassette, ATPase domain of AAA proteins; ClpB, ClpB from T. thermophilus; DnaK system, chaperone system consisting of DnaK, DnaJ, and GrpE from T. thermophilus; GPDH, glucose-6-phosphate dehydrogenase; Ni-NTA, nickel-nitrilotriacetic acid; LDH, lactate dehydrogenase; MOPS, 4-morpholinepropanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; HPLC, high pressure liquid chromatography; AMPPCP, adenosine 5′-(β,γ-methylene)triphosphate. 4The abbreviations used are: AAA, ATPases associated with a variety of cellular activities; AAA cassette, ATPase domain of AAA proteins; ClpB, ClpB from T. thermophilus; DnaK system, chaperone system consisting of DnaK, DnaJ, and GrpE from T. thermophilus; GPDH, glucose-6-phosphate dehydrogenase; Ni-NTA, nickel-nitrilotriacetic acid; LDH, lactate dehydrogenase; MOPS, 4-morpholinepropanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; HPLC, high pressure liquid chromatography; AMPPCP, adenosine 5′-(β,γ-methylene)triphosphate. is conserved (9Bochtler M. Hartmann C. Song H.K. Bourenkov G.P. Bartunik H.D. Huber R. Nature. 2000; 403: 800-805Crossref PubMed Scopus (375) Google Scholar, 10Lenzen C.U. Steinmann D. Whiteheart S.W. Weis W.I. Cell. 1998; 94: 525-536Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). AAA cassettes consist of a core nucleotide binding domain and a C-terminal subdomain. The core domain contains the classical Walker motifs characteristic of P-loop ATPases (11Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4212) Google Scholar, 12Saraste M. Sibbald P.R. Wittinghofer A. Trends Biochem. Sci. 1991; 15: 430-434Abstract Full Text PDF Scopus (1727) Google Scholar), whereas the C-terminal subdomain has a high content of α-helices. The recently solved crystal structure of the ClpB monomer/trimer from Tetrahymena thermophilus and sequence comparisons within the Clp protein family reveal that ClpB can be divided into four distinct domains. An N-terminal domain precedes the first AAA cassette, which has an insertion called middle (linker) region. This domain is followed by the second AAA cassette of ClpB. A number of biochemical studies give insight into the structures and functions of the individual domains of ClpB (13Lee S. Sowa M.E. Watanabe Y. Sigler P.B. Chiu W. Yoshida M. Tsai F.T. Cell. 2003; 115: 229-240Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). The N-terminal domain, which precedes the first AAA cassette, consists of two 75-amino acid residue long repeats (Fig. 1A). The structure of full-length ClpB shows that the N-terminal domain is rather mobile and does not interact tightly with the first AAA cassette. In the structure of the isolated N-domain of ClpB from E. coli a small hydrophobic patch was identified. This area was supposed to be important for interactions with substrates (14Li J. Sha B. Structure (Camb.). 2003; 11: 323-328Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). However, the N-domain is dispensable for chaperone activity in vivo and in vitro (8Beinker P. Schlee S. Groemping Y. Seidel R. Reinstein J. J. Biol. Chem. 2002; 277: 47160-47166Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 15Mogk A. Schlieker C. Strub C. Rist W. Weibezahn J. Bukau B. J. Biol. Chem. 2003; 278: 17615-17624Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 16Eriksson M.J. Clarke A.K. Cell Stress Chaperones. 2000; 5: 255-264Crossref PubMed Scopus (16) Google Scholar). In ClpA, the N-terminal domain interacts with an adaptor protein, which influences substrate specificity (17Dougan D.A. Reid B.G. Horwich A.L. Bukau B. Mol. Cell. 2002; 9: 673-683Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). An adaptor protein of ClpB has not been identified and therefore the function of the N-domain of ClpB is not completely understood. Both AAA cassettes of ClpB must be functional for chaperone activity (2Parsell D.A. Kowal A.S. Singer M.A. Lindquist S. Nature. 1994; 372: 475-478Crossref PubMed Scopus (726) Google Scholar). Mutations in the Walker A or Walker B motifs lead to a loss of chaperone activity and show a complex pattern of effects on the properties of ClpB. Mutations in the Walker A motif of the first AAA cassette affect the steady state ATPase activity of ClpB from T. thermophilus and E. coli (15Mogk A. Schlieker C. Strub C. Rist W. Weibezahn J. Bukau B. J. Biol. Chem. 2003; 278: 17615-17624Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 18Schlee S. Groemping Y. Herde P. Seidel R. Reinstein J. J. Mol. Biol. 2001; 306: 889-899Crossref PubMed Scopus (74) Google Scholar, 19Watanabe Y.H. Motohashi K. Yoshida M. J. Biol. Chem. 2002; 277: 5804-5809Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Additionally the oligomerization capacity is reduced and the chaperone function severely impaired. The mutations in the Walker A motif of the first AAA cassette do not affect the nucleotide binding of ClpB, indicating that the second AAA cassette has a high affinity for nucleotides (18Schlee S. Groemping Y. Herde P. Seidel R. Reinstein J. J. Mol. Biol. 2001; 306: 889-899Crossref PubMed Scopus (74) Google Scholar, 19Watanabe Y.H. Motohashi K. Yoshida M. J. Biol. Chem. 2002; 277: 5804-5809Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Interestingly ATP hydrolysis at the first AAA cassette is not essential for oligomerization. Proteins with mutations in the Walker B motif show reduced ATPase activity but still bind nucleotides and do not exhibit assembly defects (15Mogk A. Schlieker C. Strub C. Rist W. Weibezahn J. Bukau B. J. Biol. Chem. 2003; 278: 17615-17624Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 19Watanabe Y.H. Motohashi K. Yoshida M. J. Biol. Chem. 2002; 277: 5804-5809Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The importance of the first AAA cassette for oligomerization can be explained with residues of adjacent subunits within the ClpB hexamer interacting with the nucleotide bound to the preceding AAA cassette (13Lee S. Sowa M.E. Watanabe Y. Sigler P.B. Chiu W. Yoshida M. Tsai F.T. Cell. 2003; 115: 229-240Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). A unique feature of ClpB is an insertion in the first AAA cassette. This middle domain formerly also called the linker domain is not found in other Clp proteins. It is inserted in a loop of the helical subdomain of the first AAA cassette. In the ClpB structure this insertion forms a long coiled-coil structure composed of two leucine-rich segments (13Lee S. Sowa M.E. Watanabe Y. Sigler P.B. Chiu W. Yoshida M. Tsai F.T. Cell. 2003; 115: 229-240Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). The domain protrudes widely from the core of the molecule, has relatively few contacts to the rest of the molecule, and shows a high degree of flexibility in the structure. The middle region and its flexible conformation were shown to be essential for the chaperone activity of ClpB in vitro (13Lee S. Sowa M.E. Watanabe Y. Sigler P.B. Chiu W. Yoshida M. Tsai F.T. Cell. 2003; 115: 229-240Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 15Mogk A. Schlieker C. Strub C. Rist W. Weibezahn J. Bukau B. J. Biol. Chem. 2003; 278: 17615-17624Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Following this insertion the first AAA cassette continues with an α-helix connecting the two AAA cassettes of ClpB. Mutations in the Walker A motif of the second AAA cassette affect the ATPase activity of ClpB from T. thermophilus, however, their effect on oligomerization is less pronounced than for the first AAA cassette (18Schlee S. Groemping Y. Herde P. Seidel R. Reinstein J. J. Mol. Biol. 2001; 306: 889-899Crossref PubMed Scopus (74) Google Scholar, 19Watanabe Y.H. Motohashi K. Yoshida M. J. Biol. Chem. 2002; 277: 5804-5809Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Similar results are published for ClpB from E. coli, but interestingly in Hsp104 from S. cerevisiae the functions of the AAA cassettes seem to be reversed. Here mutations in the first AAA cassette eliminate ATP hydrolysis but do not effect oligomerization, whereas mutations in the P-loop of AAA cassette 2 severely impair oligomerization and reduce ATPase activity (15Mogk A. Schlieker C. Strub C. Rist W. Weibezahn J. Bukau B. J. Biol. Chem. 2003; 278: 17615-17624Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 20Schirmer E.C. Queitsch C. Kowal A.S. Parsell D.A. Lindquist S. J. Biol. Chem. 1998; 273: 15546-15552Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). One observation is conserved between the ClpB proteins from different organisms: ClpB variants with mutations in the Walker A motifs of either AAA cassette have severely reduced steady state ATPase activities and are inactive in chaperone activity assays (15Mogk A. Schlieker C. Strub C. Rist W. Weibezahn J. Bukau B. J. Biol. Chem. 2003; 278: 17615-17624Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 18Schlee S. Groemping Y. Herde P. Seidel R. Reinstein J. J. Mol. Biol. 2001; 306: 889-899Crossref PubMed Scopus (74) Google Scholar, 20Schirmer E.C. Queitsch C. Kowal A.S. Parsell D.A. Lindquist S. J. Biol. Chem. 1998; 273: 15546-15552Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). These findings show that for full steady state ATPase activity as well as chaperone function of ClpB cooperativity of the two AAA cassettes is necessary. In this study we examined the biochemical properties and the interactions of the two nucleotide binding domains of ClpB from T. thermophilus. A comparison of nucleotide binding and ATPase properties of the individual fragments and the chaperoning competent reassembled complex indicates that: 1) covalent linkage between the two nucleotide domains of ClpB is not necessary for functional coupling and 2) the presence of NBD1 strongly suppresses an intrinsically high ATPase activity of NBD2, which indicates a tentative assignment for the role of this hydrolysis step. Mutagenesis—Constructs for the two fragments (ClpB-(141-519) and ClpB-(519-854)) were produced by PCR-directed mutagenesis using petRS-ClpB as template (18Schlee S. Groemping Y. Herde P. Seidel R. Reinstein J. J. Mol. Biol. 2001; 306: 889-899Crossref PubMed Scopus (74) Google Scholar). The PCR products were purified and digested with restriction enzymes NdeI and EcoRI. The DNA fragments were cloned into NdeI-EcoRI-restricted vector pet28a (Novagen) coding for proteins with an N-terminal His tag and the sequences of the constructs were verified by DNA sequencing. Protein Expression and Purification—ClpB, ClpBΔN, DnaK, DnaJ, and GrpE from T. thermophilus were expressed and purified as described previously (8Beinker P. Schlee S. Groemping Y. Seidel R. Reinstein J. J. Biol. Chem. 2002; 277: 47160-47166Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 18Schlee S. Groemping Y. Herde P. Seidel R. Reinstein J. J. Mol. Biol. 2001; 306: 889-899Crossref PubMed Scopus (74) Google Scholar, 21Klostermeier D. Seidel R. Reinstein J. J. Mol. Biol. 1999; 287: 511-525Crossref PubMed Scopus (36) Google Scholar, 22Groemping Y. Klostermeier D. Herrmann C. Veit T. Seidel R. Reinstein J. J. Mol. Biol. 2001; 305: 1173-1183Crossref PubMed Scopus (43) Google Scholar). The proteins with N-terminal His tags were expressed in E. coli Rosetta DE3 cells (Novagen) in 2× YT medium at 37 °C. At an A600 of 0.6 the expression of the recombinant proteins was induced by isopropyl 1-thio-β-d-galactopyranoside (1 mm) and the cells were harvested after 3 h of incubation at 37 °C. Proteins with N-terminal His tags were purified using a Ni-NTA matrix (Qiagen) according to the manufacturer's protocol. After Ni-NTA chromatography protein containing fractions were pooled and dialyzed against thrombin cleavage buffer (50 mm Tris/HCl, pH 7.5, 300 mm KCl, 5mm MgCl2, 10% glycerol). His tags were removed by thrombin cleavage using 1 unit of thrombin per 10 mg of recombinant protein and incubation at 37 °C. The cleaved off His tags and undigested protein were removed by chromatography with Ni-NTA. Remaining impurities were removed by gel filtration chromatography using a Superset 75 26/60 column (Amersham Biosciences) equilibrated with gel filtration buffer (50 mm Tris, pH 7.5, 50 mm KCL, 5 mm MgCl2, 2 mm EDTA; 2 mm dithioerythritol, 10% glycerol). After thrombin cleavage four additional amino acids (GSHM) remained on the N terminus of the recombinant proteins as a consequence of cloning in pet28a. Protein concentrations were measured using the methods of Bradford (23Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar) and Ehresmann et al. (24Ehresmann B. Imbault P. Weil J.H. Anal. Biochem. 1973; 54: 454-463Crossref PubMed Scopus (212) Google Scholar). Lactate dehydrogenase (LDH) from Bacillus stearothermophilus containing a N-terminal His tag was expressed in E. coli Rosetta cells (Novagen) using LB medium at 37 °C and purified as described (25Halliwell C.M. Morgan G. Ou C.P. Cass A.E. Anal. Biochem. 2001; 295: 257-261Crossref PubMed Scopus (59) Google Scholar). If not noted otherwise, all protein concentrations refer to monomers. Gel Filtration Experiments—Oligomerization of the proteins was analyzed by gel filtration experiments at 25 °C. The experiments were performed using a Superdex 200 10/30 column (Amersham Biosciences) with a Waters HPLC system (Waters). ClpB-(141-519), ClpB-(519-854), ClpBΔN, and the complex of ClpB-(141-519)-ClpB-(519-854) were injected (50 μl, 5 mg/ml) with a flow rate of 0.2 ml/min (running buffer: 50 mm Tris/HCl, pH 7.5, 20 mm MgCl2, 1 mm EDTA, 1 mm dithioerythritol, 10% glycerol, and 1-500 mm KCl). If indicated, nucleotides (1 mm) were added to the running buffer and the protein sample. Calibration of the gel filtration column was performed using a gel filtration standard (Bio-Rad) and established the exponential relation between the molecular mass and retention time of subsequently analyzed proteins (data not shown). The oligomeric forms of ClpBΔN (80.9 kDa), ClpB-(141-519) (43.2 kDa), and ClpB-(519-854) (38.3 kDa) were defined within the following limits (kDa): ClpBΔN, monomer (M) 70-130, dimer (D) 130-200, trimer (T) 200-290, tetra-hexamer (H) 290-530; ClpB-(141-519), M 30-60, D 60-100, T 100-150, H 150-270; ClpB-(519-854), M 25-55, D 55-90, T 90-140, H 140-250. For analysis of the oligomerization behavior of the complex consisting of ClpB-(141-519)-ClpB-(519-854) the limits of ClpBΔN were used. Circular Dichroism—CD spectra were recorded (10-fold accumulation) with a Jasco-J710 Spectropolarimeter in 0.02-cm cuvettes at a scan rate of 20 nm/min, 0.2-nm resolution, 1 nm bandwidth, a time constant of 1 s, and a sensitivity of 20 mdeg at 25 °C. Fluorescence Measurements—Nucleotide affinities were determined in equilibrium titration experiments using a SLM 8100 Photon-counting spectrofluorimeter at 25 °C (18Schlee S. Groemping Y. Herde P. Seidel R. Reinstein J. J. Mol. Biol. 2001; 306: 889-899Crossref PubMed Scopus (74) Google Scholar). The relative fluorescence was plotted against the protein concentration and the data were analyzed with the quadratic equation using the program Grafit, version 3.01 (26Reinstein J. Vetter I.R. Schlichting I. Rösch P. Wittinghofer A. Goody R.S. Biochemistry. 1990; 29: 7440-7450Crossref PubMed Scopus (89) Google Scholar). The equilibrium displacement of the fluorescent nucleotide by ADP were fitted to a cubic equation (27Thrall S.H. Reinstein J. Wöhrl B.M. Goody R.S. Biochemistry. 1996; 35: 4609-4618Crossref PubMed Scopus (46) Google Scholar). Chaperone-assisted Reactivation of Substrate Proteins—Substrate proteins were denatured in reaction buffer (50 mm MOPS/NaOH, pH 7.5, 150 mm KCl, 10 mm MgCl2, 5 mm ATP, 2 mm dithioerythritol) and chaperones were added (ClpB variants 0.5 μm, DnaK system: DnaK, 1.6 μm; DnaJ, 0.4 μm; GrpE, 0.2 μm) prior to refolding at 55 °C. α-Glucosidase (0.1 μm) from B. stearothermophilus (Sigma) was denatured 10 min at 75 °C (5Motohashi K. Watanabe Y. Yohda M. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7184-7189Crossref PubMed Scopus (221) Google Scholar, 8Beinker P. Schlee S. Groemping Y. Seidel R. Reinstein J. J. Biol. Chem. 2002; 277: 47160-47166Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). α-Glucosidase activity was determined at 40 °C at the indicated time points of the refolding reaction by diluting the reaction mixture into the assay solution (50 mm sodium phosphate, 2mm p-nitrophenyl-α-d-glucopyranoside). Glucose-6-phosphate dehydrogenase (GPDH) (0.2 μm) from B. stearothermophilus (Sigma) was denatured 7.5 min at 75 °C (5Motohashi K. Watanabe Y. Yohda M. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7184-7189Crossref PubMed Scopus (221) Google Scholar). GPDH activity was determined at 40 °C at the indicated time points of the refolding reaction by diluting the reaction mixture into assay buffer (100 mm Tris/HCl, pH 8.8, 40 mm MgCl2, 3 mm glucose 6-phosphate, 1 mm NADPH, 0.1 mg/ml bovine serum albumin). LDH from B. stearothermophilus (0.2 μm) was incubated 30 min at 80 °C (5Motohashi K. Watanabe Y. Yohda M. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7184-7189Crossref PubMed Scopus (221) Google Scholar, 8Beinker P. Schlee S. Groemping Y. Seidel R. Reinstein J. J. Biol. Chem. 2002; 277: 47160-47166Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). LDH activity was determined at 25 °C at the indicated time points of the refolding reaction by diluting the reaction mixture into assay buffer (25 mm BisTris/HCl, pH 6.5, 50 mm KCl, 10 mm pyruvate, 0.25 mm NADH). Activity of the native substrate proteins was determined before the denaturation step. Concentrations refer to substrate protein solutions prior to aggregation by heat shock. Isothermal Calorimetry—Isothermal calorimetric titrations were performed with a MicroCal MCS isothermal titration calorimeter (MicroCal LLC, Northampton, MA) to measure the binding affinity of the ClpB fragments. The heat of binding was detected upon the titration of ClpB-(141-519) (90 μm) into a cell containing ClpB-(519-854) (10 μm) with 50 mm HEPES, pH 7.5, 100 mm KCl, and 5 mm MgCl2 as buffer system. Experiments were done at 45 °C at an injection rate of 0.5 μl/s. 29 injections were performed with a spacing of 240 s between each. The data were analyzed using the manufacturer's software to calculate the reaction enthalpy, stoichiometry of binding, and association constant. Steady State ATPase Measurements—Steady state ATP hydrolysis was measured with a coupled colorimetric assay (18Schlee S. Groemping Y. Herde P. Seidel R. Reinstein J. J. Mol. Biol. 2001; 306: 889-899Crossref PubMed Scopus (74) Google Scholar, 28Bergmeyer H.U. Colorimetric Assays. 1962; (Adam, H., ed) pp. , Verlag Chemie, Weinheim, Germany: 573-577Google Scholar). ClpB variants (10 μm) were incubated at 25 °C with different concentrations of Mg·ATP in assay buffer (50 mm Tris/HCl, pH 7.5, 100 mm KCl, 5 mm MgCl2, 2 mm EDTA, 2 mm dithioerythritol, 0.4 mm phosphoenolpyruvate, 0.5 mm NADH, 20 μg/ml LDH, 50 μg/ml pyruvate kinase). The observed rate constants were determined from the decrease of A340 during the reaction. The data were analyzed with the program Grafit, version 3.01, using the Hill equation describing cooperative binding. k=kcat⋅[S]HKmH+[S]H Single Turnover ATPase Measurements and Nucleotide Analysis—The single turnover ATPase activity of the proteins was determined at 25 °C. Proteins (100 μm) were incubated with Mg·ATP (50 μm) in assay buffer (50 mm Tris/HCl, pH 7.5, 5 mm MgCl2, 2 mm EDTA, 2 mm dithioerythritol, 100 mm KCl). For ClpB wild-type, ClpBΔN, ClpB-(141-519), and the complex formed by ClpB-(141-519)-ClpB-(519-854), the reaction was stopped at the indicated time points by the addition of trichloroacetic acid (10% w/v) and incubated on ice. The precipitated protein was removed by centrifugation and the supernatant neutralized. The nucleotide content was analyzed by HPLC analysis using a Waters HPLC system (Waters) with an ODS-Hypersil reverse phase C-18 column (Bischoff, Leonberg) in isocratic running buffer (50 mm KPi, pH 6.8). The single turnover ATPase activity of ClpB-(519-854) was studied using the KinTek RQF-3 Quench-Flow apparatus (KinTek Corporation, University Park, PA). Reactions were started by rapidly mixing the two reactants (15 μl of each) and then quenched with 0.6% trifluoroacetic acid at the defined time intervals. All concentrations reported are final concentrations after mixing. The nucleotide content was analyzed as described above. Design of ClpB Constructs—ClpB fragments were constructed based on sequence alignments of ClpB proteins from different organisms and the crystal structure of ClpB (Fig. 1A) (13Lee S. Sowa M.E. Watanabe Y. Sigler P.B. Chiu W. Yoshida M. Tsai F.T. Cell. 2003; 115: 229-240Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). The constructs used in this study are shown in Fig. 1B. For stability reasons, the N-terminal domain of ClpB, which is dispensable for function, was not included in the construct coding for the first AAA cassette. This construct started at position 141, where an alternative start codon is present in the E. coli mRNA being responsible for the natural occurrence of ClpBΔN (29Park S.K. Kim K.I. Woo K.M. Seol J.H. Tanaka K. Ichihara A. Ha D.B. Chung C.H. J. Biol. Chem. 1993; 268: 20170-20174Abstract Full Text PDF PubMed Google Scholar, 30Squires C.L. Pedersen S. Ross B.M. Squires C. J. Bacteriol. 1991; 173: 4254-4262Crossref PubMed Google Scholar). Because the ClpB-specific middle region inserts within the first AAA cassette it was included in the first construct. The C-terminal end of the first construct was behind the insertion of the coiled-coil domain and close to the beginning of the second AAA cassette. We introduced a stop codon after position 519. Hence the last 15 amino acids (519-534) of the first AAA cassette were not present in this construct. The second construct started at position 519 and ended with the naturally used stop codon after amino acid 854. The last 15 amino acids (519-535) of the first AAA cassettes as seen in the structure were included in the construct for the second AAA cassette. The additional N-terminal amino acids from the first AAA cassette allowed this construct to fold in its native structure and facilitated interdomain communication. Both proteins were recombinantly expressed in E. coli and purified by affinity chromatography and gel filtration. The correct folding of the constructs was assessed by CD spectroscopy (Fig. 1C). The CD spectra of both fragments had local minima at 208 and 222 nm typical of folded proteins with a relatively high content of α-helices. The first construct contains a coiled-coil domain and shows a typical strong peak at 208 nm. This is in agreement with the secondary structure of ClpB observed in the crystal structure and shows that the independently expressed ClpB fragments are folded correctly, which is a prerequisite for the functional characterization of the constructs. Oligomerization of the ClpB Variants—ClpB can form hexamers in the presence of ATP and this oligomerization is essential for the chaperone functi
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