From the
Biochemisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland,
¶ F. Hoffmann-La Roche Ltd., Pharmaceutical Research, CH-4070 Basel, Switzerland
Received for publication, January 28, 2003
, and in revised form, March 10, 2003.
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ABSTRACT |
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INTRODUCTION |
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EXPERIMENTAL PROCEDURES |
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PeptidesThe peptide ala-p5 (ALLLSAPRR) was purchased with a purity of >90% from Chiron. The peptide was dissolved in 0.1% (v/v) acetic acid, 10% (v/v) acetonitrile and stored at 20 °C. The two D-peptides RI117 (EALWKSTSVLARYLVQHV) and RI110 (VLARYLVQHV) were synthesized by Dr. S. Klauser in our Institute with an ABI 430 A Peptide Synthesizer (Applied Biosystems) and purified on a fast protein liquid chromatography system (Pharmacia Corp.) by gel filtration (Fractogel EMD BIOSEC 650 S, from Merck). The molecular masses were confirmed by electrospray mass spectrometry, and the concentrations were determined by amino acid analysis. The peptides were dissolved and stored at 20 °C in 50% (v/v) acetonitrile. Control experiments excluded the possibility that the chaperone-inhibitory effect was due to contaminating acetonitrile or acetate in the refolding mixture. Both peptides were labeled with the environmentally sensitive fluorophor acrylodan (Molecular Probes) and purified as described (3). Three peaks were separated, corresponding to labeling at the -amino group, the
-amino group of the D-lysine residue, and double labeling at both groups. The fractions of the first peak corresponding to uniquely
-labeled RI117 or RI110 were used for the experiments.
Other MaterialsNuclease- and protease-free bovine serum albumin (fraction V) was obtained from Calbiochem-Novabiochem. The stock solutions of serum albumin were filtrated with a 0.2-µm membrane; ATP was from Fluka, and D-luciferin was from Sigma. [2,5',8-3H]Adenosine 5'-triphosphate ammonium salt (41.0 Ci/mmol) was purchased from Amersham Biosciences. All of the buffers were filtrated with a 0.2-µm membrane.
Analytic ProceduresThe ATPase activity of DnaK was determined with a single-turnover assay (5). The radioactive ADP product was separated from radioactive ATP by thin layer chromatography, and the radioactivity of ADP and ATP was determined with a liquid scintillation counter. The binding rate constants of DnaJ for acrylodan-labeled peptides were determined with a Spex Fluorolog spectrofluorimeter as described (7). The excitation wavelength was set at 370 nm (band pass, 4.6 nm) with the emission wavelength set at 520 nm (band pass, 4.6 nm). For fluorescence titration, the reactions were followed at an emission wavelength of 520 nm (band pass, 4.6 nm) until equilibrium was reached.
Preparation of LuciferaseLuciferase of Photinus pyralis was purchased from Roche Molecular Biochemicals. For denaturation, 1 mg of lyophilized material was dissolved in 1.56 ml of refolding buffer (50 mM Tris/HCl, 55 mM KCl, 5.5 mM dithiothreitol, pH 7.7), filtrated with a 0.2-µm membrane, and precipitated by adding five volumes of acetone (20 °C, 30 min). After centrifugation for 10 min at 10000 x g and 4 °C, the pellet was redissolved in 1.56 ml of denaturing buffer (6 M guanidine HCl, 100 mM Tris/HCl, 10 mM dithiothreitol, pH 7.7) and after 0.2-µm filtration was stored at 80 °C in 20-µl portions.
Refolding AssayThe chaperone activity of the DnaK/DnaJ/GrpE system was assessed by its effect on the refolding of guanidine HCl-denatured luciferase. Under the conditions used, denatured luciferase refolds to the active enzyme only in the presence of the chaperones under consumption of ATP. In the presence of O2, luciferase catalyzes the conversion of D-luciferin and ATP to oxiluciferin, CO2, AMP, PPi, and h. Generated photons were counted with a luminometer (Lumac Biocounter M 1500 from Lumac bv).
DnaJ (0.1 µM) was preincubated in refolding buffer at 25 °C for 20 min with the peptide to be tested. DnaK (1 µM), GrpE (0.5 µM), ATP (2 mM), and MgCl2 (5 mM) were then added; the total protein concentration was set to 0.5 mg/ml by supplementation with bovine serum albumin. The refolding reaction was performed at 25 °C and started by adding 2 µl of denatured luciferase (0.1 µM) to the refolding solution with a final dilution factor of 1:100. All of the concentrations in parentheses given above are the final concentrations in the total 200 µl of the refolding reaction mixture. The activity was measured in triplicate every 22.5 min according to a modified previously published procedure (4, 12). Samples of the refolding mixture (10 µl) were added to 190 µl of refolding buffer containing 0.5 mg/ml bovine serum albumin. A 10-µl sample of this 1:20-diluted solution was added to 990 µl of luciferase assay buffer (50 mM Tris/HCl, 15 mM MgCl2, pH 7.7), and subsequently 300 µl of 5 µM D-luciferin in 50 mM Tris/HCl, 15 mM MgCl2, 5 mM ATP, pH 7.7, were added. After a delay of 90 s, the intensity of the light generated by the luciferase reaction was measured for 10 s.
Programs and CalculationsFor graphical evaluation of the peptide-induced inhibition, we used the program GRAFIT 3.0 (Erithacus Software Ltd.), applying a one-site competition algorithm. The EC50 values were estimated from the fitted curves in the plots of yield of luciferase reactivation versus concentration of the peptide inhibitor.
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RESULTS AND DISCUSSION |
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We examined the binding of the acrylodan-labeled peptides a-RI117 and a-RI110 to DnaJ by following the change in fluorescence (3). The kinetic traces followed a double-exponential function (Fig. 1A). The rates of the first phases were found to be a linear function of the concentration of DnaJ (Fig. 1B), and the kon and koff values were obtained from the slope and intercept, respectively, of the plot (Table I). The rate of the second, smaller phase was independent of the concentration of DnaJ (data not shown) and thus may reflect an isomerization of the complex formed in the first phase. In accord with previous reports (7, 8), both D-peptides did not bind to DnaK, as indicated by the fact that they did not stimulate the ATPase activity of DnaK, whereas the control peptide ala-p5, a known substrate for DnaK (13), increased the ATPase activity by a factor of 20 (Table II). The peptide binding properties of DnaK are different from those of DnaJ. DnaK interacts with both the backbone and side chains of the peptides (14) and thus shows binding polarity that admits only D-peptide (7, 8). DnaJ apparently interacts exclusively with the side chains of the substrate and thus can also bind D-peptides.
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Inhibition of the Chaperone Effect by D-PeptidesThe refolding of denatured luciferase provides an often used in vitro assay for the chaperone action of the DnaK/DnaJ/GrpE system (15). Previous studies have shown that for efficient refolding the presence of all three chaperones is necessary (15) and that denatured luciferase forms ternary complexes with DnaK and DnaJ (6). However, it has remained unclear whether the conjoint binding of DnaK and DnaJ to different sites of the same target polypeptide chain is prerequisite for the chaperone action. The finding that D-peptides bind exclusively to DnaJ and compete with L-peptides for the same binding site in DnaJ (7, 8) provides an approach to explore this question.
We tested the effect of the D-peptides on the refolding of denatured luciferase in the presence of DnaK, DnaJ, GrpE, and ATP. Upon preincubation of DnaJ with RI117 or RI110, the yield of refolding decreased with increasing concentration of the D-peptide (Fig. 2A). Both RI117 and RI110 were effective in inhibiting the chaperone-assisted refolding of luciferase, with EC50 values of 2 µM (Fig. 2B) and
1 µM (Fig. 2C), respectively. The EC50 values correlated with their binding affinities for DnaJ (Table I). In a control experiment, the addition of either peptide alone to denatured luciferase did not affect its spontaneous refolding (data not shown). Because both D-peptides bind exclusively to DnaJ and compete with L-peptide ligands for the same binding site (7), the observed inhibition appears to be due to the formation of DnaJ·D-peptide complexes that renders DnaJ incapable to bind to denatured luciferase and to form ternary (ATP·DnaK)m·luciferase·DnaJn complexes (the indices point out that more than one molecule of either chaperone might simultaneously interact with luciferase). Addition of the inhibitory D-peptide RI117 at different times after initiation of the refolding of luciferase arrests the refolding process without apparent delay (Fig. 3). Apparently, binding of the D-peptide to DnaJ disrupts the chaperone action.
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Conjoint binding of DnaK and DnaJ to one and the same polypeptide chain increases the ATPase activity of DnaK by 1 order of magnitude over and above the activity observed in the presence of DnaJ and a small peptide (16). This effect provides an additional means to examine the formation of ternary DnaK· substrate·DnaJ complexes. In the presence of denatured luciferase, DnaJ stimulated the ATPase activity of DnaK 100-fold (Table II and Ref. 17), whereas preformed DnaJ·RI117 or DnaJ·RI110 stimulated the ATPase activity of DnaK only 10-fold (Table II). The binding of peptides RI117 or RI110 to DnaJ does not affect the interaction of the J-domain of DnaJ with the ATPase domain of DnaK; the stimulatory effect of DnaJ on the ATPase activity of DnaK in the presence of either D-peptide was the same as that in the absence of peptide inhibitor (Table II). Apparently, the ineffectiveness of DnaJ·D-peptide complexes in stimulating the ATPase activity of DnaK in the presence of denatured luciferase is due to the incapability of the DnaJ·D-peptide complexes to bind to denatured luciferase. These results corroborate the notion that D-peptides inhibit the chaperone action of the DnaK/DnaJ/GrpE system by precluding DnaJ from engaging in ternary (ATP· DnaK)m·substrate·DnaJn complexes.
Conceivably, the conjoint binding of DnaJ and DnaK to one and the same polypeptide chain facilitates the interaction of the J-domain of DnaJ and the ATPase domain of DnaK by a propinquity effect (16). The cis-interaction of DnaJ with DnaK seems not only to be necessary for efficient chaperone-mediated refolding of target proteins but has also been reported to be required for the activation of latent P1 RepA dimer into its active monomer (18). Why is the binding of DnaK and DnaJ to one and the same unfolded target polypeptide necessary for the chaperone action of the DnaK system? Rapid triggering of the hydrolysis of DnaK-bound ATP might be essential for feeding the substrate protein into the chaperone cycle. Hydrolysis of ATP converts DnaK from its low affinity state to its high affinity ADP-liganded state. Without the cis-action of DnaJ, the substrate would cycle on and off ATP·DnaK, without chaperone action being effected.
Concluding RemarksThe exclusive binding of D-peptides to the substrate-binding site of DnaJ (7, 8) specifically allows inhibition of the association of DnaJ with a substrate polypeptide. Substrate-induced effects in the DnaK/DnaJ/GrpE system that are due to the binding of substrate to DnaJ may thus be discriminated from effects that are due to the binding of substrate to DnaK. Using this approach, we provided evidence that the chaperone action of the DnaK/DnaJ/GrpE system depends on the formation of ternary (ATP·DnaK)m·substrate·(DnaJ)n complexes. The acceleration of the chaperone cycle consequent to the formation of ternary complexes, perhaps together with an as yet unknown topological or conformational constraint inherent in the cis-interaction of DnaK and DnaJ, appears to be a prerequisite for efficient chaperone action.
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FOOTNOTES |
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Both authors contributed equally to this work.
|| To whom correspondence should be addressed. Tel.: 41-1-6355511; Fax: 41-1-6355907; E-mail: christen{at}bioc.unizh.ch.
1 The abbreviations used are: Hsp70, 70-kDa heat shock protein; RI117, retro-inverso rhodanese peptide 117; RI110, retro-inverso rhodanese peptide 110; acrylodan, 6-acrylolyl-2-dimethylaminonaphthalene; a-, acrylodan-labeled.
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ACKNOWLEDGMENTS |
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REFERENCES |
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