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Originally published In Press as doi:10.1074/jbc.M406795200 on August 30, 2004

J. Biol. Chem., Vol. 279, Issue 44, 45737-45743, October 29, 2004
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BeFx Stops the Chaperonin Cycle of GroEL-GroES and Generates a Complex with Double Folding Chambers*

Hideki Taguchi{ddagger}§, Keigo Tsukuda{ddagger}, Fumihiro Motojima{ddagger}, Ayumi Koike-Takeshita{ddagger}, and Masasuke Yoshida{ddagger}||

From the {ddagger}Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama, 226-8503, Japan, §Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, 332-0012, Japan

Received for publication, June 17, 2004 , and in revised form, August 26, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Coupling with ATP hydrolysis and cooperating with GroES, the double ring chaperonin GroEL assists the folding of other proteins. Here we report novel GroEL-GroES complexes formed in fluoroberyllate (BeFx) that can mimic the phosphate part of the enzyme-bound nucleotides. In ATP, BeFx stops the functional turnover of GroEL by preventing GroES release and produces a symmetric 1:2 GroEL-GroES complex in which both GroEL rings contain ADP·BeFx and an encapsulated substrate protein. In ADP, the substrate protein-loaded GroEL cannot bind GroES. In ADP plus BeFx, however, it can bind GroES to form a stable 1:1 GroEL-GroES complex in which one of GroEL rings contains ADP·BeFx and an encapsulated substrate protein. This 1:1 GroEL-GroES complex is converted into the symmetric 1:2 GroEL-GroES complex when GroES is supplied in ATP plus BeFx. Thus, BeFx stabilizes two GroEL-GroES complexes; one with a single folding chamber and the other with double folding chambers. These results shed light on the intermediate ADP·Pi nucleotide states in the functional cycle of GroEL.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Chaperonins are a class of molecular chaperones that promote protein folding in the cell and are found in bacteria, chloroplasts, mitochondria, Archaea, and eukaryotic cytosol (1, 2). The best characterized of these is the Escherichia coli chaperonin, GroEL, and its partner GroES (1–5). GroEL is a large cylindrical protein complex comprising two heptameric rings of 57-kDa identical subunits, and these rings are stacked back to back (6, 7). GroES contains seven identical 10-kDa subunits assembled as a heptamer ring (7, 8). Current understanding of the major productive pathway of an ATP hydrolysis-coupled GroEL function starting from the substrate protein-loaded GroEL is as follows. (i) For cis-ternary complex formation, the binding of ATP to the substrate protein-loaded heptamer ring of GroEL permits the binding of GroES to this ring (cis-ring) generating an enlarged, closed central cavity (cis-cavity) in which the substrate protein is discharged (cis-ATP complex) (7). The cis-cavity underneath GroES provides a protein chamber for folding without the risk of aggregation (e.g. Refs. 9–11). (ii) For trans-ring activation, ATP hydrolysis in the cis-ring leads to produce the cis-ADP complex, and the GroEL heptamer ring opposite to the cis-ring (trans-ring) becomes ready to bind ATP (12, 13). (iii) In cis-ternary complex decay, the binding (not hydrolysis) of ATP to the trans-ring, in return, induces the release of GroES and ADP from the cis-ring allowing the substrate protein, whether folded or not, to escape from the cis-cavity (12–14). Binding of the next substrate protein and GroES to the previous trans-ring changes this ring to the next cis-ring and the second cycle of reaction starts (12–14).

According to the above scheme, the step (ii), the trans-ring activation involves the hydrolysis of ATP in the cis-ATP complex to generate a transient intermediate cis-ADP·Pi complex, which is followed by rapid Pi release (15). To investigate what happens if Pi release is prevented, we have adopted fluoroberyllate (BeFx)1 that can mimic the hydrolysis-product Pi or {gamma}-phosphate part of the enzyme-bound nucleotide and can form a stable complex with ADP (or GDP) in a variety of ATP (or GTP)-utilizing enzymes (16) including the chaperonin family (17–19). When we added BeFx, the GroEL functional cycle was stopped immediately and a symmetric 1:2 GroEL-GroES complex2 was formed in which both two GroEL rings contained a folding competent encapsulated substrate protein. In ADP + BeFx, a stable 1:1 GroEL-GroES complex with a substrate protein in the cis-cavity was generated and the further addition of GroES in ATP + BeFx produced the symmetric 1:2 GroEL-GroES complex. The implication of these results in the mechanism of the chaperonin functional cycle is discussed.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Proteins and Reagents—BeCl2 was purchased from Aldrich. NaF and AlCl3 were obtained from Wako (Osaka, Japan). Malate dehydrogenase (porcine), proteinase K, ATP, ADP, and NADH were obtained from Roche Applied Science. Calcium-depleted bovine {alpha}-lactalbumin (Type III), pyruvate kinase, lactate dehydrogenase, and hexokinase were from Sigma. Cy3-NHS (Fluorolink Cy3 monofunctional dye) was from Amersham Biosciences. GroEL, GroES, and bovine mitochondrial rhodanese were purified as described (20). GroEL purified by the procedures including gel-filtration column chromatography in the presence of methanol contained only a very small amount of contaminated proteins (< 0.1 Trp residues/GroEL 14-mer). GroES labeled with Cy3 (GroESCy3) was prepared as described (14) in a stoichiometry of ~0.7 Cy3 dye molecules/GroES 7-mer. GroEL saturated with denatured rhodanese was prepared as described (21). Briefly, 4 µM rhodanese was heat-denatured at 60 °C for 15 min in HKM buffer (20 mM HEPES-KOH, pH 7.4, 100 mM KCl, 5 mM MgCl2) containing 1 mM dithiothreitol (DTT) and 1 µM GroEL. After the heat denaturation of rhodanese, the temperature was shifted down to 25 °C, and the GroEL-denatured rhodanese complex was isolated by a 100-kDa cut ultrafiltration (Microcon YM-100, Amicon). Approximately 2.6 mol of rhodanese were bound to 1.0 mol of GroEL. Trace amounts of contaminating ATP in the ADP solution was eliminated by a hexokinase/glucose treatment as described (21). Protein concentrations were determined spectrophotometrically (14, 15) or by the Bradford protein assay (Bio-Rad). Protein concentration was expressed as oligomer (GroEL, 14-mer; GroES, 7-mer) throughout this paper. 15% of SDS-gels was used for electrophoresis and stained with Coomassie Brilliant Blue-R250.

ATPase Assay—The release of ADP from GroEL was measured spectrophotometrically with an ATP-regenerating system as described (15). The assay mixture contained 0.2 mM NADH, 5 mM phosphoenolpyruvate, 100 µg/ml pyruvate kinase, 100 µg/ml lactate dehydrogenase, 5 mM DTT, 10 mM NaF, and 0.2 µM GroEL in HKM buffer. When indicated, 0.8 µM GroES, 4.0 µM lactalbumin reduced with 5 mM DTT for at least 1 h (rLA) (22–25), 1.0 mM BeCl2 were included.3 The reaction was initiated by injection of ATP (final concentration, 1 mM) into the vigorously stirred solution. The decreases in the absorbance at 340 nm, due to oxidation of NADH, were monitored continuously with a spectrophotometer (V-550, Jasco, Japan).

GroES Exchange—ATP was rapidly injected (0.5 mM final concentration) into 1.3 ml of HKM buffer containing 5 mM DTT, 10 mM NaF, 100 nM GroEL, 50 nM GroESCy3, and 10 µM rLA in the absence or presence of BeCl2 at 25 °C. Changes in the Cy3 fluorescence (excitation at 545 nm, emission at 563 nm) were monitored continuously with a fluorometer (F-4500, Hitachi, Japan). When indicated, unlabeled GroES (1.0 µM final concentration) was injected into the mixtures.

Gel Filtration—The solution containing 5 mM DTT, 10 mM NaF, 1 mM BeCl2, 1.4 µM GroEL, 5.6 µM GroESCy3, 10 µM rLA, and 1 mM nucleotide (ATP or ADP) in HKM buffer was incubated at 25 °C for 2 min, and applied to a gel filtration HPLC column (G3000SWXL, Tosoh, Japan) equilibrated with HKM buffer containing 50 mM Na2SO4, 10 mM NaF, and 1 mM BeCl2. Elution was monitored by an in-line fluorometer (excitation at 545 nm, emission at 563 nm) with a flow rate of 0.5 ml/min. The peak containing the GroEL-GroES complex was collected for the further analyses (see below). In some experiments, the peak fractions containing the GroEL-GroES complexes were concentrated by ultrafiltration (YM-100) and then applied again to a gel-filtration column under the conditions indicated.

Quantitation of Bound GroES and Nucleotide—To know the amount of the bound GroES, the GroEL complexes isolated by gel-filtration HPLC were analyzed by SDS-PAGE. The Coomassie Brilliant Bluestained bands were quantitated by a public domain software package (NIH Image). The determination of bound nucleotides was as follows. The isolated GroEL complexes were treated with perchloric acid (final 1.0%), and the supernatant was neutralized with K2CO3. The supernatants were applied to a reverse-phase HPLC column (ODS-80Ts, Tosoh, Japan), which can separate ATP and ADP with monitoring by absorbance at 260 nm (26). Amount of nucleotide was calculated by the integrated peak area.

Electron Microscopy—The solution containing 1 mM ATP, 10 mM NaF, 1 mM BeCl2, 5 mM DTT, 1.0 µM GroEL, 4.0 µM GroES, and 20 µM rLA in HKM buffer was subjected to ultrafiltration to remove free GroES and rLA. An aliquot of the solution was applied on an electron microscope specimen grid covered with a carbon support film. The specimen was immediately stained with 2.0% uranyl acetate and observed with an electron microscope (JEM-1230, JEOL) at an accelerating voltage of 80 kV. Images were recorded onto electron image films at a magnification of 40,000.

Rhodanese Folding Assay—To initiate folding reactions, the solution containing the GroEL saturated with rhodanese and GroES in HKM buffer was mixed with a 3-fold volume of the solution containing nucleotide (ATP or ADP), 10 mM NaF, and 1 mM BeCl2. When indicated, hexokinase was included in the ADP solutions. Final concentrations of the components in the reaction mixtures were as follows: 1 mM nucleotides, 0.5 µM GroEL saturated with rhodanese, 1.0 µM GroES, 200 mM glucose, 1 mM DTT, 20 mM Na2S2O3, and when indicated, 0.04 units/µl hexokinase. For the single turnover ATP hydrolysis experiment, excess ATP was quenched by adding hexokinase (final concentration, 0.04 units/µl) to the reaction mixture at 3 s after initiation of the reaction. We confirmed that 1 mM ATP in the reaction mixture was quenched completely within the next 3 s (21). To know the time course of folding of rhodanese, aliquots (5 µl) were taken out at indicated times, mixed with 750 µl of the solution containing 100 mM KH2PO4, 150 mM Na2S2O3, and 1 mM EDTA, and recovered rhodanese activities were determined according to the standard protocol (27). To know the amount of GroES and the rhodanese in the folding chambers, released GroES and rhodanese were removed by the ultrafiltration at 1 h after initiation of the reaction, and subsequently proteinase K (final concentration, 1 µg/ml) was added. After a 30-min incubation at 25 °C, phenylmethanesulfonyl fluoride was added at final concentration 1 mM, and the components with molecular mass smaller than 100 kDa were removed by ultrafiltration. An aliquot of the resulting solution (~20 µg) was applied to 15% SDS gel. Other aliquots of proteinase K-treated, ultrafiltered samples (~4 µg protein) were used for the assays of the recovered activities of rhodanese. To release all of the proteins from the folding chambers, GroES was detached from GroEL by addition of equal volume of 20 mM CDTA. After a 90-min incubation at 25 °C, the activities of rhodanese were measured.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of GroEL ATPase Turnover by BeFx—The effect of BeFx on GroEL ATPase activity in the presence of GroES and rLA was examined. Upon the addition of 1 mM BeCl2 in the medium containing excess NaF, the turnover of ATP hydrolysis by GroEL was inhibited almost completely and immediately (<2 s) (Fig. 1A and inset). Because single turnover of the ATPase cycle of GroEL under these conditions takes ~8 s, BeFx stops the action of GroEL within a single reaction cycle. AlFx was also tested but, unlike BeFx, several minutes were required to gain the maximum inhibition (data not shown). Another phosphate analogue, NaVO4, did not show an inhibitory effect on GroEL ATPase, as reported previously (18, 19). Therefore, we focused on the effect of BeFx. When BeFx concentrations in the assay mixtures were changed by varying BeCl2 concentrations in 10 mM NaF, 30 µM BeCl2 gave a 50% inhibition of ATPase, 100 µM BeCl2 90%, and 250 µM BeCl2 99%. Accordingly, we adopted 1 mM BeFx in the following experiments to ensure the maximum inhibition. Above measurements were carried out for GroEL + rLA + GroES, but ATPase activity of GroEL + rLA or GroEL alone was also severely inhibited by BeFx (Fig. 1B). An alternative ATPase assay using an inorganic phosphate determination (Malachite Green assay) also gave indistinguishable results (data not shown).



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  		FIG. 1.
Inhibition of GroEL ATPase by BeFx. A, time course of ATP hydrolysis. ATP hydrolysis by GroEL in the presence of GroES and rLA was monitored as the oxidation of NADH at 340 nm in an ATP-regenerating system. The vertical bar denotes absorbance unit at 340 nm. BeFx was formed by adding BeCl2 (final concentration 1.0 mM) to the buffer containing 10 mM NaF. The trace marked with none represents an experiment in the absence of BeCl2. Inset, an expanded time-scale to show immediate inhibition of ATPase upon addition of BeCl2. B, ATPase activities of GroEL under various conditions. Other experimental conditions are described under "Experimental Procedures."

 
Stable Binding of GroES to GroEL in BeFx—The fluorescently labeled GroESCy3 is functionally intact, and the fluorescence increases upon binding to GroEL (14). The addition of ATP to the mixture of GroEL, GroESCy3, and rLA initiated the functional chaperonin cycle and induced the increase of the fluorescence of GroESCy3 (Fig. 2A). The dilution of GroESCy3 by the addition of a large excess of unlabeled GroES resulted in a decrease in fluorescence, indicating an exchange of GroEL-bound GroESCy3 with unlabeled free GroES. In the presence of BeFx, however, the fluorescence was not changed by the addition of unlabeled GroES (Fig. 2B). Therefore, BeFx inhibits the functional cycling of GroEL by preventing the release of GroES from GroEL.



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  		FIG. 2.
Release of fluorescently labeled GroES from GroEL. Fluorescence of Cy3 attached to GroES (GroESCy3) was monitored. The solution contained 0.1 µM GroEL, 0.05 µM GroESCy3, and 10 µM rLA in the absence (A) or presence (B) of BeFx. ATP and a 20-fold excess of unlabeled GroES were injected at 20 and 100 s, respectively. a.u., arbitrary units. Other experimental conditions are described under "Experimental Procedures."

 
GroEL-GroES Complexes Formed in BeFx—To analyze the GroEL-GroES complex formed in BeFx, we incubated GroEL, rLA, ATP, and GroESCy3 for 2 min, added BeFx, incubated for 2 min, subjected the mixture to gel-filtration HPLC, and eluted the mixture with the BeFx-containing buffer. The GroESCy3 eluted at the position of the GroEL shown by an arrow (Fig. 3A) represents the GroEL-GroESCy3 complex. The amount of GroESCy3 in the complex was calculated to be ~1.8 mol/mol of GroEL based on the fraction in the total eluted fluorescence. When ADP was included instead of ATP in the incubation mixture, ~0.9 mol of GroESCy3/mol of GroEL were co-eluted with GroEL (Fig. 3A). The stability of the GroEL-GroESCy3 complex formed in ATP + BeFx was dependent on the presence of BeFx in the medium, because when the peak fraction of the GroEL-GroESCy3 complex was applied to a second gel-filtration column and eluted with the buffer that did not contain BeFx, approximately one-half of the GroESCy3 was dissociated from GroEL during elution, leaving the complex with ~0.9 mol of GroESCy3/mol of GroEL (Fig. 3B). The released GroESCy3 was eluted as a long tailing of the major peak but not an isolated peak, indicating that dissociation occurred gradually in BeFx-free buffer. The complex made in ADP + BeFx was stable; it was not dissociated during the elution with the BeFx-free buffer (data not shown). When BeFx was absent in both the reaction mixtures and the elution buffer, ATP and ADP gave similar results; ~0.3 mol of GroESCy3/mol of GroEL were found at the position of GroEL (Fig. 3C).



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  		FIG. 3.
Gel filtration analyses of the GroEL-GroESCy3 complexes. Fluorescence of GroESCy3 was monitored with an in-line fluorometer. a.u., arbitrary units. A, mixtures of GroEL, GroESCy3, rLA, and ATP (solid line) or ADP (dashed line) were incubated for 2 min and analyzed with gel-filtration HPLC eluted with a buffer containing BeFx. B, the isolated GroEL-GroESCy3 complex formed in the presence of ATP + BeFx (the 12.2-min peak fraction of solid line in A) was applied to a second gel-filtration HPLC eluted with a buffer that did not contain BeFx. C, the same as in A, except that BeFx was absent in both the reaction mixtures and the elution buffer.

 
Analysis of the GroEL-GroES Complexes Formed in BeFx— The peak fraction of the GroEL-GroES complex in gel-filtration HPLC was isolated and further analyzed. The densitometry of the bands of GroEL and GroES in SDS-PAGE of the complexes confirmed the GroEL:GroES molar ratios estimated from the elution profiles above described; 1:1.7 for the complex made in ATP + BeFx and 1:0.8 for the complex made in ADP + BeFx (Table I). The analysis of nucleotides extracted from the complexes indicated that ADP occupied all of the nucleotide binding sites of GroEL in the complex formed in ATP + BeFx and half of the nucleotide binding sites in the complex formed in ADP + BeFx. From these results we concluded that the complex made in ATP + BeFx was the 1:2 GroEL-GroES complex in which the nucleotide binding sites of two GroEL rings were filled by ADP and that the complex made in ADP + BeFx was the 1:1 GroEL-GroES complex in which the nucleotide binding sites of one GroEL ring were filled by ADP. Bound ADP in these complexes should be coordinated with BeFx, because the complexes were isolated only in the presence of BeFx. It appeared, however, that the BeFx moiety of ADP·BeFx in one of the GroEL rings of the 1:2 GroEL-GroES complex was exchangeable with BeFx medium and, in the absence of BeFx medium, ADP·BeFx (and GroES) dissociated from one of GroEL rings to produce the 1:1 GroEL-GroES complex (see also Fig. 6B).


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  		TABLE I
Stoichiometry of GroES, nucleotide, and rhodanese in the isolated GroEL complexes

 



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  		FIG. 6.
Binding of the second GroES to the 1:1 GroEL-GroES complex. A, the isolated 1:1 GroEL-GroES complex formed in ADP + BeFx was incubated with GroESCy3 in the absence (trace 1) or the presence of ATP (trace 2) or ADP (trace 3) and applied to gel-filtration HPLC eluted with a buffer containing BeFx. Inset, SDS-PAGE analysis of the isolated 1:1 GroEL-GroES complex formed in ADP + BeFx before and after proteinase K treatment. Proteins were stained by Coomassie Brilliant Blue. The molar ratios of GroEL:GroES:rhodanese, determined by the band intensity, are 1:0.8:2.1 (left lane) and 1:0.8:1.3 (right lane). B, the 1:2 GroEL-GroES complex isolated from the trace 2 in A was concentrated and applied to gel-filtration HPLC eluted with a buffer containing BeFx (trace 1) or absence of (trace 2) BeFx. For the complete dissociation of GroEL-GroES complex, 10 mM of CDTA was included in the elution buffer (trace 3).

 
Electron Micrograph of the GroEL-GroES Complexes Formed in BeFx—We observed the images of the complexes formed in ATP + BeFx under an electron micrograph. Complexes formed in ATP + BeFx showed symmetrical football-like shapes, and GroES associated to both ends of GroEL (Fig. 4). On the contrary, bullet-like shapes with GroES associated to one end of GroEL were seen for the complexes formed in ADP + BeFx (data not shown), a familiar shape for the usual GroEL-GroES complex made in the absence of BeFx.(e.g. Ref. 28).



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  		FIG. 4.
Electron micrograph of GroEL-GroES complex formed in the presence of ATP + BeFx. The sample was negatively stained with 1.0% uranyl acetate. Lower gallery shows selected typical images of the football-shaped GroEL-GroES complex. Bar represents 50 nm.

 
Folding in the Presence of BeFx—All of the above experiments were carried out in the presence of substrate proteins, rLA. To know whether substrate protein(s) can fold in the 1:1 and 1:2 GroEL-GroES complexes formed in BeFx, we used rhodanese as a substrate protein. Rhodanese is a stringent substrate protein that can fold efficiently only with the aid of GroEL, GroES, and ATP (e.g. Refs. 29 and 30). The reaction was started by mixing GroES, ATP, ± BeFx, and GroEL whose substrate protein binding sites of two rings were saturated previously with denatured rhodanese (21). Without BeFx, rhodanese activity was gradually recovered until it finally reached a value corresponding to 1.8 mol of rhodanese/mol of GroEL (Fig. 5A). In this case, unfolded rhodanese was repeatedly encapsulated into (and escaped from) the cis-cavity during multiple turnovers of the chaperonin functional cycle (31–33). In the presence of BeFx, 1.9 mol of rhodanese were folded. We also examined the folding of rhodanese in ADP ± BeFx. It should be noted that, even with taking special precautions to remove contaminating ATP from commercial ADP (21), no recovery of rhodanese activity was observed in ADP. However, when BeFx was present, 1.1 mol of rhodanese/mol of GroEL were folded in ADP. In these folding experiments, the final folding yields were different from each other, but the folding rates were nearly the same, reflecting the inherent folding characteristics of rhodanese. We also examined the folding of another stringent substrate protein, malate dehydrogenase (34, 35). Consistent with the results of rhodanese, activity recovered in ADP + BeFx was 54% of that recovered in ATP + BeFx (data not shown).4 The above results, together with the GroEL:GroES stoichiometry, indicated that the 1:2 GroEL-GroES complex generated in ATP + BeFx contains two substrate proteins, whereas the 1:1 GroEL-GroES complex generated in ADP + BeFx contains one. These substrate proteins are assumed to be encapsulated in the folding chambers underneath GroES and allowed to fold. Next, we further confirmed this assumption.



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  		FIG. 5.
GroEL-GroES-dependent folding of rhodanese in the presence of BeFx. A, recovery of rhodanese activities. Recovered yields were expressed as mol of recovered enzyme/mol of GroEL. At time 0, ATP or ADP was added to the mixtures containing GroEL saturated with denatured rhodaneses and GroES. B and C, protection of the encapsulated rhodaneses from proteinase K. The aliquots after the folding assay (A) were subjected to the following procedures: ultrafiltration (100-kDa cut), proteinase K treatment, second ultrafiltration (100-kDa cut), and SDS-PAGE (B) or measurements of remaining rhodanese activity (C). Molar ratios of GroEL:GroES:rhodanese, determined by the band intensity (B), are 1:2.0:1.9 (ATP + BeFx) and 1:1.4: 1.4 (ADP + BeFx). Other experimental conditions are described under "Experimental Procedures."

 

Accessibility of Protease—The mixtures incubated for 60 min as in Fig. 5A were subjected to ultrafiltration to remove free GroES, treated with proteinase K, and again subjected to ultrafiltration to remove proteinase K and digestion products. The resultant solutions were used for SDS-PAGE analysis and for the rhodanese activity assay. The densitometry of the bands in Fig. 5B showed that the complexes generated in ATP + BeFx and ADP + BeFx contained 1.9 and 1.4 mol of proteinase K-resistant rhodanese/mol of GroEL, respectively (Table I). Also, the rhodanese activity roughly corresponding to the amount of rhodanese in SDS-PAGE was detected (Fig. 5C). Without BeFx, a rhodanese band in SDS-PAGE and rhodanese activity was not detected in the complexes generated in ATP or ADP. The reasons why no rhodanese was recovered in the absence of BeFx are explained as follows. With the ATP present in the absence of BeFx, the folding of rhodanese was assisted by GroEL-GroES and released to a bulk solution and thus removed in the ultrafiltration process. However with the ADP present in the absence of BeFx, rhodanese was bound to GroEL but no or little GroES bound to the rhodanese-bound GroEL ring under this condition, the non-native rhodanese was susceptible to proteolytic digestion. The 1:2 and 1:1 GroEL:GroES molar ratios for the complexes made in ATP + BeFx and ADP + BeFx, respectively, were also seen in Fig. 5B. These results showed clearly that rhodanese molecules in these complexes after the protease treatment were really accommodated in the folding chambers but not associated to the trans-ring or peripheral surface of GroEL.

GroESCy3 Binding to the 1:1 GroEL-GroES Complex—The 1:1 GroEL-GroES complex formed in ADP + BeFx was isolated by gel-filtration HPLC with the buffer containing BeFx. The isolated complex contained ~2 mol of rhodanese/mol of GroEL, one in the cis-cavity and the other at the trans-ring as shown by sensitivity to proteinase K digestion (Fig. 6A, inset). The isolated complex in the BeFx buffer was incubated with GroESCy3 + ATP, GroESCy3 + ADP, or GroESCy3 alone and applied again to the same gel-filtration column eluted with the BeFx buffer. As shown in Fig. 6A, binding of GroESCy3 to the 1:1 GroEL-GroES complex in the BeFx buffer was observed only in ATP but not in ADP nor in the absence of nucleotide. SDS-PAGE analysis of the GroEL complexes confirmed that the amount of GroES (sum of GroESCy3 and unlabeled GroES) relative to GroEL increased from 1.0 to 2.3 after incubation with ATP + BeFx (data not shown).

Release of GroESCy3 from the 1:2 GroEL-(ES + ESCy3) Complex—Taking advantage of the isolation of the 1:2 GroEL-(ES + ESCy3) complex, we tested whether the secondary bound GroESCy3 in the complex is equivalent to the previously bound GroES. To address the question, we applied the 1:2 GroEL-(ES + ESCy3) complex to a gel-filtration column eluted in the absence of BeFx where the 1:2 GroEL-GroES complex should release one of two GroES to produce the 1:1 GroEL-GroES complex (Fig. 3B). If the secondary bound GroESCy3 is preferentially released, all of the GroESCy3 should be eluted as unbound GroESCy3 leaving a non-fluorescent 1:1 GroEL-GroES complex. However, this was not the case; half of GroESCy3 remained associated with GroEL, and another half was eluted as unbound GroESCy3 (Fig. 6B, trace 2). As expected, with BeFx in the elution buffer, the original 1:2 GroEL-(ES + ESCy3) complex was recovered, and with CDTA in the elution buffer, all the GroESCy3 was released from GroEL (Fig. 6B, traces 1 and 3). These results demonstrate that the two GroES heptamers in the 1:2 GroEL-GroES complex are equivalent.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study are summarized in Fig. 7. In the presence of BeFx, the 1:1 GroEL-GroES complex is formed in ADP. This complex is converted into the 1:2 GroEL-GroES complex in the presence of BeFx when GroES and ATP are supplied. The same 1:2 GroEL-GroES complex is also formed in ATP in the presence of BeFx. The 1:2 GroEL-GroES complex is unstable in the absence of BeFx medium and is changed into the 1:1 GroEL-GroES complex when exposed to the BeFx-free buffer. The 1:1 GroEL-GroES complex is stable in the BeFx-free buffer. Encapsulated substrate protein(s) in these complexes is free to fold.



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  		FIG. 7.
The complexes formed in the presence of BeFx. In the presence of BeFx, two kinds of GroEL complexes are formed, the 1:1 GroEL-GroES complex in ADP and the 1:2 GroEL-GroES complex in ATP. The 1:1 GroEL-GroES complex is converted into the 1:2 GroEL-GroES complex when GroES and ATP + BeFx are supplied. Conversely the 1:2 GroEL-GroES complex is changed into the 1:1 GroEL-GroES complex when exposed to the BeFx-free buffer. Encapsulated substrate protein(s) in these complexes are allowed to fold.

 
As described in the Introduction, the functional cycle of GroEL is comprised of three steps,5 (i) cis-ternary complex formation, (ii) trans-ring activation, and (iii) cis-ternary complex decay. The experiments using BeFx as an inhibitor provide some insight into the nucleotide states of cis-ring required for the progress of these steps (Table II). It has been known that BeFx can form stable ADP·BeFx at the catalytic sites of various ATP-metabolizing enzymes by mimicking the Pi part of ADP·Pi or {gamma}-P part of ATP (16). Indeed, recent x-ray crystallography of the 1:1 GroEL-GroES formed in AlFx showed that AlF3 is coordinated to ADP at each of the catalytic sites in the cis-ring of the complex (19). X-ray crystallography, as well as x-ray scattering analysis, also suggested that the 1:1 GroEL-GroES complexes formed in metal fluorides resemble the usual 1:1 GroEL-GroES complex formed in the absence of metal fluorides (18, 19). We recently reported that ADP with minimum ATP contamination can mediate the binding of GroES to the substrate protein-free GroEL but not to the substrate protein-loaded GroEL (21). Previously reported cis-folding supported by ADP is most likely because of the trace amount of ATP contained in or generated from ADP. However, in BeFx, authentic ADP can promote GroES binding to the substrate protein-loaded GroEL to generate the cis-ternary complex in which folding can proceed efficiently (Fig. 5). It has been known that ATP binding, but not hydrolysis, is required for the step (i), cis-ternary complex formation, because a slow ATP-hydrolysis mutant GroEL (D398A) can generate a similar 1:1 GroEL-GroES complex when ATP is provided (12). In this context, ADP·BeFx, but not ADP alone, can substitute ATP in the cis-ternary complex formation (Table II).


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  		TABLE II
Nucleotide states of cis-ring

 
If nucleotides at the cis-ring are unhydrolyzed ATP, step (ii) trans-ring activation does not occur even when ATP medium is present (12). It occurs when the nucleotides at the cis-ring are ADP. However, the 1:1 GroEL-GroES complex with ADP·BeFx in the cis-ring can undergo step (ii) when ATP is available (Fig. 6A). Thus, it appears that ATP hydrolysis of the cis-ATP complex is the prerequisite for the trans-ring activation, but Pi release from the cis-ADP·Pi complex is not. In the case when the reaction is started by ATP in the presence of BeFx, an intermediate cis-ADP·Pi complex would appear after the cis-ATP complex and BeFx rapidly replaces the Pi moiety of ADP·Pi to produce the 1:1 GroEL-GroES complex, which then binds ATP at the trans-ring. These results indicate that, in the cis-ADP·Pi complex, the trans-ring is already activated and ready to bind ATP.

As to the step (iii), different from the uninhibited cycle, ATP binding to the trans-ring in the presence of BeFx does not induce the release of GroES, ADP, and substrate protein from the cis-ring. Prior release of Pi from the cis-ring may be necessary for the cis-cavity opening that is induced by ATP binding to the activated trans-ring. The reactions continue in the presence of BeFx even without the decay of the cis-ternary complex and several events follow, binding of GroES to the trans-ring and hydrolysis of ATP in the trans-ring to produce ADP·Pi, which is then converted to ADP·BeFx. These events result in the production of the symmetric 1:2 GroEL-GroES complex with two folding chambers and with 14 ADP·BeFx. The contention above described is our preferred explanation, but of course, other possibilities are not excluded. For example, if the symmetric 1:2 GroEL-GroES complex would be a naturally occurring intermediate of the GroEL functional cycle as argued previously (31, 36–38), the complex formed in ATP + BeFx might represent that intermediate.


   FOOTNOTES
 
* This work was supported in part by grants-in-aid for Scientific Research on Priority Areas (to H. T. and M. Y.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Present address: Dept. of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8562, Japan. Back

|| To whom correspondence should be addressed: Chemical Resources Laboratory, R1-7, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama, 226-8503, Japan. Tel.: 81-45-924-5233; Fax: 81-45-924-5277; E-mail: myoshida{at}res.titech.ac.jp.

1 The abbreviations used are: BeFx, fluoroberyllate, BeF3 or BeF4; rLA, reduced {alpha}-lactalbumin; DTT, dithiothreitol; GroESCy3, Cy3-labeled GroES; HPLC, high pressure liquid chromatography; CBB, Coomassie Brilliant Blue-R250; CDTA, 1,2-cyclohexylenedinitrilotetraacetic acid. Back

2 Concentrations of GroEL and GroES are all expressed as tetradecamer and heptamer, respectively. The 1:1 GroEL-GroES complex is composed of tetradecamer GroEL and heptamer GroES, and the 1:2 GroEL-GroES complex is composed of tetradecamer GroEL and two heptamer GroESs. Back

3 The presence of BeFx did not affect the ATP-regenerating system. The rate of ATP formation catalyzed by pyruvate kinase in the presence of BeFx was indistinguishable from that in the absence of BeFx (data not shown). Back

4 Malate dehydrogenase is a dimeric enzyme. For the dimerization, GroES was partially detached from the BeF complexes by the addition of CDTA under an on-ice condition (12) to xrelease the encapsulated malate dehydrogenase monomer. Therefore, an accurate mol of folded malate dehydrogenase/mol of GroEL was not obtained. Back

5 We recently proposed a two timer mechanism of the GroEL functional cycle that is significantly different from the conventional single timer model (15). However, the functional cycle expressed in the text is compatible with either of the two models. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Kaoru Mitsuoka for the electron microscopic observation, Yukie Kakiyama for technical assistance, and Dr. Tomohiro Mizobata for helpful advice.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bukau, B., and Horwich, A. L. (1998) Cell 92, 351–366[CrossRef][Medline] [Order article via Infotrieve]
  2. Hartl, F. U., and Hayer-Hartl, M. (2002) Science 295, 1852–1858[Abstract/Free Full Text]
  3. Sigler, P. B., Xu, Z., Rye, H. S., Burston, S. G., Fenton, W. A., and Horwich, A. L. (1998) Annu. Rev. Biochem. 67, 581–608[CrossRef][Medline] [Order article via Infotrieve]
  4. Thirumalai, D., and Lorimer, G. H. (2001) Annu. Rev. Biophys. Biomol. Struct. 30, 245–269[CrossRef][Medline] [Order article via Infotrieve]
  5. Saibil, H. R., and Ranson, N. A. (2002) Trends Biochem. Sci. 27, 627–632[CrossRef][Medline] [Order article via Infotrieve]
  6. Braig, K., Otwinowski, Z., Hegde, R., Boisvert, D. C., Joachimiak, A., Horwich, A. L., and Sigler, P. B. (1994) Nature 371, 578–586[CrossRef][Medline] [Order article via Infotrieve]
  7. Xu, Z., Horwich, A. L., and Sigler, P. B. (1997) Nature 388, 741–750[CrossRef][Medline] [Order article via Infotrieve]
  8. Hunt, J. F., Weaver, A. J., Landry, S. J., Gierasch, L., and Deisenhofer, J. (1996) Nature 379, 37–45[CrossRef][Medline] [Order article via Infotrieve]
  9. Weissman, J. S., Hohl, C. M., Kovalenko, O., Kashi, Y., Chen, S., Braig, K., Saibil, H. R., Fenton, W. A., and Horwich, A. L. (1995) Cell 83, 577–587[CrossRef][Medline] [Order article via Infotrieve]
  10. Mayhew, M., da Silva, A. C., Martin, J., Erdjument-Bromage, H., Tempst, P., and Hartl, F. U. (1996) Nature 379, 420–426[CrossRef][Medline] [Order article via Infotrieve]
  11. Sakikawa, C., Taguchi, H., Makino, Y., and Yoshida, M. (1999) J. Biol. Chem. 274, 21251–21256[Abstract/Free Full Text]
  12. Rye, H. S., Burston, S. G., Fenton, W. A., Beechem, J. M., Xu, Z., Sigler, P. B., and Horwich, A. L. (1997) Nature 388, 792–798[CrossRef][Medline] [Order article via Infotrieve]
  13. Rye, H. S., Roseman, A. M., Chen, S., Furtak, K., Fenton, W. A., Saibil, H. R., and Horwich, A. L. (1999) Cell 97, 325–338[CrossRef][Medline] [Order article via Infotrieve]
  14. Taguchi, H., Ueno, T., Tadakuma, H., Yoshida, M., and Funatsu, T. (2001) Nat. Biotechnol. 19, 861–865[CrossRef][Medline] [Order article via Infotrieve]
  15. Ueno, T., Taguchi, H., Tadakuma, H., Yoshida, M., and Funatsu, T. (2004) Mol. Cell 14, 423–434[CrossRef][Medline] [Order article via Infotrieve]
  16. Chabre, M. (1990) Trends Biochem. Sci. 15, 6–10[Medline] [Order article via Infotrieve]
  17. Melki, R., and Cowan, N. J. (1994) Mol. Cell. Biol. 14, 2895–2904[Abstract/Free Full Text]
  18. Inobe, T., Kikushima, K., Makio, T., Arai, M., and Kuwajima, K. (2003) J. Mol. Biol. 329, 121–134[CrossRef][Medline] [Order article via Infotrieve]
  19. Chaudhry, C., Farr, G. W., Todd, M. J., Rye, H. S., Brunger, A. T., Adams, P. D., Horwich, A. L., and Sigler, P. B. (2003) EMBO J. 22, 4877–4887[CrossRef][Medline] [Order article via Infotrieve]
  20. Motojima, F., Makio, T., Aoki, K., Makino, Y., Kuwajima, K., and Yoshida, M. (2000) Biochem. Biophys. Res. Commun. 267, 842–849[CrossRef][Medline] [Order article via Infotrieve]
  21. Motojima, F., and Yoshida, M. (2003) J. Biol. Chem. 278, 26648–26654[Abstract/Free Full Text]
  22. Okazaki, A., Ikura, T., Nikaido, K., and Kuwajima, K. (1994) Nat. Struct. Biol. 1, 439–446[CrossRef][Medline] [Order article via Infotrieve]
  23. Hayer-Hartl, M. K., Ewbank, J. J., Creighton, T. E., and Hartl, F. U. (1994) EMBO J. 13, 3192–3202[Medline] [Order article via Infotrieve]
  24. Aoki, K., Taguchi, H., Shindo, Y., Yoshida, M., Ogasahara, K., Yutani, K., and Tanaka, N. (1997) J. Biol. Chem. 272, 32158–32162[Abstract/Free Full Text]
  25. Aoki, K., Motojima, F., Taguchi, H., Yomo, T., and Yoshida, M. (2000) J. Biol. Chem. 275, 13755–13758[Abstract/Free Full Text]
  26. Hisabori, T., Muneyuki, E., Odaka, M., Yokoyama, K., Mochizuki, K., and Yoshida, M. (1992) J. Biol. Chem. 267, 4551–4556[Abstract/Free Full Text]
  27. Sorbo, B. (1953) Acta Chem. Scand. 7, 1129–1136
  28. Langer, T., Pfeifer, G., Martin, J., Baumeister, W., and Hartl, F. U. (1992) EMBO J. 11, 4757–4765[Medline] [Order article via Infotrieve]
  29. Mendoza, J. A., Rogers, E., Lorimer, G. H., and Horowitz, P. M. (1991) J. Biol. Chem. 266, 13044–13049[Abstract/Free Full Text]
  30. Brinker, A., Pfeifer, G., Kerner, M. J., Naylor, D. J., Hartl, F. U., and Hayer-Hartl, M. (2001) Cell 107, 223–233[CrossRef][Medline] [Order article via Infotrieve]
  31. Todd, M. J., Viitanen, P. V., and Lorimer, G. H. (1994) Science 265, 659–666[Abstract/Free Full Text]
  32. Weissman, J. S., Kashi, Y., Fenton, W. A., and Horwich, A. L. (1994) Cell 78, 693–702[CrossRef][Medline] [Order article via Infotrieve]
  33. Taguchi, H., and Yoshida, M. (1995) FEBS Lett. 359, 195–198[CrossRef][Medline] [Order article via Infotrieve]
  34. Peralta, D., Hartman, D. J., Hoogenraad, N. J., and Hoj, P. B. (1994) FEBS Lett. 339, 45–49[CrossRef][Medline] [Order article via Infotrieve]
  35. Ranson, N. A., Burston, S. G., and Clarke, A. R. (1997) J. Mol. Biol. 266, 656–664[CrossRef][Medline] [Order article via Infotrieve]
  36. Azem, A., Kessel, M., and Goloubinoff, P. (1994) Science 265, 653–656[Abstract/Free Full Text]
  37. Sparrer, H., Rutkat, K., and Buchner, J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1096–1100[Abstract/Free Full Text]
  38. Sparrer, H., and Buchner, J. (1997) J. Biol. Chem. 272, 14080–14086[Abstract/Free Full Text]

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