Molecular chaperonins assist protein folding in the cell, during which the chaperonin recognizes and binds the unfolded protein substrate(1, 2, 3) . The bound protein assumes a molten globule-like state and changes conformation in a poorly understood process, resulting in the formation of its native state(4, 5, 6, 7, 8, 9) . Recently published x-ray structure and mutational analysis of the bacterial tetradecameric GroEL protein from Escherichia coli suggest that chaperonin's principal role is to provide an interactive surface for unfolded proteins and their cofactors(10, 11) . The interaction with this surface presumably limits the possible polypeptide conformations to those that are committed to fold into the native structure. The mechanism by which this is achieved and the exact role of chaperonin is the subject of extensive study(12) . However, it is generally accepted that in the process of folding, both protein and chaperonin undergo major conformational changes(3, 4, 7, 8, 9, 13, 14, 15) .

Much less is known about protein folding mediated by the TCP1/TRiC family of chaperonins, which includes the cytosolic chaperonins of eukaryota and archaea. As many as nine different subunits of eukaryotic TCP1 protein can be associated in a double-ring structure(12, 16) . Assisting protein factors, analogous to GroES, have been identified, but their role in protein folding remains unclear(17) . Archaeal chaperonins share a high sequence similarity with eukaryotic TCP1 chaperonins, but they are composed of two subunits(18, 19, 20) . These chaperonins also form double-ring structures with 8/2 or 9/2 symmetry (18, 19, 20, 21) . Archaeal chaperonins have ATPase activity, recognize and bind unfolded proteins(22, 23) , and can contribute to the thermostability and folding of enzymes(22) . Furthermore, chaperonins isolated from hyperthermophilic organisms show remarkable chemical and thermal stability(18, 22, 23) .

We report here that the major heat shock protein in S. shibitae (called also TF55 (23) and rosettasome (1a)), unlike the bacterial GroEL, exists in two distinct, stable conformational states (closed and open) that appear to be part of the protein folding pathway. This protein will be referred to here as the ``archaeosome.'' The closed archaeosome complex binds ATP and forms an open complex, which upon ATP hydrolysis specifically dissociates into subunits. Free subunits reassemble to an 18-subunit bitoroidal complex and complete the cycle. The equilibrium between complex and subunits is affected by ATP and temperature. Heat-denatured proteins associate with both conformational states. The subunits also bind denatured proteins and form an intermediate complex.

MATERIALS AND METHODS

Reagents and Proteins

()

Dephosphorylated bovine -S1-casein and pure and subunits of the archaeosome were labeled with P using [-P]ATP and human casein kinase II. Excess of proteins over [-P]ATP were used to ensure a single labeling event per polypeptide chain. Human casein kinase II was inactivated by heat and P-labeled proteins were purified from unreacted [-P]ATP on excellulose GF5 gel filtration columns from Pierce. The specific radioactivity of radiolabeled proteins was determined by liquid scintillation counting and UV spectroscopy. Protein concentration was determined by spectroscopy using calculated extinction coefficients. Molar extinction coefficients of chaperonin and its subunits were calculated from the protein sequence using the method of Gill and von Hippel (24) and were 9.08 10M cm at 278 nm for the subunit, 3.26 10M cm for the subunit at 280 nm, and 3.75 10M cm for the / archaeosome at 280 nm.

Purification of archaeosome and glutamate dehydrogenase from Sulfolobus shibatae was done using the following procedure. S. shibatae (DSM strain 5389) was grown at 80 °C in liquid medium (containing yeast extract and Brock's salts) in a 160-liter fermenter as described previously(23) . Cells were lysed by raising the pH to 7.5 with 0.1 N NaOH in the presence of 0.25% Triton X-100 (v/v) in 50 mM Tris/HCl, pH 7.5, 10 mM 2-mercaptoethanol, and 1 mM EDTA. Soluble crude cell extract was chromatographed on a FastQ FPLC column, and proteins were eluted with 20 mM Tris/HCl, pH 7.5, 1 mM DTT, and 0-500 mM NaCl gradient. Fractions containing chaperonin were concentrated on YM100 membrane from Amicon, and chaperonin was further purified by gel permeation chromatography on a Sephacryl 300 column in 20 mM Tris/HCl pH 7.5 buffer containing 1 mM DTT and 250 mM NaCl. A protein peak that eluted close to void volume comprised the archaeosome. Finally, chaperonin was separated on a high resolution MonoQ (16/10) column using FPLC and eluted with 20 mM Tris/HCl pH 7.5 buffer, 1 mM DTT, and 150-400 mM NaCl gradient. Chaperonin was at least 95% pure as judged by gel electrophoresis and staining with silver. Protein sequence was confirmed by partial sequencing of a chymotrypsin-resistant 30-kDa protein fragment (W. M. Keck Foundation, Biotechnology Resource Laboratory, Yale University). Pure chaperonin was shown to prevent heat-induced aggregation of S. shibatae glutamate dehydrogenase and stimulate folding of guanidinium/HCl denatured glutamate dehydrogenase under conditions described previously(22) .

Archaeosome subunits were purified using the following procedure. S. shibatae crude cell extract was chromatographed on a FastQ FPLC column, and proteins were eluted with 20 mM Tris/HCl pH 7.5 buffer, 1 mM DTT, and 0-500 mM NaCl gradient. Fractions containing and subunits were concentrated on YM30 membrane from Amicon and were further purified by gel permeation chromatography on a Sephacryl 300 column in 20 mM Tris/HCl pH 7.5 buffer, 1 mM DTT, and 250 mM NaCl. A protein peak that eluted close to two void volumes contained archaeosome subunits. Finally, and subunits were separated on a high resolution MonoQ (16/10) column using FPLC and eluted with 20 mM Tris/HCl, pH 7.5, 1 mM DTT, and 0-150 mM NaCl gradient. Under these conditions and subunits can be separated from each other. Alternatively, pure archaeosome was dissociated to subunits by a 1-h incubation at 75 °C with 2 mM ATP, and and subunits were separated on a high resolution MonoQ (16/10) column using FPLC, as described above. Subunits were at least 90% pure as judged by gel electrophoresis and staining with silver. Thermus aquaticus chaperonin was purified using a procedure similar to that described for the archaeosomes.

Glutamate dehydrogenase from S. shibatae (DSM strain 5389) was purified using the modified procedure of Robb et al.(25) , in which Sepharose CL-6B column was replaced with Sephacryl 300 column and Phenyl-Sepharose CL-6B column was replaced with MonoQ (16/10). The purity and the identity of the protein were confirmed by sequencing of the N terminus (W. M. Keck Foundation, Biotechnology Resource Laboratory, Yale University) and by activity assay(22) .

Native Gel Electrophoresis

Electron Micrograph Images

Circular Dichroism Spectroscopy

Archaeosome Reconstitution Experiments

Two-dimensional Gel Electrophoresis

RESULTS

Composition and Properties of Archaeosome Complexes

Figure 1: Separation of archaeosome complexes on polyacrylamide gel electrophoresis under native conditions. Lane 1, pure archaeosome obtained from freshly grown culture of S. shibatae separated on 6% native gels; lane 2, top band (TB, open complex); lane 3, bottom band (BB, closed complex). Protein bands were stained with silver. 2-DE panel, archaeosome bands were excised, electroeluted, and run on two-dimensional gel electrophoresis under denaturing conditions, as indicated by arrows; both complexes show identical subunit composition. Micrographs panel, electron micrographs of open (TB) and closed (BB) complexes. Symmetry panel, rotationally averaged electron micrograph of seven open complexes (from an electron micrograph similar to that shown in the top micrograph panel). This panel shows archaeosome complexes with a well defined central cavity and 9-fold symmetry.

The two polypeptides are sequence-related and have molecular masses of 59.8 and 59.9 kDa, respectively, as derived from amino acid sequences of their genes(19, 23) . The 59.8 kDa protein () is identical to the TF55 protein(23) , and the 59.9 kDa protein () will be characterized in detail elsewhere(19) . Pure top and bottom complexes were examined by two-dimensional gel electrophoresis and showed identical protein composition (Fig. 1, 2-DE panel). In both complexes, and subunits were present in roughly stoichiometric amounts (19 and below). Both complexes are free of nucleotides, as indicated by their UV spectra (not shown).

Using electron microscopy and circular dichroism (CD), we examined the possibility that conformational differences in the chaperonin complex could account for the observed difference in gel mobility. Electron micrographs showed that the pure top band complex forms a nine-fold, double-ring structure that has an electron dense core, i.e. it stains preferentially in the center with uranyl acetate (Fig. 1, micrographs panel). This complex appears identical to the TF55 structures published earlier(23) . We describe top band as an ``open'' complex because it appears to contain an open cavity when viewed down the nine-fold axis of the particle (Fig. 1, symmetry panel). The side view shows the characteristic four-band striation pattern reported for other hsp60 chaperonins having 7/2, 8/2, and 9/2 symmetries(3, 20, 21, 23) . In contrast, the faster moving BB complex showed no obvious symmetry on electron micrographs and forms a ``closed'' complex with a poorly defined central cavity (Fig. 1, micrographs panel), although it is similar to the open complex in size and appearance both in top and side views. CD spectra of the purified closed and open complexes show quite remarkable differences (Fig. 2). The archaeosome closed complex show ellipticity typical of proteins with high -helix contents. The CD spectrum of the open complex is similar but the minimum at 224 nm shifts to 220 nm perhaps suggesting lower -helical content.

Figure 2: CD spectra of closed and open complexes of archaeosome. CD spectra of the polyacrylamide gel purified open complex (dotted line) and closed complex (solid line) were recorded in 50 mM sodium phosphate, pH 7.5 (the distorted CD spectra at 200 nm may be due to high optical density at this wavelength).

Relative Amount of Closed and Open Complexes Varies During Heat Shock

Figure 3: Properties of open and closed archaeosome complexes. A, heat shock affects the relative amounts of closed and open complexes of the archaeosome. Lane 1, total protein extract obtained from cells grown at 75 °C separated on 6-10% native PAGE; lane 2, extract obtained from cells grown at 75 °C and then heat shocked at 88 °C for 1 h. B, binding of heat-denatured P--S1-casein to open and closed archaeosome complexes. The archaeosome was incubated for 15 min with P--S1-casein at 75 °C, cooled to 4 °C, and loaded onto 4% polyacrylamide gels. Lane 1, free P--S1-casein at 0.5 µM. Lanes 2-6 contain P--S1-casein at 0.5 µM and increasing concentration of the archaeosome (as indicated on the figure). The amounts of open and closed complex were approximately the same at the start of incubation. Specific radioactivity of P--S1-casein is 25.2 kcpm/pmol. C, binding of P--S1-casein to GroEL-like chaperonin from thermophilic bacteria T. aquaticus is shown as a control (28) . Binding and gel conditions are like in B. Lane 1, free P--S1-casein at 0.5 µM; lane 2, contain P--S1-casein at 0.5 and 1 µM chaperonin from T. aquaticus.

Trent et al.(23) reported previously that TF55 protein binds guanidine/HCl-denatured dihydrofolate reductase and unspecified E. coli proteins. We found that both the open and closed complexes bind heat-denatured P--S1-casein (Fig. 3B). The binding of casein is temperature-dependent and occurs only above 50 °C. The association of casein with the archaeosome complexes is weak, as compared with the binding of P--S1-casein to thermophilic GroEL-like chaperonin from T. aquaticus (Fig. 3C and (28) ), and the ternary complex appears to be formed transiently. Similar results have been reported recently for binding of -actin to c-cpn60 cytosolic chaperonin of eukaryota(29) . C-cpn60 chaperonin binds -actin ten times more weakly than E. coli GroEL, but in contrast to GroEL c-cpn60 supports effective folding of -actin.

Specific Dissociation of Archaeosome Complexes

Figure 4: Dissociation and reconstitution of archaeosome open and closed complexes. A, pure closed complex was incubated for 15 min at 75 °C in the absence or presence of Mg-ATP, Mg-ADP, and Mg-ATPS, and 5% TFE as indicated on the figure. Samples were cooled on ice and separated on 6% native polyacrylamide gels (as described under ``Materials and Methods'' and the legend to Fig. 3). Proteins were stained with silver. Arrows and chaperonin icons mark the positions of the closed and open complexes and / subunits. Under these gel conditions the and subunits do not separate. Lane 1 shows native archaeosome purified from S. shibatae. B, reconstitution of the archaeosome from free subunits. Left panel, lane 1, native archaeosome complexes; lane 2, mixture of and archaeosome subunits purified from S. shibatae extracts; lane 3, complexes reconstituted from 10 µM subunits in 50 mM phosphate buffer, pH 7.5, at 20 °C (samples were separated on 6% polyacrylamide gels as described in legend to Fig. 3). The micrograph panel shows an electron micrograph of the reconstituted complexes shown in lane 3. Right panel, reconstitution of stoichiometric amounts of and at 10 µM with the P- casein. Lane 1, P--S1-casein at 0.5 µM; lane 2, stoichiometric amounts of unlabeled and at 10 µM reconstituted with P--S1-casein at 0.5 µM. C, binding P--S1-casein to and subunits. Left panel, lane 1, free P--S1-casein at 0.5 µM; lanes 2-6, P--S1-casein at 0.5 µM incubated at 75 °C with increasing concentrations of subunits (as indicated on the figure); lanes 7-11, P--S1-casein at 0.5 µM incubated at 75 °C with increasing concentrations of subunit (as indicated on the figure). Complexes were separated on 10% native PAGE as described under ``Materials and Methods'' and in the legend to Fig. 3. Specific radioactivity of radiolabeled proteins was: P--S1-casein, 25.2 kcpm/pmol; subunit, 3.3 kcpm/pmol; and subunit, 2.0 kcpm/pmol.

In the presence of ATP, ADP or ATPS, the closed complex can be partly converted to the open complex (Fig. 4A, lanes 4, 5, 8, and 9). The open complex appears to be stabilized by the addition of ADP or ATPS (Fig. 4A, lanes 4, 5, 8, and 9). The binding of ATP and subsequent hydrolysis of the phosphoester bond lead to complex dissociation (Fig. 4A, lanes 6 and 7). As expected, the addition of 5% TFE further destabilizes the complexes, causing dissociation of the complex to subunits (Fig. 4A, lanes 3 and 7). More complex dissociates in the presence of ATP.

The observed effect of ATP is very specific, because the closed complex resists treatment with 7 M urea, and both complexes are stable for weeks at ambient temperatures with or without bound nucleotide (data not shown and (18) ).

Reconstitution of Archaeosome from Free Subunits

To study the composition of the archaeosome, purified and subunits were P-labeled, and the relative composition of the reconstituted archaeosome was determined. When a stoichiometric mixture of and subunits was reconstituted with a small amount of P-labeled subunit, 18.4 ± 3.2% of P-label was found in the reconstituted chaperonin. Under identical conditions 15.0 ± 3.3% P-labeled subunit reconstituted into the chaperonin complex. This result suggests an apparent 1:1 stoichiometry for and subunits in archaeosome. Each subunit alone, however, can also reconstitute a high molecular weight complex; these complexes have gel mobilities slightly different from that of the heteroligomeric archaeosome(19) . subunits form a complex both in the presence and absence of ATP, whereas subunits only assemble in the absence of ATP. In the presence of both and subunits, only the stable - heteroligomeric complex is formed with and without ATP(19) . Hence, the bitoroidal archaeosome seems to be asymmetric in respect to ATP-dependent ring stability.

When and subunits reconstitute in the presence of heat-denatured P--casein, the P-casein is found mainly bound to subunits and to an intermediate complex, whereas only small amounts are associated with the open and closed forms of full-size archaeosome (Fig. 4B, right panel). Similar weaker binding of denatured proteins to TCP-1-like chaperonin was reported by Tian et al.(29) . We evaluated the relative affinity of and subunits for P--casein in titration experiments (Fig. 4C). Both subunits bind P-casein, but the subunit appears to bind casein 5-10 times more strongly than the subunit. The subunits-P-casein complex migrated in gels slower than free subunits but faster than the archaeosome complex, suggesting that this complex may represent an intermediate. As judged by its gel mobility, it could represent a single ring composed of identical subunits with bound -casein. Single rings of thermophilic chaperonin have been reported recently(31) , and the equilibria between chaperonin, single rings, and subunits have been observed for three different chaperonins including GroEL(32) .

DISCUSSION

Extensive efforts are underway in many laboratories to fully characterize the eukaryotic chaperonin TCP1/TRiC. In contrast to bacterial GroEL, where the underlying mechanisms of protein binding and folding have been studied in great depth, less is known about the mechanism by which the eukaryotic TCP1 chaperonin folds proteins. The most obvious difference is the presumed multisubunit complexity of the TCP1 chaperonin. Archaebacterial chaperonins show extensive sequence similarity to eukaryotic cytosolic chaperonins (TCP1) and represent a simpler model with which to study folding in eukaryotic cells because the archaeal chaperonin complex is composed of just two subunits(18, 19) .

We have shown that the S. shibatae chaperonin exists in vitro and presumably also in vivo as two distinct complexes that can be separated by native PAGE. We believe that these two complexes can be detected because the complex is frozen in these two states by lowering the temperature from 75 °C to room temperature. Two distinct conformations of TCP1 chaperonin from mouse testis have been observed by Hynes et al.(16) using specific antibodies. These authors suggested that binding or hydrolysis of ATP acts as a switch between two conformational forms of chaperonin. Knapp et al.(18) observed similar complexes of S. solfataricus chaperonin on electron micrographs. Guagliardi et al. (22) reported that the chaperonin from S. solfataricus (termed Ssocpn) in the presence of Mg-ATP undergoes a large conformational rearrangement (as observed by change in tryptophan fluorescence). Conformational changes in E. coli GroEL (33) and complexes of GroEL with bound ATP, GroES, and protein-substrate (3) have been reported. The magnitude of the conformational changes in the archaeosome structure can be pertinent to the structure of chaperonin from thermophilic archaebacteria obtained with electron microscopy by Phipps et al.(21) . This structure shows a large mass of protein blocking the entrance to the chaperonin central cavity. These EM reconstructions could represent the structure of closed complex described here or complex with bound protein substitute.

The CD spectra of purified complexes showed remarkable differences, implying that the open complex is structurally altered (Fig. 2). Both the electron microscopy and the CD results suggest that there is a major conformational difference between the two complexes. The structural changes in the open complex that allow uranyl acetate to bind within the central cavity also increase the effective cross-section of the complex, which reduces its mobility in native polyacrylamide gels. The change in the CD spectrum reflects more extensive structural rearrangement than just domain movement. If the domain organization of archaeosome is similar to that of E. coli GroEL (as suggested by limited but significant sequence similarity), then our data would imply conformational changes in the apical region. This domain was proposed by Horwich and co-workers (11) to be involved in protein binding and folding.

We have shown here that the conformational changes are related to conversion of closed complex to open complex and dissociation to subunits. The equilibrium between three states, open and closed complexes and free subunits, is affected in vitro by temperature and by Mg-ATP and its derivatives, ADP and ATPS. It appears that in vivo under mild heat shock, the amount of open complex increases, suggesting a response to a stress. Under lethal heat shock conditions only the closed complex remains. Our data suggest that this dissociation is controlled by ATP hydrolysis. A large concentration of free subunits may provide an advantage to the cell by capturing unfolded polypeptides under heat shock conditions and arresting protein aggregation. The full role of free subunits in the protein folding cycle has yet to be established, and an hsp70-like function of the and subunits cannot be excluded. A similar function in heat shock response has been attributed to yeast hsp104 protein, which is believed to assist protein solubilization rather than folding. Parsell et al.(34) recently reported that yeast hsp104 can rescue proteins from aggregates once they have formed. Strikingly, it has been noted earlier that the stability of hsp104 hexamer in vitro, similar to the archaeosome, is ATP-dependent(35) . Thus the dissociation of the archaeosome to subunits could be an important part of a functional cycle that links protein-mediated thermotolerance with protein folding.

Several protein folding cycles have been proposed for bacterial chaperonin(7, 8, 9, 13, 14) . These cycles postulate the formation of specific bi-, ter-, and quaternary complexes that facilitate protein folding and chaperonin regeneration. The cycle that we propose in Fig. 5includes a change in conformation and in oligomerization state. The cell maintains all the chaperonin components (closed complex, open complex, and subunits) in equilibrium. Both complexes and subunits appear to bind denatured proteins. Our data imply that in the archaea, unfolded protein enters the cycle by binding to subunits, proceeds through an intermediate that is composed of individual subunits, and continues to the double-ring complex. We believe that the folded protein is released when the open complex dissociates into subunits (Fig. 5).

Figure 5: Conformational cycle of archaeosome. Proposed model for conformational cycle and protein binding of the S. shibatae chaperonin during thermotolerance and protein folding.

There is a direct analogy between the archaeosome open complex and the GroEL-GroES complex as high energy states and the free subunits and GroEL as the low energy states(7) . The dissociation of the archaeosome to subunits is the ultimate relaxation of the high energy state. The closed complex appears to represent an intermediate energy state. We suggest that as previously reported for GroEL(7) , the thermodynamic barriers separating protein-bound and free archaeosome states are overcome by ATP hydrolysis. The dissociation of bound protein is most likely accomplished by a change in the binding affinities of the chaperonin for a non-native protein. The extreme way to achieve this is to break up the structure of the complex into its subunits. Clearly the chaperonin complex and free subunits must present different interactive surfaces for unfolded proteins.

We propose that, as an unfolded protein assumes its native structure, the archaeosome undergoes conformational changes. The high entropy of the unfolded protein is assimilated by the chaperonin as the protein folds, the archaeosome acting as an ``entropic sink.'' After ATP hydrolysis, the ternary complex dissociates, releasing folded protein and subunits (Fig. 5). Free subunits reassemble into complexes, completing the cycle. It is likely that the archaeosome folds proteins in a quite different way than the GroEL-like chaperonins. In fact, Cowan and co-workers (29) showed recently that a distinct set of folding intermediates is released from different chaperonins. The hyperthermophilic archaebacteria must protect and fold proteins that are already quite thermostable, and therefore archaeosome may require higher energy to overcome thermodynamic barrier in folding of these proteins. It is likely that proteins and chaperonins coevolved to optimize folding requirements of the cell. This is reflected in the properties of chaperonin and the dynamics and degree of structural changes during chaperonin-mediated protein folding.

FOOTNOTES

*

This project was supported by the U. S. Department of Energy, Office of Health and Environmental Research, under Contract W-31-109-Eng-38. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Argonne National Laboratory, 9700 S. Cass Ave., Argonne, IL 60439.

()

The abbreviations used are: TFE, trifluoroethanol; DTT, dithiothreitol; FPLC, fast protein liquid chromatography; PAGE, polyacrylamide gel electrophoresis; CD, circular dichroism; ATPS, adenosine 5`-O-(3-thiotriphosphate).

ACKNOWLEDGEMENTS

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