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J. Biol. Chem., Vol. 275, Issue 47, 37212-37218, November 24, 2000

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Chaperone Activity and Homo- and Hetero-oligomer Formation of Bacterial Small Heat Shock Proteins^*

Sonja Studer and Franz Narberhaus

From the Institute of Microbiology, Eidgenössische Technische Hochschule, Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland

Received for publication, May 31, 2000, and in revised form, August 23, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Rhizobia are the only bacteria known to induce a multitude of small heat shock proteins (sHsps) upon temperature upshift. The sHsps of Bradyrhizobium japonicum fall into two different classes, class A and class B. Here, we studied the chaperone activity and oligomeric features of two representative members of each class. The purified sHsps were efficient chaperones, as demonstrated by their ability to prevent thermally induced aggregation of citrate synthase in vitro. Homo-oligomer formation of all four sHsps was demonstrated by gel filtration and by two independent co-purification approaches. Mixed oligomers were readily observed between members of the same class, even when these proteins originated from different species such as Escherichia coli and B. japonicum. The chaperone activity of purified hetero-oligomers was indistinguishable from the activity of homo-oligomers. Heteromeric complexes were never obtained between class A and class B sHsps, indicating that hetero-oligomer formation is restricted to sHsps of the same class.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

All organisms respond to a sudden temperature upshift with the induction of a similar set of highly specialized proteins. Many of these heat shock proteins function as molecular chaperones, suppressing the aggregation or assisting in the refolding of partially denatured proteins. The small heat shock proteins (sHsps)¹ are one of the five major families of molecular chaperones. sHsps are widely distributed in both prokaryotes and eukaryotes. Members of this diverse protein family are characterized by a conserved stretch of approximately 100 amino acids (1, 2). This so-called -crystallin domain displays sequence similarity to the vertebrate eye lens protein -crystallin, which prevents protein precipitation and cataract formation in the eye lens.

The function and chaperoning mechanism of sHsps are still poorly understood. Various authors have reported that overproduction of sHsps endows cells with increased thermotolerance (3-5). Nevertheless, many sHsps are dispensable for cell viability and growth under physiological conditions, and several mutants lacking sHsps do not exhibit a severe growth defect (4, 6).

Chaperone activity in vitro has been demonstrated for numerous sHsps. They protect various substrate proteins such as citrate synthase, malate dehydrogenase, -lactalbumin, or insulin from thermally or chemically induced aggregation (7-9). Upon binding to sHsps, these substrates can subsequently be refolded by other chaperones. Unlike chaperones of the Hsp70 and Hsp60 families, sHsps are unable to refold their substrates in an ATP-dependent manner. The main function of sHsps is to bind denatured proteins and to maintain them in a folding-competent state (10, 11). Thereby they minimize the risk of protein aggregation by decreasing the free concentration of partially denatured proteins.

As the name indicates, sHsps are characterized by their low molecular weight. The molecular mass of sHsp monomers ranges between 12 and 43 kDa. These monomers assemble into high molecular weight complexes in vivo. Many sHsps are known to form well ordered oligomers with a strictly defined number of subunits. Hsp16.5 from Methanococcus jannaschii, so far the only sHsp whose crystal structure has been solved (12), forms a hollow spherical complex of 24 subunits. Other sHsps reportedly assemble into more ill-defined complexes consisting of a variable number of subunits (13-15). The formation of oligomeric complexes is a prerequisite for chaperone activity of sHsps. Upon heat shock, these oligomeric complexes undergo distinct structural changes. Various sHsps have been reported to form large complexes of over 1000 kDa when incubated with substrate proteins under heat shock conditions (10, 11, 16). In addition, in the absence of substrate proteins, alterations of the quaternary structure of sHsps are observed upon heat shock. Hsp26 from Saccharomyces cerevisiae, which forms a 24-mer chaperone complex of 550 kDa at room temperature, dissociates into species of lower molecular weight when incubated at elevated temperatures (16). This reversible dissociation of the large oligomeric sHsp complexes upon heat shock seems to be essential for chaperone activity. sHsps that are unable to form oligomers generally exhibit weak or no chaperone activity (17).

Although the oligomerization principles of selected sHsps are being extensively studied, knowledge on the formation and function of heteromultimers consisting of different members of the sHsp family is restricted. In bacteria, oligomer formation of sHsps has not been investigated so far. This is not surprising considering the fact that most bacteria appear to have a very limited number of sHsps. Escherichia coli, for example, encodes two sHsps (18, 19), the thermophilic archaeon M. jannaschii encodes one (20), and many other bacteria do not possess any gene encoding an sHsp (21). The only bacteria known to encode multiple sHsps so far are the rhizobia. Bradyrhizobium japonicum, the nitrogen-fixing root nodule symbiont of soybean, responds to a heat shock with the induction of at least 10 sHsps (21).

Although the presence of multiple sHsps is rather uncommon in bacteria, it is a well known feature in plants. Depending on the plant species, up to 30 sHsps are induced after a temperature upshift. These proteins can be assigned to six different classes and are localized in different cellular compartments (22).

Similarly, the B. japonicum sHsps can be assigned to two distinct classes, A and B, based on their primary amino acid sequences. Class A consists exclusively of proteins that are typical for prokaryotes, the most prominent being the E. coli sHsps IbpA and IbpB. Class B proteins are much more divergent in sequence and phylogenetic origin. Apart from some bacterial sHsps sharing little sequence similarity with IbpA and IbpB, this class includes plant sHsps (21). In contrast to their counterparts in plants, the rhizobial proteins all occur in the same compartment, i.e. the cytoplasm, thus principally having a chance to interact.

In the present publication, we show that oligomerization between different B. japonicum sHsps is possible but that interaction is restricted to members of the same class.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmid Construction-- The hspB, hspC, hspF, and hspH genes of B. japonicum were cloned into the expression vectors pET21b and pET24b (Novagen) after introduction of appropriate restriction sites by polymerase chain reaction.

Plasmids for the production of small heat shock proteins carrying a hexahistidine tag at the carboxyl terminus were constructed by cloning the corresponding genes into the NdeI and NotI sites of the pET vectors. pET24b, confering kanamycin resistance, was used for generating plasmids pRJ5304 to pRJ5307. pET21b, which carries an ampicillin resistance marker, provided the basis for plasmids pRJ5318 to pRJ5321. A vector-encoded stretch of five amino acids (AAALE) was introduced between the carboxyl-terminal amino acid of each sHsp and the histidine tag.

Plasmids pRJ5300 to pRJ5303 (based on pET24b) and pRJ5314 to pRJ5317 (based on pET21b) for producing proteins without hexahistidine tag were obtained by cloning the hspB, hspC, hspF, and hspH genes into the NdeI/XhoI sites of the pET vectors. Thereby, a stretch of three alanines was introduced at the carboxyl-terminal end of the sHsp. The plasmid pBF3-IbpB for the production of the E. coli sHsp IbpB was obtained from Helmut Burtscher (Roche Diagnostics GmbH).

Protein Expression and Purification-- Small heat shock proteins both with and without hexahistidine tag were overexpressed in the host strains E. coli BL21(DE3)pLysS and E. coli JGT14. Strain JGT14, a BL21(DE3) derivative carrying a ibp1::kan null mutation, was obtained from François Baneyx, University of Washington, Seattle, Washington (23).

Overexpression cultures were grown at 30 °C. Once the cultures had reached an A₆₀₀ of 0.6, production of the recombinant proteins was induced by addition of isopropyl--D-thiogalactopyranoside to a final concentration of 0.5 mM. 2-3 h after induction, bacterial cells were harvested and resuspended in binding buffer (500 mM KCl, 20 mM Tris-HCl, 5 mM imidazole, pH 7.9) containing 1 mM phenylmethylsulfonyl fluoride and DNaseI. Lysis was performed in a french pressure cell at 1000 p.s.i. Soluble crude extracts were prepared by centrifugation at 12,000 × g for 30 min at 4 °C.

Purification of hexahistidine-tagged proteins was performed by Ni-NTA affinity chromatography under native conditions with Ni-NTA resin from Qiagen. The column was pre-equilibrated with binding buffer. After application of the crude extract, the column was washed with washing buffers (500 mM KCl, 20 mM Tris-HCl, pH 7.9) with increasing imidazole concentrations (5-60 mM). Purified proteins were eluted by raising the imidazole concentration to 250-300 mM. To obtain pure proteins, the eluted fractions were diluted in binding buffer and applied to the Ni-NTA column again. Eluted proteins were analyzed by SDS-PAGE on a 15% polyacrylamide gel.

Protein fractions were dialyzed against storage buffer (400 mM KCl, 20 mM Tris-HCl, pH 8) containing 20% glycerol and stored at 20 °C and 80 °C. Protein concentrations were determined by using the Bio-Rad protein assay. All protein concentrations reported in this study are expressed in terms of protomers.

Gel Filtration-- Gel filtration chromatography was performed both with E. coli crude extracts containing overexpressed B. japonicum sHsps and with purified sHsps. 500 µl of filtered (0.22 µm) crude extract was injected on a Superdex 200 HR 30/10 gel filtration column (Amersham Pharmacia Biotech) pre-equilibrated with binding buffer. Separation was performed on a GradiFrac system (Amersham Pharmacia Biotech) at room temperature at a flow rate of 0.3 ml/min. 1-ml fractions were collected and analyzed by SDS-PAGE. Purified hexahistidine-tagged sHsps in binding buffer were also applied to the Superdex 200 HR 30/10 column and analyzed on a Biocad perfusion chromatography system at room temperature at a flow rate of 0.6 ml/min. The following standards were used to calibrate the column: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), and aldolase (158 kDa).

Chaperone Activity Assay-- Citrate synthase (CS, Sigma) was heat-denatured in the presence or absence of various amounts of sHsps. Samples for the aggregation assay were prepared in 1 ml of 50 mM sodium phosphate, pH 6.8. The buffer was pre-incubated at 43 °C for at least 20 min before addition of CS (in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to a final concentration of 600 nM. Purified small heat shock proteins were added to the solution in varying concentrations. Light scattering at 360 nm was monitored for 30 min at 43 °C in an Ultrospec 3000 spectrophotometer (Amersham Pharmacia Biotech). Control measurements were performed with bovine -crystallin (StressGen) and BSA as well as with purified sHsps in the absence of CS.

Co-purification of Oligomers-- Oligomer formation of sHsps was studied by co-purification assays, taking advantage of the Ni-NTA affinity chromatography technique. Two alternative assays were used. In the first assay, crude extracts were denatured by adding urea to a final concentration of 8 M to disrupt pre-existing sHsp oligomers. A crude extract containing an sHsp carrying a hexahistidine tag was mixed with a crude extract containing an untagged protein. After mixing, the denaturing agent was removed by dialysis against binding buffer for 2 h, thus allowing for the formation of oligomers. The resulting protein mixture was applied to a Ni-NTA affinity column under native conditions as described above. The presence of two bands on Coomassie-stained SDS-polyacrylamide gels was indicative of mixed oligomers consisting of both tagged and untagged sHsps. This procedure was performed with all possible combinations of histidine-tagged and untagged proteins.

In the second assay, oligomer formation was allowed in vivo by co-expressing an sHsp with a hexahistidine tag together with an untagged sHsp in E. coli. Two plasmids carrying different antibiotic markers were co-transformed into E. coli BL21(DE3)pLysS. Protein expression and purification were then performed as outlined above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification of sHsps from B. japonicum-- Four representative B. japonicum sHsps belonging to two distinct classes (class A, HspB and HspH; class B, HspC and HspF) were overproduced in E. coli JGT14. This strain, a ibpAB mutant of BL21 (6), was used to eliminate the background of the E. coli sHsps IbpA and IbpB. All four B. japonicum sHsps were produced both with and without a carboxyl-terminal hexahistidine tag.

Overproduced sHsps represented the major band on SDS-polyacrylamide gels with extracts of E. coli JGT14 bearing the pET-derived overexpression plasmids (Fig. 1). More than 50% of the overproduced protein was present in soluble form, as shown by centrifugation of cell lysates at 20,000 × g (data not shown). Protein purification was complicated by the fact that all four sHsps were extremely prone to aggregation at elevated protein concentrations (1 mg/ml). In particular the class A proteins HspB and HspH tended to precipitate, leading to a significant loss of protein.

View larger version (76K): [in this window] [in a new window]   Fig. 1.   SDS-PAGE analysis of overproduction and purification of B. japonicum sHsps. sHsps carrying a carboxyl-terminal hexahistidine tag were overproduced in E. coli JGT14 and purified by Ni-NTA affinity chromatography. The Coomassie Brilliant Blue-stained SDS-polyacrylamide gel shows the high level of overproduction reached 2 h after induction with isopropyl--D-galactopyranoside (crude extracts on lanes 1, 3, 5, and 7) and the purified proteins after two subsequent applications onto a Ni-NTA affinity column (3-4 µg of protein, lanes 2, 4, 6, and 8). Molecular mass markers (in kDa) are indicated adjacent to the gel.

The hexahistidine-tagged proteins were purified by affinity chromatography on a Ni-NTA column. Two purification steps were necessary to obtain proteins of 95% purity, as judged by SDS-PAGE (Fig. 1). Purified sHsps migrated as bands with an apparent molecular mass between 18 and 20 kDa, in agreement with the molecular masses calculated from the deduced amino acid sequences of the hexahistidine-tagged proteins. These were 18,449 Da for HspB-His₆, 18,377 Da for HspH-His₆, 19,881 Da for HspC-His₆, and 19,924 Da for HspF-His₆. The apparent molecular masses of HspH and HspH-His₆ were significantly higher than their calculated molecular masses, as had been observed previously (21).

HspB, HspH, HspC, and HspF Exhibit Chaperone Activity-- The chaperone activity of purified hexahistidine-tagged HspB, HspH, HspC, and HspF was assessed by determining their ability to prevent the thermally induced aggregation of CS at 43 °C. CS, a dimer of 49-kDa subunits, aggregates spontaneously upon incubation at elevated temperatures. Aggregation of CS can be monitored by measuring the increase in absorbance at a wavelength of 360 nm. If CS was incubated at 43 °C in the absence of sHsps, it rapidly aggregated within 30 min. In contrast, the solubility of the B. japonicum sHsps was not affected by prolonged incubation at 43 °C (Fig. 2).

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Chaperone activity of B. japonicum small heat shock proteins. Thermally induced aggregation of CS at 43 °C is depicted as a function of time in the presence of various amounts of B. japonicum HspB-His6 (A), HspH-His6 (B), HspC-His6 (C), HspF-His6 (D), BSA (E), and bovine -crystallin (F). Aggregation was monitored by the increase in absorbance at 360 nm. Proteins were incubated at 43 °C in a total volume of 1.0 ml of 50 mM phosphate buffer, pH 6.9. The CS concentration was 600 nM. CS was incubated in the absence of sHsps () and in the presence of sHsps, BSA, or -crystallin at final concentrations of 150 nM (), 300 nM (), 600 nM (×), and 1.2 µM (*). The absorbance of B. japonicum sHsps, BSA, or -crystallin in the absence of CS was also monitored ().

All four B. japonicum sHsps efficiently inhibited the thermally induced aggregation of CS in vitro in a concentration-dependent manner (Fig. 2, A-D). No major differences were observed between the four sHsps regarding their chaperone activity. The addition of sHsps at a molar ratio of CS:sHsp (monomers) of 1:0.5 reduced CS aggregation significantly. At a molar ratio of CS:sHsp (monomers) of 1:2, precipitation of CS was prevented almost completely.

In control reactions, CS was incubated with either BSA or -crystallin. A small concentration-dependent, general protective effect of BSA was observed, which is presumably not due to chaperone activity (Fig. 2E) and which was much less pronounced than the effect of the sHsps. -Crystallin, on the other hand, exhibited chaperone activity that was comparable with the B. japonicum sHsps (Fig. 2F).

B. japonicum sHsps Form Oligomers-- The oligomeric structure of heterologously expressed sHsps was determined by gel filtration chromatography. Experiments were preferentially performed with E. coli crude extracts containing an overproduced sHsp rather than with purified proteins because a substantial fraction of the purified proteins remained irreversibly bound to the column. Similar difficulties have been described for IbpB (23). To localize the sHsp peak within the elution profile, elution fractions were collected and analyzed by SDS-PAGE (Fig. 3).

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Gel filtration profile (A) and SDS-PAGE analysis (B) of a crude extract containing overproduced HspF-His6. 500 µl of an E. coli crude extract were applied on a Superdex 200 HR 30/10 column. 1-ml fractions were collected and analyzed by SDS-PAGE. The highest concentration of HspF-His6 was found in the fractions 11 to 13, corresponding to a molecular mass of 400-500 kDa, as determined by calibration of the column with high molecular mass standards. The HspF-His6 protein on the SDS-polyacrylamide gel and the corresponding peak on the elution profile are indicated by arrows. Molecular mass markers (in kDa) are indicated adjacent to the gel.

Gel filtration chromatography was performed both with extracts containing hexahistidine-tagged sHsps and with extracts containing untagged sHsps. The elution profiles of hexahistidine-tagged proteins were similar to those of untagged proteins (data not shown). This finding indicates that the hexahistidine tag does not influence oligomerization.

All four sHsps led to a similar elution pattern. As a representative example, the elution profile of a crude extract containing HspF-His₆ is depicted in Fig. 3. The sHsps eluted predominantly as 400-500-kDa complexes, as estimated by comparison with high molecular mass marker proteins. Deduced from a monomeric mass of 20 kDa, this peak corresponds to an oligomer consisting of 20 to 25 subunits. Detectable amounts of sHsps were not found in the molecular mass range of 20-50 kDa, implying that the major portion of these proteins exists in a high molecular state. Although the majority of each sHsp was present in the 400-500-kDa range, the proteins were distributed over a broad size range, extending from approximately 100 kDa to the void volume of the column. This suggests that sHsps form oligomers of heterogenous size, a finding that is consistent with results obtained by native PAGE. We observed that all examined B. japonicum sHsps migrated as a broad smear rather than as a distinct band in native gels (data not shown).

To exclude the possibility that substrate proteins bound to sHsps affected the oligomeric mass of the complexes in the crude extracts, gel filtration chromatography was also performed with purified hexahistidine-tagged sHsps. Although only a small fraction of these purified proteins could be recovered from the column, they clearly displayed a comparable mass distribution as the sHsps present in crude extracts (data not shown).

Homo-oligomer Formation between sHsps in Vitro-- The fact that B. japonicum is able to induce a surprisingly high number of different sHsps raises the question of whether these proteins assemble into homo- or hetero-oligomers. We investigated the interaction between sHsps using a co-affinity purification approach. Mixtures of crude extracts containing sHsps both with and without carboxyl-terminal hexahistidine-tag were applied to a Ni-NTA affinity column, and elution fractions were analyzed by SDS-PAGE. Co-purification of the untagged sHsp was observed as an additional band on SDS gels and indicated the formation of mixed oligomers containing sHsps with and without hexahistidine tag.

To provide optimal conditions for the de novo formation of oligomers, pre-existing sHsp homo-oligomers present in the crude extracts were disrupted by the addition of 8 M urea. Denaturation, renaturation, and co-purification were performed as described under "Experimental Procedures" and as outlined in Fig. 4A. We performed purifications with untreated extracts containing the individual proteins, as a control. In each case, the tagged protein was retained by the Ni-NTA column, whereas the untagged protein alone was unable to bind (Fig. 4, B and C, lanes 2 and 4, respectively).

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Formation of sHsp homo-oligomers. A, outline of the denaturation-renaturation strategy applied for the detection of oligomer formation. Two crude extracts containing an overproduced sHsp, one with and one without a carboxyl-terminal hexahistidine tag, were denatured, mixed, renatured by dialysis, and eventually applied to a Ni-NTA affinity column and analyzed by SDS-PAGE. B and C, SDS-PAGE analysis of crude extracts and purified proteins. Lane 1, crude extract containing the hexahistidine-tagged sHsp; lane 2, hexahistidine-tagged sHsp after purification; lane 3, tagged and untagged sHsps after co-purification; lane 4, untagged sHsp does not bind to the column; lane 5, crude extract containing the untagged sHsp. Note that lanes 2 and 4 in the right gel of panel C were loaded in reverse order.

Following the denaturation-renaturation protocol with two extracts providing a hexahistidine-tagged sHsp and the same protein without tag (e.g. HspB-His₆ and HspB), we observed two bands on SDS-polyacrylamide gels. The upper and lower bands corresponded to the tagged and the untagged proteins, respectively (Fig. 4, B and C, lane 3). As one might have expected, both proteins had interacted to form homo-oligomers.

To exclude the risk of observing artificial complex formation due to the denaturing step, all co-purification experiments were repeated in an independent experimental setup. In this alternative approach, a hexahistidine-tagged and an untagged sHsp were co-expressed in E. coli BL21(DE3). The resulting extract was applied to Ni-NTA affinity chromatography. Again, the appearance of two bands, one corresponding to the hexahistidine-tagged and one to the untagged chaperone, was clearly indicative of homo-oligomer formation (data not shown).

sHsps of the Same Class Can Form Hetero-oligomers, Which Are Active Chaperones-- Having confirmed the formation of homo-oligomers, possible formation of heteromers was assessed by co-purifying various combinations of two different sHsps. Crude extracts containing two sHsps of the same class were subjected to both the denaturation-renaturation approach (Fig. 5) and the co-expression approach (Fig. 6). In all cases analyzed, a protein corresponding to the size of the untagged protein was co-purified with the tagged protein (Figs. 5 and 6B, lane 3). Thus, sHsps of the same class (HspB and HspH, or HspC and HspF) are able to form hetero-oligomers.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   Formation of class A-class A (A) and class B-class B (B) hetero-oligomers. Crude extracts were denatured, mixed, and renatured by dialysis before being applied to the Ni-NTA column, as outlined in Fig. 4A. Crude extracts and purified proteins were analyzed by SDS-PAGE. Lane 1, crude extract containing the hexahistidine-tagged sHsp; lane 2, hexahistidine-tagged sHsp after purification; lane 3, tagged and untagged sHsps after co-purification; lane 4, untagged sHsp is not retained by the column; lane 5, crude extract containing the untagged sHsp.

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Formation of class A-class A and class B-class B hetero-oligomers demonstrated by a co-expression approach. A, outline of the strategy applied for the detection of oligomer formation without prior denaturation. Two sHsps, one with and one without a carboxyl-terminal hexahistidine tag, were co-expressed in E. coli BL21(DE3). Crude extracts were then directly applied to a Ni-NTA affinity column and analyzed by SDS-PAGE. B, SDS-PAGE analysis of purified proteins. Lane 1, crude extract with hexahistidine-tagged sHsp; lane 2, crude extract containing both tagged and untagged sHsps; lane 3, tagged and untagged sHsps after co-purification; lane 4, crude extract with untagged sHsp.

The chaperone activity of heteromeric sHsp complexes was tested using the citrate synthase assay. sHsp hetero-oligomers acted as efficient chaperones (Fig. 7; compare with Fig. 2). As observed with their homo-oligomeric counterparts, CS aggregation was reduced significantly when CS and sHsp heteromers were incubated at a ratio of CS:sHsp (monomers) of 1:1. The addition of sHsp heteromers in a ratio of CS:sHsp (monomers) of 1:2 led to an almost complete prevention of CS aggregation.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Chaperone activity of small heat shock protein hetero-oligomers. Chaperone activity assays using CS as a substrate were performed as outlined in the legend of Fig. 2. Depicted are the chaperone activity of the class A hetero-oligomer HspHHis-HspB (A) and of the class B hetero-oligomer HspCHis-HspF (B). Shown is CS aggregation in the absence of sHsps () and in the presence of sHsps at final concentrations of 150 nM (), 300 nM (), 600 nM (×), and 1.2 µM (*); absorbance of sHsps in the absence of CS () is also shown.

No Hetero-oligomers Are Formed with sHsps Belonging to Different Classes-- Further co-purification experiments were performed with mixtures of two sHsps originating from two different classes, i.e. a hexahistidine-tagged class A protein and an untagged class B protein, or vice versa. Regardless of the assay used, SDS-PAGE of the purified proteins always revealed the presence of only one single protein band, which migrated to a position reflecting the apparent molecular mass of the hexahistidine-tagged protein (Fig. 8, A-D, lane 3). Co-purification of the untagged chaperone was never observed. We therefore conclude that hetero-oligomer formation is not possible with members of different sHsp classes.

View larger version (69K): [in this window] [in a new window]   Fig. 8.   Class A-class B hetero-oligomer formation does not occur. Four representative examples are shown. A, SDS-PAGE analysis of co-expression and purification of sHsps without prior denaturation of the crude extracts. Only one band, migrating at the molecular mass of the hexahistidine-tagged sHsp, was present after purification (lane 3). Lane 1, crude extract with hexahistidine-tagged sHsp; lane 2, crude extract containing both tagged and untagged sHsps; lane 3, only the hexahistidine-tagged sHsp was detected after purification; lane 4, crude extract with untagged sHsp. B-D, results consistent with A were obtained when two crude extracts were mixed, denatured, renatured, applied to the Ni-NTA affinity column, and analyzed by SDS-PAGE. Lane 1, crude extract containing the hexahistidine-tagged sHsp; lane 2, hexahistidine-tagged sHsp after purification; lane 3, only the hexahistidine-tagged sHsp was detected after the co-purification procedure; lane 4, untagged sHsp does not bind to the column; lane 5, crude extract containing the untagged sHsp.

E. coli IbpB Interacts with sHsps of Class A but Not with Class B Proteins-- To support our conclusions on hetero-oligomer formation, we analyzed whether E. coli IbpB would interact with B. japonicum sHsps. On the basis of its primary amino acid sequence, IbpB itself can be assigned to class A (21). Like the B. japonicum sHsps, IbpB was overproduced in E. coli JGT14, and the crude extract was mixed with extracts containing either HspB-His₆, HspH-His₆, HspC-His₆, or HspF-His₆ for co-purification following the denaturation-renaturation protocol.

It is evident that IbpB was co-purified with the class A proteins HspB-His₆ and HspH-His₆ (Fig. 9A, lane 2). The IbpB band was never observed after purification of mixtures together with HspC-His₆ and HspF-His₆ (Fig. 9B, lane 2). This clearly indicates that IbpB does form hetero-oligomers with class A proteins but is unable to form oligomers with class B sHsps.

View larger version (84K): [in this window] [in a new window]   Fig. 9.   Formation of E. coli IbpB-B. japonicum sHsp hetero-oligomers. Crude extracts were denatured, mixed, and renatured by dialysis before being applied to the Ni-NTA column, as outlined in Fig. 4. Crude extracts and purified proteins were analyzed by SDS-PAGE. The E. coli sHsp IbpB was co-purified with sHsps belonging to class A (A) but not class B (B). Lane 1, crude extract containing the hexahistidine-tagged B. japonicum sHsp; lane 2, B. japonicum sHsp and E. coli IbpB after co-purification; lane 3, crude extract with E. coli IbpB.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Small heat shock proteins must assemble into oligomeric complexes to gain chaperone activity. The precise composition of these complexes is largely unknown, in particular when different sHsps occur in the same cellular compartment. Very few cases of interactions between different sHsps have been described. In many organisms, the question of homo- or hetero-oligomer formation does not even arise, because they encode only one single sHsp. The thermophilic archaeon M. jannaschii, the only organism whose sHsp structure has been resolved (12), belongs to these organisms.

Hetero-oligomer formation has been studied most intensively for the vertebrate eye lens protein -crystallin, which consists of two closely related sHsps, A- and B-crystallin. A- and B-crystallins were isolated from the mammalian eye lens as mixed oligomers with a subunit ratio of approximately 3:1. Both subunits are also able to form homopolymers that exhibit chaperone activity (7, 24). When human B-crystallin occurs in the same tissue with Hsp27, the two sHsps can be isolated as heteromeric complexes (25).

In plants, a remarkable number of sHsps belonging to six different classes is induced upon a temperature upshift (22). As most of these classes are localized in different cellular compartments, the corresponding sHsps do not have the opportunity to interact. However, members of two sHsp families, designated class I and class II, are both located in the cytosol. So far, only homo-oligomeric complexes have been reported for these cytosolic plant sHsps (26).

In bacteria other than M. jannaschii, oligomer formation of sHsps has not been analyzed yet. The E. coli sHsps IbpA and IbpB are found to be associated with inclusion bodies (18). Evidence for the formation of IbpAB hetero-oligomers is lacking. To our knowledge, the present study is the first report addressing the formation of sHsp hetero-oligomers in bacteria.

The soil bacterium B. japonicum is exceptional in inducing at least 10 sHsps belonging to two different classes upon a temperature upshift (21, 27). Because all these proteins presumably occur in the cytoplasm, they have, in principal, a chance of interacting. B. japonicum sHsps are therefore perfect model proteins for addressing homo- and hetero-oligomer formation.

In the present study, we purified four representative B. japonicum sHsps by Ni-NTA affinity chromatography and analyzed their chaperone activity and oligomer formation. Purified proteins were obtained in a functionally active form, indicating that the carboxyl-terminal hexahistidine tag affected neither their chaperone activity nor their ability to form oligomers. A carboxyl-terminal histidine tag was chosen, because an intact amino terminus might be required for the assembly of oligomers (28).

All four purified sHsps act as efficient chaperones in vitro, as demonstrated by their ability to protect CS from thermally induced aggregation. Apparently, the sHsps have a high substrate binding capacity. Equimolar amounts of sHsp and CS (all calculated as monomers) efficiently reduced the aggregation of the latter enzyme. A molar ratio of sHsp:CS of 2:1 was sufficient to virtually prevent CS precipitation. These values are in agreement with the chaperone activities reported for various other sHsps (9, 11, 16, 29). No major differences were observed between the chaperone activities of the B. japonicum sHsps. This finding suggests that, at least if CS is used as a model substrate, all sHsps have a similar affinity for the unfolded protein.

A prerequisite for chaperone activity is the formation of sHsp oligomers. Many sHsp oligomers have well defined quaternary structures. Hsp16.5 of the hyperthermophilic archaeon M. jannaschii forms a 24-mer (12), as does Hsp16 from Synechococcus vulcanus (30). Hsp16.3 of Mycobacterium tuberculosis is organized as a trimer of trimers (9). Hsp18.1 from the pea is a dodecamer (10); murine Hsp25 is a hexadecamer (31).

Other sHsps assemble into complexes of heterogenous size. Human Hsp27 forms 200-800-kDa complexes (32). -Crystallin is mainly isolated as a 600-800-kDa oligomer (33), but complexes may range from 280 kDa to 10 MDa. The mass of recombinant B-crystallin ranges from 510 (corresponding to a 25-mer) to 790 kDa (39-mer), with 650 kDa or a 32-mer average (14). A recent study based on gel filtration chromatography and cryo-electron microscopy supports the notion that both Hsp27 and -crystallin assemblies form polydisperse complexes with highly variable quaternary structures (15). E. coli IbpB also exhibits pronounced size heterogeneity. The basic oligomers appear to be roughly spherical 600-kDa structures, which interact to form larger complexes (23).

Gel filtration chromatography performed with tagged and untagged B. japonicum proteins revealed in each case a similar elution pattern, indicating that oligomerization is not affected by the presence of a carboxyl-terminal hexahistidine tag. All four sHsps form oligomers of rather heterogenous size. They eluted predominantly as complexes with a molecular mass of 400-500 kDa, corresponding approximately to a 24-mer. Thus, chaperone activity and homo-oligomer formation of HspB, HspH, HspC, and HspF provide evidence that B. japonicum sHsps share the typical properties of sHsps belonging to the -crystallin family.

The fact that multiple sHsps are expressed simultaneously in heat-shocked B. japonicum cells raises the fascinating question of whether they have the ability to interact. We demonstrated by two independent assays that such an interaction can indeed be observed, provided that both partners originate from the same class. sHsps from different classes were unable to interact. This finding is in agreement with experiments performed previously with the two classes of plant cytosolic sHsps (26). Although oligomer formation between members of the same class has not been addressed in plants, the formation of mixed oligomers between class I and class II cytosolic sHsps has been excluded.

Formation of mixed oligomers occurs between sHsps from different bacterial species. IbpB, a class A protein from E. coli, interacted with class A proteins of B. japonicum but not with class B chaperones. Thus, hetero-oligomer formation is principally possible across species barriers, provided that the interacting partners belong to the same class of sHsps. The molecular basis for the strict discrimination between class A and class B proteins remains to be elucidated.

To follow up on these studies it will be interesting to learn whether different sHsp assemblies have distinct substrate specificities or additional functions. As judged from citrate synthase assays, the chaperone activity of both homo-oligomeric and hetero-oligomeric sHsp complexes was seemingly very similar. However, it has been described recently that the physical properties of hetero-oligomeric sHsp complexes can differ from the corresponding homopolymers. Native -crystallin complexes containing A- and B-subunits in a 3:1 molar ratio differ in their membrane binding properties from homopolymeric A-crystallin and B-crystallin (33). Even very similar homo-oligomers consisting of A- or B-crystallin have slightly different properties. It has been reported that B-crystallin exhibits higher chaperone capacity at physiological temperatures, whereas A-crystallin is more heat-stable (34). It is tempting to speculate that the activity and substrate specificity of -crystallin-type chaperones can be modulated by changing the subunit composition of oligomeric complexes.

	ACKNOWLEDGEMENTS

We are indebted to Hauke Hennecke for continuous support and stimulating comments throughout the work. We thank Helmut Burtscher for providing plasmid pBF3-IbpB and François Baneyx for the E. coli strain JGT14.

	FOOTNOTES

^* This study was supported by a grant from the Swiss National Foundation for Scientific Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 41-1-632-2586; Fax: 41-1-632-1148; E-mail: fnarber@micro.biol.ethz.ch.

Published, JBC Papers in Press, September 7, 2000, DOI 10.1074/jbc.M004701200

	ABBREVIATIONS

The abbreviations used are: sHsp, small heat shock protein; PAGE, polyacrylamide gel electrophoresis; CS, citrate synthase; BSA, bovine serum albumin; NTA, nickel-nitrilotriacetic acid.

	REFERENCES

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