A critical motif for oligomerization and chaperone activity of bacterial a-heat shock proteins Sonja Studer, Markus Obrist, Nicolas Lentze and Franz Narberhaus Institute of Microbiology, Eidgeno ¨ ssische Technische Hochschule, Zu ¨ rich, Switzerland Oligomerization into multimeric complexes is a prerequisite for the chaperone function of almost all a-crystallin type heat shock proteins (a-Hsp), but the molecular details of complex assembly are poorly understood. The a-Hsp pro- teins from Bradyrhizobium japonicum are suitable bacterial models for structure-function studies of these ubiquitous stress proteins. They fall into two distinct classes, A and B, display chaperone activity in vitro and form oligomers of  24 subunits. We constructed 19 derivatives containing truncations or point mutations within the N- and C-terminal regions and analyzed them by gel filtration, citrate synthase assay and coaffinity purification. Truncation of more than the initial few amino acids of the N-terminal region led to the formation of distinct dimeric to octameric structures devoid of chaperone activity. In the C-terminal extension, integrity of an isoleucine-X-isoleucine (I-X-I) motif was imperative for a-Hsp functionality. This I-X-I motif is one of the characteristic consensus motifs of the a-Hsp family, and here we provide experimental evidence of its structural and functional importance. a-Hsp proteins lacking the C-termi- nal extension were inactive, but still able to form dimers. Here, we demonstrate that the central a-crystallin domain alone is not sufficient for dimerization. Additional residues at the end of the N-terminal region were required for the assembly of two subunits. Keywords: a-crystallin; a-heat shock protein; small heat shock protein; chaperone; oligomerization. Heat shock or other forms of stress induce the expression of a-heat shock proteins (a-Hsp proteins) in a broad range of prokaryotic and eukaryotic organisms [1–5]. a-Hsp proteins take part in the cellular multichaperone protein-folding network by binding to partially denatured proteins, thereby creating a reservoir of unfolded proteins for subsequent refolding by other chaperones such as DnaK and GroEL [6–8]. Virtually all a-Hsp proteins examined display chap- erone activity in vitro, measured by their ability to protect model substrates from thermally or chemically induced aggregation [8–13]. a-Hsp proteins are named after their most prominent representative, a-crystallin, which prevents protein precipi- tation in the vertebrate eye lens. They are mostly referred to as small heat shock proteins (sHsp), because their mono- meric molecular mass ranges between 12 and 43 kDa. However, the term sHsp is somewhat misleading, as several other small heat-inducible proteins bear no resemblance to a-Hsp proteins. Moreover, native a-Hsp proteins are among the largest protein particles in the cell, as they assemble into complexes whose molecular mass often exceeds 500 kDa. Many of these complexes have been reported to consist of  24 subunits [13–16], but both larger and smaller structures have also been described [11,12,17,18]. The quaternary structure of a-Hsp proteins is highly dynamic, which is often reflected by pronounced size heterogeneity or rapid subunit exchange [19]. Mammalian members of the a-Hsp family are remarkably polydisperse [20,21], and some bacterial proteins such as Escherichia coli IbpB also display pro- nounced size heterogeneity [22]. Particularly rigid structures are of prokaryotic origin, such as the 24-meric Hsp16.5 from Methanococcus jannaschii or nonameric Hsp16.3 from Mycobacterium tuberculosis [11,14]. a-Hsp proteins are widely distributed but poorly con- served. One major characteristic of this protein family is the presence of a central a-crystallin domain, flanked by a N-terminal region and a C-terminal extension [23]. The highest degree of amino-acid similarity is found within the a-crystallin domain, while the N-terminal region and the C-terminal extension are variable in length and sequence. Naturally occurring a-Hsp proteins lacking these flanking regions acquire monomeric to tetrameric structures and are poor or inactive chaperones [24,25]. In the last few years, systematic a-Hsp structure–function studies have mainly been focused on the mammalian representatives aA- and aB-crystallin. These proteins proved remarkably resistant against mutational alterations. For example, a wide variety of truncations and point mutations within the N-terminal region had no consequence on protein structure and function [26–30]. However, some modifications in the N-terminal region of eukaryotic a-Hsp proteins were reported to affect chaperone activity [31] or complex formation [26,32]. The C-terminal extension, being located on the outer surface of the a-Hsp oligomers, is generally assumed to contribute to complex solubility [33], but its further functional implication remains unclear. The recently resolved crystal structures of a plant and an archaeal a-Hsp show that in these proteins, the C-terminal extensions are involved in subunit–subunit interactions by strapping around the outer surface of the complex [14,34]. The most Correspondence to F. Narberhaus, Institute of Microbiology, ETH-Zentrum, Schmelzbergstrasse 7, CH-8092 Zu ¨ rich, Switzerland. Fax: + 41 1632 1148, Tel.: + 41 1632 2586, E-mail: fnarber@micro.biol.ethz.ch Abbreviations: a-Hsp, a-crystallin type heat shock protein; sHsp, small heat shock protein; CS, citrate synthase. (Received 1 March 2002, revised 25 April 2002, accepted 17 June 2002) Eur. J. Biochem. 269, 3578–3586 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03049.x striking feature of the poorly conserved C-terminal exten- sion is the presence of an isoleucine-X-isoleucine (I-X-I) motif in the majority of a-Hsp proteins. This I-X-I (or, more generally, I/V-X-I/V) motif was recognized as one of the three main consensus regions of the a-Hsp family [5]. There is little experimental evidence to circumstantiate the role of the C-terminal extension, and even less concerning the conserved I-X-I motif. Various modifications within the C-terminal extension decreased chaperone activity of a-crystallin and other vertebrate a-Hsp proteins [27,31,33,35], but only in the case of one plant a-Hsp were C-terminal truncations observed to reduce complex size [36]. Altogether, the oligomerization principles of a-Hsp proteins, and especially of their bacterial representatives, are still poorly understood. We found in previous studies that the soil bacterium Bradyrhizobium japonicum is a suitable model organism for investigating prokaryotic a-Hsp proteins [4,13,37]. It contains at least 10 a-Hsp proteins, which are highly induced upon heat shock [4]. The sequences of seven B. japonicum a-Hsp genes are available and can be assigned to two distinct classes, A and B. These two classes are not restricted to B. japonicum, but also occur in other rhizobia [38]. Members of both classes have been shown to prevent citrate synthase aggregation in vitro,to form 400- to 500-kDa complexes and to interact with other a-Hsp proteins of the same class [13]. The present study demonstrates that chaperone activity of B. japonicum a-Hsp proteins is stringently coupled to multimerization, and that both the N- and C-terminal regions are required for the formation of chaperone- competent complexes. In particular, we show that integrity of the I-X-I motif is critical for assembly of functional a-Hsp proteins, and that the isolated a-crystallin domain is unable to dimerize. EXPERIMENTAL PROCEDURES Plasmid construction Plasmids for the expression of B. japonicum hspF (pRJ5306) and hspH (pRJ5307) provided with a C-terminal His 6 tag have been described previously [13]. N- or C-terminal truncations of hspF and hspH were constructed by PCR, using pRJ5306 and pRJ5307 as templates. For subsequent cloning, we took advantage of two unique restriction sites, namely BsaIonhspF and SfiIonhspH. PCR products were digested with either BsaIorSfiIandNdeI(N-terminal truncations) or NotI (C-terminal truncations). The purified fragments were used to replace the corresponding wild-type fragments in pRJ5306 or pRJ5307. hspH variants encoding the isolated a-crystallin domain or the a-crystallin domain including either the N-terminal region or the C-terminal extension were constructed in a similar manner. Mutations leading to single and double amino-acid exchanges were introduced into hspH by means of the QuickChange TM Site- Directed Mutagenesis Kit (Stratagene). Expression vector pRJ5307 was used as a template, and mutagenesis was performed according to the manufacturer’s instructions. All resulting plasmids encoded a-Hsp versions carrying a His 6 tag at the C-terminus. A plasmid for the expression of an untagged hspH variant lacking the C-terminal extension was constructed by introducing a stop codon followed by an XhoI site by PCR. The amplification product was digested with NdeIandXhoI and ligated into a pET24b vector digested with the same endonucleases. The correct nucleo- tide sequence of all inserts was confirmed by automated DNA sequencing. Protein expression and purification Freshly transformed E. coli BL21(DE3)pLysS strains were used for protein expression. Overexpression cultures were grown at 30 °CtoD 600 ¼ 0.6, induced by addition of isopropyl thio-b- D -galactoside to a final concentration of 0.5 m M , and grown for a further 2–3 h. After harvesting, cells were resuspended in binding buffer (500 m M KCl, 20 m M Tris/HCl, 5 m M imidazole, pH 7.9) containing 1m M phenylmethanesulfonyl fluoride and 10 lgÆmL )1 DNaseI. Lysis was performed in a French pressure cell at 1000 p.s.i, and soluble crude extracts were prepared by centrifugation at 12 000 g for 30 min at 4 °C. Proteins were purified by Ni-nitrilotriacetic acid affinity chromatography (Ni-nitrilotriacetic acid resin from Qiagen) under native conditions essentially as described previously [13]. The column was pre-equilibrated with binding buffer and then washed with washing buffer (500 m M KCl, 20 m M Tris/HCl, pH 7.9) containing increasing imidazole concen- trations (5–50 m M ). The imidazole concentration was finally raised to 250 m M in order to elute bound proteins. If protein purity was not satisfactory, the eluate was diluted to an imidazole concentration below 50 m M andappliedtoa second column, either Ni-nitrilotriacetic acid/agarose (Qia- gen) or Co-PDC/agarose (Acros Organics). Whenever possible, proteins were analyzed by gel filtration and citrate synthase assay immediately after purification. Otherwise, eluates were supplemented with 20% glycerol and stored at )20 or )80 °C. Protein concentrations were determined by the Bradford assay. All protein concentrations reported in this study are expressed in terms of protomers. Chaperone activity assay Chaperone activity was determined by the citrate synthase (CS) assay. a-Hsp proteins were preincubated at 43 °Cin 1mL of 50m M sodium phosphate, pH 6.8, for at least 15 min. The assay was started by addition of CS to a final concentration of 600 n M . CS aggregation was measured by monitoring light scattering at 360 nm for 30 min in an Ultrospec 3000 spectrophotometer (Amersham Pharmacia Biotech). Prior to use, CS (Sigma) was dialyzed against Tris/ EDTA buffer (10 m M Tris/HCl, 1 m M EDTA, pH 8.0). For each protein, chaperone activity assays were performed at least twice with preparations from independent purifica- tions. Gel filtration Analytical size exclusion chromatography of purified pro- teins was performed at room temperature on a Superdex 200 HR 30/10 column (Amersham Pharmacia Biotech) using a BioCAD perfusion chromatography system (PerSeptive Biosystems). After equilibrating the column with elution buffer (500 m M KCl, 20 m M Tris/HCl, 250 m M imidazole, pH 7.9), 200-lL protein samples were injected and separated at a flow rate of 0.6 mLÆmin )1 . Absorbance was recorded at 280 nm. The following standards were used Ó FEBS 2002 A cricital motif for a-Hsp oligomerization (Eur. J. Biochem. 269) 3579 for calibration: thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), albumin (67 kDa), and ribonuclease A (13.7 kDa), all from Amersham Pharmacia Biotech. Gel filtrations were performed at least twice with protein obtained from independent preparations. Co-affinity purification Copurification of untagged a-Hsp proteins with His 6 -tagged variants by Ni-nitrilotriacetic acid affinity chromatography was used to investigate protein–protein interactions of certain truncated a-Hsp proteins. The applied denaturation–renat- uration protocol has been described previously [13]. RESULTS Construction of HspH and HspF derivatives for structure-function studies In order to study the principles of a-Hsp assembly and the relation between complex formation and chaperone activity, we constructed a series of truncated and point-mutated a-Hsp variants. A total of 19 a-Hsp derivatives were analyzed in the course of this study. An overview of these constructs is given in Fig. 1. The alignment presents the seven known a-Hsp sequences from B. japonicum, i.e. five class A (HspA, HspB, HspD, HspE and HspH) and two class B proteins (HspC and HspF), as well as the class A E. coli proteins IbpA and IbpB. A representative example from each class, A (HspH) and B (HspF), was chosen for further analysis. The N-terminal region of HspH and HspF was gradually shortened by eliminating an increasing number of amino- acid residues. The resulting proteins were named HspH (D3N), HspH(D9N), HspH(D15N), HspH(D20N), and HspF(D5N), HspF(D30N), HspF(D40N). In HspH(D20N) and HspF(D40N), approximately half of the N-terminal region was eliminated, including a proline and an arginine residue conserved in all seven B. japonicum a-Hsp proteins (Fig. 1). To examine the importance of these two particular residues, we constructed two HspH derivatives in which the proline (P8A) or the arginine (R18A) was replaced by an alanine. The characteristic I-X-I motif in the C-terminal extension is present in all B. japonicum and E. coli a-Hsp proteins. To investigate its role in oligomerization, we constructed two a-Hsp derivatives lacking one [HspF(D5C)] or both [HspH(D20C)] isoleucine residues. Two further constructs, HspH(D5C) and HspH(D15C), contained the entire I-X-I motif. To assess the role of the conserved motif in the context of the full-length protein, three HspH variants were constructed, in which the isoleucines were replaced by alanine, either individually, resulting in HspH(I133A) and HspH(I135A), or simultaneously, leading to HspH(II133,135AA). Finally, we searched for the minimal fragment required for dimer formation. For this purpose HspH variants lacking entire subregions were constructed. Three proteins, Fig. 1. Amino-acid alignment of B. japonicum and E. coli a-Hsp proteins. The alignment includes the seven known sequences of B. japonicum a-Hsp proteins, belonging to class A [HspH (accession number O86110), HspA (P70917), HspB (P70918), HspD (O69241) and HspE (O69242)] and B [HspC (AAC44757) and HspF (CAA05837)]. IbpA (P29209) and IbpB (G65170) from E. coli (both class A) were added for comparison. The alignment was created with CLUSTAL W [48]. Amino acids that are identical in all analyzed proteins are shown in white letters shaded in black. White letters and black letters shaded in grey indicate amino acids that are identical in at least 80 or 60% of all proteins, respectively. Arrows mark truncations introduced to HspH and HspF, and asterisks indicate alanine exchange mutations in HspH. Note that four N-terminal residues were included in HspH(a) and HspH(aC) to avoid disturbance of potential secondary structures. 3580 S. Studer et al. (Eur. J. Biochem. 269) Ó FEBS 2002 consisting of the a-crystallin domain alone [HspH(a)], the a-crystallin domain plus the N-terminal region [HspH(Na)], or the a-crystallin domain followed by the C-terminal extension [HspH(aC)], were analyzed. Alterations in the N-terminal region affect chaperone activity and oligomerization As demonstrated previously [13], full-length class A and class B a-Hsp proteins of B. japonicum act as efficient chaperones in vitro (Fig. 2H,I). They form oligomers with an apparent molecular mass of 400–500 kDa, which corresponds to a complex of  24 subunits ([13]; for comparison with mutated variants see gel filtration profiles in Fig. 3H–J). These features were not affected if the N-terminus was shortened by just a few amino-acid residues. HspH(D3N) and HspF(D5N) could not be distin- guished from native HspH and HspF with regard to chaperone activity and oligomeric state. They prevented CS aggregation (Fig. 2A,E), and their apparent molecular mass of 350–480 kDa was consistent with oligomers of 20–30 subunits (Fig. 3A,E). Thus, the integrity of the extreme N-terminus is not relevant for the assembly of functional a-Hsp complexes. Removal of additional amino acids, however, drastically altered the characteristics of either protein. HspH(D9N), HspH(D15N) and HspH(D20N) were devoid of chaperone activity (Fig. 2B–D) and unable to assemble into large oligomers. Instead, they formed complexes consisting of approximately eight subunits (Fig. 3B–D). Oligomerization was only partially compromised, whereas chaperone activity was completely abolished. Note that in many gel filtration runs [e.g. HspH or HspH(D3N)], a considerable portion of the protein eluted after 12–13 min in the void volume of the column, reflecting the tendency of all B. japonicum a-Hsp proteins to form large aggregates. Extended truncations in the N-terminal region of HspF caused similar, but not identical effects as in HspH. When 30 or more amino-acid residues were removed from the N-terminus of HspF, chaperone activity was completely lost (Fig. 2F,G) and oligomer formation was impaired even more drastically than in the HspH derivatives. Gel filtration analysis of HspF(D30N) and HspF(D40N) suggested that both pro- teins were only dimers (Fig. 3F,G), whereas HspH deri- vatives devoid of the first half of the N-terminal region [HspH(D20N)] appeared as octameric complexes. In either case, it is evident that a largely intact N-terminal region is strictly required for the formation of active a-Hsp oligomers. In an attempt to narrow down the critical determinants to the amino-acid level, we tested the effect of two single amino-acid exchanges (P8A and R18A) on chaperone activity and oligomeric state of HspH. Although these residues are conserved throughout class A and class B proteins of B. japonicum (Fig. 1), neither exchange inhibited chaperone activity and oligomerization (data not shown). Fig. 2. Effect of truncations in the N-terminal region on chaperone activity of HspH and HspF. Thermally induced aggregation of citrate synthase at 43 °Cisdepictedasa function of time in the presence of various amounts of a-Hsp proteins. (A–D), N-termi- nally truncated variants of HspH (class A); (E–G), N-terminally truncated variants of HspF (class B). Chaperone activity of full-length HspH and HspF is shown for comparison (H, I). Proteins were incubated at 43 °C in a total volume of 1.0 mL of 50 m M phosphate buffer, pH 6.9. CS aggregation was measured by the increase of absorbance at 360 nm in the absence (r) and in the presence of a-Hsp proteins at a final concentration of 150 n M (j), 300 n M (m), 600 n M (·)and 1.2 l M (d). The CS concentration was 600 n M . Absorbance of a-Hsp proteins in the absenceofCSisalsoshown(S). Ó FEBS 2002 A cricital motif for a-Hsp oligomerization (Eur. J. Biochem. 269) 3581 Two conserved isoleucines are essential for a-Hsp functionality When assessing the impacts of C-terminal truncations on oligomerization and chaperone function, we focused our attention on the conserved I-X-I motif. First, the C-terminal extension of HspH was shortened by five and 15 amino acids [HspH(D5C) and HspH(D15C), respectively], leaving the I-X-I motif intact. These alterations had no influence on chaperone activity. Both proteins efficiently protected CS from thermally-induced aggregation (Fig. 4A,B) and as- sembled into large complexes (Fig. 5A,B). But while HspH(D5C) appeared similar to full-length HspH in terms of oligomer formation, HspH(D15C) formed larger aggre- gates with an apparent molecular mass exceeding 2 MDa. All C-terminally truncated a-Hsp derivatives exhibited an increased tendency to precipitate. Elimination of the entire I-X-I motif in HspH(D20C) led to a complete loss of chaperone activity (Fig. 4C) and a protein that was unable to multimerize. The only species encountered in the gel filtration profile was a dimer of  29 kDa (Fig. 5C). Removal of the C-terminal isoleucine from the I-X-I motif in HspF(D5C) was sufficient to completely abolish chaperone activity (Fig. 4D) and Fig. 3. Oligomerization of N-terminally truncated HspH and HspF derivatives. The oligomeric state of purified a-Hsp variants was determined by gel filtration over a Superdex 200 column at a flow rate of 0.6 mLÆmin )1 . N-Terminally truncated derivatives of HspH (A–D) and HspF (E–G) were analyzed and compared with native HspH (H) and HspF (I). The minor peak at 12–13 min that is observed in all gel filtration profiles represents large protein aggregates eluting in the column’s void volume. J, calibration curve for the gel filtration profiles in (A–I). K av ¼ (V e ) V 0 )/(V t ) V 0 )is depicted as a function of log molecular mass (V e ¼ elution volume of the protein, V 0 ¼ column void volume, V t ¼ total bed volume). Open circles represent standard proteins listed in Experimental procedures (molecular masses given in kDa), filled circles indicate HspH and HspF variants. Only major peaks aside from the void volume are indicated. Fig. 4. Effect of truncations and single amino acid exchanges in the C-terminal extension on chaperone activity of HspH and HspF. Chap- erone activity of purified a-Hsp proteins was determined by the CS aggregation assay as outlined in the legend of Fig. 2. (A–C) C-Terminally truncated variants of HspH. (D) C-Terminally truncated variant of HspF; E-G, point mutated variants of HspH. Depicted is CS aggregation in the absence (r)andinthe presence of a-Hsp proteins at final concen- trations of 150 n M (j), 300 n M (m), 600 n M (·)and1.2l M (d); as well as the absorbance of a-Hsp proteins in the absence of CS (S). 3582 S. Studer et al. (Eur. J. Biochem. 269) Ó FEBS 2002 severely impaired oligomer formation (Fig. 5D). The apparent molecular mass of 44–48 kDa probably repre- sents a dimer. As both truncations affecting the I-X-I motif impaired chaperone activity and oligomer formation, we replaced the two isoleucine residues in HspH by alanines and analyzed whether these mutations had a similar effect. In fact, HspH(I133A), HspH(I135A) and HspH(II133,135AA) were devoid of chaperone activity (Fig. 4E–G) and appeared as small, dimeric structures of 43–50 kDa (Fig. 5E–G). Summarizing the gel filtration data, the calibration curve in Fig. 5H illustrates that all modifications affecting the I-X-I motif resulted in the formation of small, presumably dimeric complexes. Taken together, these results demonstrate that the isoleucine motif plays a crucial structural and functional role in the assembly of functional HspH or HspF oligomers. The a-crystallin domain alone is not sufficient for dimer formation All HspF and HspH variants investigated so far acquired dimeric structures, indicating that the dimerization motif remained untouched. In order to determine the a-Hsp region responsible for dimerization, we initially attempted to purify the isolated a-crystallin domain. As the protein repeatedly showed a strong tendency to precipitate and could only be recovered in concentrations too low for size exclusion chromatography, we used an alternative approach instead. The interaction of full-length HspH with a series of truncated His 6 -tagged HspH variants, i.e. HspH(a)–His 6 , HspH(Na)–His 6 ,HspH(aC)–His 6 and HspH(HD20N)– His 6 was tested. Crude extracts containing tagged and untagged protein were mixed, denatured and renatured before being applied to Ni-nitrilotriacetic acid affinity columns. The eluted proteins were subsequently analyzed by SDS/PAGE. Co-elution of the untagged protein together with the His 6 -tagged species was indicative of protein– protein interactions, whereas proteins that did not interact with the His 6 -tagged species were not retained on the column [13]. Untagged HspH did not coelute with the His 6 -tagged a-crystallin domain (Fig. 6A), suggesting that the isolated a-crystallin domain was unable to dimerize. The a-crys- tallin domain carrying the C-terminal extension appeared to enable weak interactions with native HspH, because after copurification of HspH(aC)–His 6 with HspH, a faint band corresponding to HspH was visible on SDS gels. In contrast, HspH(Na)–His 6 , an HspH derivative consisting of the a-crystallin domain plus the N-terminal region, strongly interacted with native HspH. After co-affinity purification, both proteins were detected on SDS gels in a 1 : 1 ratio. A similarly efficient copurification of HspH was observed with HspH(D20N)–His 6 . This suggests that the a–Hsp interaction is in part mediated by a portion of the N-terminal region in vicinity of the a-crystallin domain. To ensure that efficient dimerization requires solely a-crystallin domain and N-terminal region and not the C-terminal extension, HspH(Na)–His 6 was also subjected to a copurification assay with untagged HspH(Na). The latter protein alone did not bind to the Ni-nitrilotriacetic acid resin (Fig. 6B, lane 5). However, it co-eluted with HspH(Na)– His 6 (Fig. 6B, lane 4). Size exclusion chromatography confirmed that HspH(Na) was present as small, presumably dimeric species, which were, as expected, devoid of chaper- one activity (data not shown). Thus, dimer formation does not depend on the integrity of the C-terminal extension but requires part of the N-terminal region. DISCUSSION Up to now, systematic a-Hsp structure–function studies have almost exclusively focused on a-crystallins from higher organisms. Introduction of mutations often had little impact on oligomerization and chaperone activity, as mammalian a-Hsp proteins are very resistant to mutational changes owing to their intrinsic plasticity [20,26–30,39]. In compar- Fig. 5. Oligomerization of HspH and HspF derivatives with alterations in the C-terminal extension. Gel filtration profiles of a-Hsp derivatives with truncations or point mutations in the C-terminal extension are shown. (A–C) C-Terminally truncated variants of HspH; (D) C-terminally truncated variant of HspF; E-G, point mutated variants of HspH. See Fig. 3H,I for the gel filtration profiles of HspH and HspF. The minor peak at 12–13 min observable in most gel filtration profiles represents large protein aggregates eluting in the column’s void volume. (H) calibration curve for the gel filtration profiles depicted in (A–G). Standard proteins (molecular masses given in kDa) are represented by open circles, HspH and HspF variants by filled circles. HspH(D15C) eluting in the void volume of the column was not included. Ó FEBS 2002 A cricital motif for a-Hsp oligomerization (Eur. J. Biochem. 269) 3583 ison with mammalian a-crystallins, the oligomerization principles of bacterial a-Hsp proteins have received little attention. Available data indicate that a-Hsp proteins from prokaryotes and plants may differ significantly from their mammalian counterparts in structure and function [40,41]. Some prokaryotic a-Hsp proteins possess remarkably rigid structures [11,14] that render them more susceptible to point mutations and truncations. We therefore chose two dis- tantly-related bacterial a-Hsp proteins from B. japonicum in order to study, in detail, the contribution of their subregions to chaperone activity and oligomerization. Tight coupling of chaperone activity and complex assembly B. japonicum HspH and HspF oligomers contain  24 subunits, as shown previously with crude extracts [13] and confirmed with purified proteins in the present study. Transmission electron microscopical analysis of both pro- teins revealed roughly spherical structures (data not shown) similar to those observed for other members of the a-Hsp family [10,12,20,21]. We have demonstrated that assembly of this native structure is severely impaired by truncations in the N-terminal region and the C-terminal extension. Interest- ingly, all of the resulting low molecular mass complexes were devoid of chaperone activity. Although we cannot rule out that the truncated proteins had lost substrate binding regions in addition to oligomerization sites, this finding illustrates that chaperone activity and oligomerization are tightly coupled. The ability to bind unfolded proteins and prevent them from aggregation clearly requires fully assembled multimeric a-Hsp complexes. Even complexes that exceed the size of the wild-type protein, e.g. HspHD15C, retained chaperone activity. Much like our a-Hsp variants, naturally occurring multimerization-incom- petent a-Hsp proteins have poor or lacking chaperone activity [24,25,42]. The chaperone activity of mammalian a-crystallins, on the other hand, appears to be much more resistant against structural modifications. Human aB-crys- tallin does not require a multimeric a-Hsp complex for chaperone activity [43]. The functional, substrate-binding entity might actually be an a-Hsp dimer rather than the fully oligomerized particle, which is regarded as a transient storage form of the chaperone [34,44]. Prerequisites for dimer formation The initial step in the oligomerization process is apparently the formation of dimers. Even the most drastic modifications within the N- and C-terminal regions of HspF and HspH did not impair dimerization. This observation argues for the presence of additional interaction sites in or near the a-crystallin domain. Crystallographic data from M. janna- schii Hsp16.5 [14] and wheat Hsp16.9 [34], as well as spin labeling studies with aA-crystallin and Hsp27 [45] indicate that the dimeric building block of many a-Hsp proteins is formed by interacting bstrands in the a-crystallin domain. Although this may also apply to the B. japonicum a-Hsp proteins, the isolated a-crystallin domain of HspH was not sufficient for dimer formation. Only the presence of a C-terminal portion of the N-terminal region enabled the a-crystallin domain to dimerize. It is conceivable that the a-crystallin domain, as it is defined on the basis of sequence similarities, does not reflect the actual functional and structural entity. This assumption is supported by the fact that a b strand overlaps the junction of N-terminal region and a-crystallin domain of M. jannaschii Hsp16.5 [14]. However, even the addition of four N-terminal amino acids to the deduced a-crystallin domain of HspH (Fig. 1) in order to avoid disruption of a predicted b strand was not sufficient for dimer formation. This argues that more residues towards the N-terminus are involved in dimeriza- tion. Importance of the N-terminal region Additional portions of the N- and C-terminal regions are required for assembly into functional multimeric complexes. Interestingly, class A (HspH) and class B (HspF) proteins exhibited similar, yet not identical assembly properties. Truncations in the N-terminal region of HspF interfered Fig. 6. Interaction of truncated HspH variants with full-length and truncated HspH demonstrated by coaffinity purification. Crude extracts with overexpressed native HspH were mixed with extracts containing truncated His 6 -tagged HspH variants according to the schematic representation on the right half of the figure. After denaturation and renaturation, the extracts were applied to Ni-nitrilotriacetic acid affinity columns, and eluates were analyzed by tricine SDS/PAGE [49]. Coomassie-stained eluates on 13% polyacrylamide gels are shown on the left half of (A) and (B). The expected positions of relevant proteins are indicated by arrows. (A) Copurification of untagged HspH with His 6 -tagged HspH derivatives. Lane 1, purification of the His 6 -tagged HspH variant; lane 2, His 6 -tagged HspH variant and untagged HspH after copurification. (B) Interaction of tagged and untagged HspH(Na). Lane 3, HspH(Na)-His after purification; lane 4, copuri- fication of HspH(Na)andHspH(Na)-His; lane 5, untagged HspH(Na) application to the Ni-nitrilotriacetic acid affinity column. 3584 S. Studer et al. (Eur. J. Biochem. 269) Ó FEBS 2002 more drastically with oligomerization than comparable alterations in HspH (compare Fig. 3D,F). In line with clearly deviating N-terminal sequences (Fig. 1), oligomer- ization of HspH and HspF might be mediated by different interacting regions. Such distinctions most likely explain why a-Hsp proteins from different classes are unable to interact [13,36,46]. In both classes, the region near, but not directly at the N-terminus is evidently critical. Likewise, in Hsp16–2 from Caenorhabditis elegans, removal of the N-terminal 15 residues was sufficient to inhibit chaperone activity and oligomerization [44]. In a-crystallin, on the other hand, numerous point mutations and even truncation of the first half of the N-terminal region did not affect complex formation and functionality [26–30]. Only complete removal of the N-terminal region resulted in the formation of an inactive low molecular mass species [26,32]. These data suggest that subunit interactions of mammalian a-Hsp proteins may differ from their counterparts in prokaryotes and plants. The crystal structure of wheat Hsp16.9 demon- strates that the N-terminal region of this protein is involved in numerous subunit contacts [34]. It is puzzling that only every other N-terminal region in the Hsp16.9 complex but all N-termini in M. jannaschii Hsp16.5 are highly disordered [14,34]. Possibly, the N-terminal regions of B. japonicum a-Hsp proteins are also disordered, which might explain why subunit contact sites in the N-terminal region could not yet be narrowed down to individual conserved residues. A crucial motif in the C-terminal extension Wheat Hsp16.9 and M. jannaschii Hsp16.5 are distantly related proteins with distinct quaternary structures. Never- theless, the role of their C-terminal extensions in oligomeric assembly is strikingly similar. The C-termini reach out to neighbouring subunits, where the I-X-I motif undergoes intramolecular hydrophobic interactions with a b strand in the a-crystallin domain [14,34]. Our mutational studies strongly suggest that the C-terminal extension and the conserved I/V-X-I/V motif in particular plays a similar structural role in both classes of bacterial a-Hsp proteins. Partial or entire removal of the two conserved isoleucines led to a dramatic reduction in complex size and solubility and a complete loss of chaperone activity. All modifications touching the motif resulted in dimers. On the other hand, truncations that left the motif intact did not inhibit multimerization and chaperone activity. Similarly, alter- ations outside of the I/V-X-I/V motif did not impair oligomer formation of vertebrate a-Hsp proteins [31,33], though some of them lowered chaperone activity [31,35]. So far, very few mutational studies have included the isoleucine motif. C-Terminally truncated C. elegans Hsp16–2 lacking the I-X-I motif still formed high molecular mass complexes and retained full chaperone activity [47]. In aA-crystallin, C-terminal truncations touching the I/V-X-I/V motif did not inhibit multimerization, but reduced chaperone activity [27]. Only in one particular plant a-Hsp, Hsp17(II) from pea, were C-terminal truncations, including the I/V-X-I/V motif, associated with a significant reduction in oligomeric mass [36]. However, whether the motif itself or other residues in the C-terminal extension were responsible for this effect was not determined. The data reported in the present study further substan- tiate that oligomerization of prokaryotic a-Hsp proteins is a complicated multistep process that differs from the assem- bly of eukaryotic a-Hsp proteins and merits further investigation. ACKNOWLEDGEMENTS We thank Hauke Hennecke for support and encouragement. This study was supported by a grant from the Swiss National Foundation. REFERENCES 1. Waters, E.R., Lee, G.J. & Vierling, E. (1996) Evolution, structure and function of the small heat shock proteins in plants. J. Exp. Bot. 47, 325–338. 2. Arrigo, A P. & Landry, J. (1994) Expression and Function of the Low-molecular-weight Heat Shock Proteins. In The Biology of Heat Shock Proteins and Molecular Chaperones (Morimoto, R.I., Tissie ` res, A. & Georgopoulos, C., eds), pp. 335–373, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 3. Ehrnsperger, M., Buchner, J. & Gaestel, M. (1998) Structure and Function of Small Heat-Shock Proteins. In: Molecular Chaperones in the Life Cycle of Proteins (Fink, A.L. & Goto, Y., eds), pp. 533– 575. Marcel Dekker, New York. 4. Mu ¨ nchbach,M.,Nocker,A.&Narberhaus,F.(1999)Multiple small heat shock proteins in rhizobia. J. Bacteriol. 181, 83–90. 5. de Jong, W.W., Caspers, G.J. & Leunissen, J.A.M. (1998) Gen- ealogy of the a-crystallin – small heat-shock protein superfamily. Int. J. Biol. Macromol. 22, 151–162. 6. Lee, G.J., Roseman, A.M., Saibil, H.R. & Vierling, E. (1997) A small heat shock protein stably binds heat-denatured model sub- strates and can maintain a substrate in a folding-competent state. EMBO J. 16, 659–671. 7. Ehrnsperger, M., Graber, S., Gaestel, M. & Buchner, J. (1997) Binding of non-native protein to Hsp25 during heat shock creates a reservoir of folding intermediates for reactivation. EMBO J. 16, 221–229. 8. Veinger, L., Diamant, S., Buchner, J. & Goloubinoff, P. (1998) The small heat-shock protein IbpB from Escherichia coli stabilizes stress-denatured proteins for subsequent refolding by a multi- chaperone network. J. Biol. Chem. 273, 11032–11037. 9. Horwitz, J. (1992) a-crystallin can function as a molecular chap- erone. Proc. Natl Acad. Sci. U.S.A. 89, 10449–10453. 10. Kim, R., Kim, K.K., Yokota, H. & Kim, S.H. (1998) Small heat shock protein of Methanococcus jannaschii, a hyperthermophile. Proc. Natl Acad. Sci. U.S.A. 95, 9129–9133. 11. Chang, Z.Y., Primm, T.P., Jakana, J., Lee, I.H., Serysheva, I., Chiu, W., Gilbert, H.F. & Quiocho, F.A. (1996) Mycobacterium tuberculosis 16-kDa antigen (Hsp16.3) functions as an oligomeric structure in vitro to suppress thermal aggregation. J. Biol. Chem. 271, 7218–7223. 12. Lee,G.J.,Pokala,N.&Vierling,E.(1995)Structureandin vitro molecular chaperone activity of cytosolic small heat shock pro- teins from pea. J. Biol. Chem. 270, 10432–10438. 13. Studer, S. & Narberhaus, F. (2000) Chaperone activity and homo- and hetero-oligomer formation of bacterial small heat shock proteins. J. Biol. Chem. 275, 37212–37218. 14. Kim, K.K., Kim, R. & Kim, S.H. (1998) Crystal structure of a small heat-shock protein. Nature 394, 595–599. 15. Roy, S.K., Hiyama, T. & Nakamoto, H. (1999) Purification and characterization of the 16-kDa heat-shock-responsive protein from the thermophilic cyanobacterium Synechococcus vulcanus, which is an a-crystallin-related, small heat shock protein. Eur. J. Biochem. 262, 406–416. 16. Michelini, E.T. & Flynn, G.C. (1999) The unique chaperone operon of Thermotoga maritima: Cloning and initial characteri- Ó FEBS 2002 A cricital motif for a-Hsp oligomerization (Eur. J. Biochem. 269) 3585 zation of a functional Hsp70 and small heat shock protein. J. Bacteriol. 181, 4237–4244. 17. Ehrnsperger, M., Lilie, H., Gaestel, M. & Buchner, J. (1999) The dynamics of Hsp25 quaternary structure – structure and function of different oligomeric species. J. Biol. Chem. 274, 14867– 14874. 18. Groenen, P.J., Merck, K.B., de Jong, W.W. & Bloemendal, H. (1994) Structure and modifications of the junior chaperone a-crystallin. From lens transparency to molecular pathology. Eur. J. Biochem. 225, 1–19. 19. Bova, M.P., Ding, L.L., Horwitz, J. & Fung, B.K.K. (1997) Subunit exchange of aA-crystallin. J. Biol. Chem. 272, 29511– 29517. 20. Haley, D.A., Horwitz, J. & Stewart, P.L. (1998) The small heat- shock protein, aB-crystallin, has a variable quaternary structure. J. Mol. Biol. 277, 27–35. 21. Haley, D.A., Bova, M.P., Huang, Q.L., Mchaourab, H.S. & Stewart, P.L. (2000) Small heat-shock protein structures reveal a continuum from symmetric to variable assemblies. J. Mol. Biol. 298, 261–272. 22. Shearstone, J.R. & Baneyx, F. (1999) Biochemical characteriza- tion of the small heat shock protein IbpB from Escherichia coli. J. Biol. Chem. 274, 9937–9945. 23. Caspers, G.J., Leunissen, J.A. & de Jong, W.W. (1995) The expanding small heat-shock protein family, and structure predic- tions of the conserved Ôa-crystallin domainÕ. J. Mol. Evol. 40,238– 248. 24. Kokke, B.P.A., Leroux, M.R., Candido, E.P.M., Boelens, W.C. & de Jong, W.W. (1998) Caenorhabditis elegans small heat-shock proteins Hsp12.2 and Hsp12.3 form tetramers and have no cha- perone-like activity. FEBS Lett. 433, 228–232. 25. Leroux, M.R., Ma, B.J., Batelier, G., Melki, R., Peter, E. & Candido, M. (1997) Unique structural features of a novel class of small heat shock proteins. J. Biol. Chem. 272, 12847–12853. 26. Bova, M.P., Mchaourab, H.S., Han, Y. & Fung, B.K.K. (2000) Subunit exchange of small heat shock proteins – analysis of oligomer formation of aA-crystallin and Hsp27 by fluorescence resonance energy transfer and site-directed truncations. J. Biol. Chem. 275, 1035–1042. 27. Andley, U.P., Mathur, S., Griest, T.A. & Petrash, J.M. (1996) Cloning, expression and chaperone-like activity of human aA-crystallin. J. Biol. Chem. 271, 31973–31980. 28. Derham, B.K., van Boekel, M.A., Muchowski, P.J., Clark, J.I., Horwitz, J., Hepburne-Scott, H.W., de Jong, W.W., Crabbe, M.J. & Harding, J.J. (2001) Chaperone function of mutant versions of aA- and aB-crystallin prepared to pinpoint chaperone binding sites. Eur. J. Biochem. 268, 713–721. 29. Muchowski, P.J., Wu, G.J.S., Liang, J.J.N., Adman, E.T. & Clark, J.I. (1999) Site-directed mutations within the core Ôa-crys- tallinÕ domain of the small heat-shock protein, human aB-crys- tallin, decrease molecular chaperone functions. J. Mol. Biol. 289, 397–411. 30. Smulders, R.H., van Boekel, M.A.M. & de Jong, W.W. (1998) Mutations and modifications support a Ôpitted-flexiballÕ model for a-crystallin. Int. J. Biol. Macromol. 22, 187–196. 31. Plater, M.L., Goode, D. & Crabbe, M.J. (1996) Effects of site- directed mutations on the chaperone-like activity of aB-crystallin. J. Biol. Chem. 271, 28558–28566. 32. Merck, K.B., De Haard-Hoekman, W.A., Oude Essink, B.B., Bloemendal, H. & De Jong, W.W. (1992) Expression and aggregation of recombinant aA-crystallin and its two domains. Biochim. Biophys. Acta. 1130, 267–276. 33. Smulders, R.H., Carver, J.A., Lindner, R.A., van Boekel, M.A.M., Bloemendal, H. & de Jong, W.W. (1996) Immobilization of the C-terminal extension of bovine aA-crystallin reduces chaperone-like activity. J. Biol. Chem. 271, 29060–29066. 34. van Montfort, R.L., Basha, E., Friedrich, K.L., Slingsby, C. & Vierling, E. (2001) Crystal structure and assembly of a eukaryotic small heat shock protein. Nat. Struct. Biol. 8, 1025–1030. 35. Fernando, P. & Heikkila, J.J. (2000) Functional characterization of Xenopus small heat shock protein, Hsp30C: the carboxyl end is required for stability and chaperone activity. Cell Stress Chaper- ones 5, 148–159. 36.Kirschner,M.,Winkelhaus,S.,Thierfelder,J.M.&Nover,L. (2000) Transient expression and heat-stress-induced co-aggrega- tion of endogenous and heterologous small heat-stress proteins in tobacco protoplasts. Plant J. 24, 397–411. 37. Mu ¨ nchbach, M., Dainese, P., Staudenmann, W., Narberhaus, F. & James, P. (1999) Proteome analysis of heat shock protein expres- sion in Bradyrhizobium japonicum. Eur. J. Biochem. 264, 39–48. 38. Nocker, A., Krstulovic, N P., Perret, X. & Narberhaus, F. (2001) ROSE elements occur in disparate rhizobia and are functionally interchangeable between species. Arch. Microbiol. 176, 44–51. 39. Koteiche, H.A. & Mchaourab, H.S. (1999) Folding pattern of the a-crystallin domain in aA-crystallin determined by site-directed spin labeling. J. Mol. Biol. 294, 561–577. 40. Kappe ´ , G., Leunissen, J.A.M. & de Jong, W.W. (2002) Evolution and diversity of prokaryotic small heat shock proteins. Prog. Mol. Subcell. Biol. 28, 1–17. 41. Narberhaus, F. (2002) a-Crystallin-type heat shock proteins: Socializing minichaperones in the context of a multichaperone network. Microbiol. Mol. Biol. Rev. 66, 64–93. 42. van de Klundert, F., Smulders, R.H., Gijsen, M.L.J., Lindner, R.A., Jaenicke, R., Carver, J.A. & de Jong, W.W. (1998) The mammalian small heat-shock protein Hsp20 forms dimers and is a poor chaperone. Eur. J. Biochem. 258, 1014–1021. 43. Feil, I.K., Malfois, M., Hendle, J., van der Zandt, H. & Svergun, D.I. (2001) A novel quaternary structure of the dimeric a-crystallin domain with chaperone-like activity. J. Biol. Chem. 276, 12024– 12029. 44. Haslbeck,M.,Walke,S.,Stromer,T.,Ehrnsperger,M.,White, H.E., Chen, S., Saibil, H.R. & Buchner, J. (1999) Hsp26: a tem- perature-regulated chaperone. EMBO J. 18, 6744–6751. 45. Berengian, A.R., Parfenova, M. & Mchaourab, H.S. (1999) Site- directed spin labeling study of subunit interactions in the a-crys- tallin domain of small heat-shock proteins – comparison of the oligomer symmetry in aA-crystallin, HSP 27, and HSP 16.3. J. Biol. Chem. 274, 6305–6314. 46. Helm, K.W., Lee, G.J. & Vierling, E. (1997) Expression and native structure of cytosolic class II small heat-shock proteins. Plant Physiol. 114, 1477–1485. 47. Leroux, M.R., Melki, R., Gordon, B., Batelier, G. & Candido, E.P. (1997) Structure-function studies on small heat shock protein oligomeric assembly and interaction with unfolded polypeptides. J. Biol. Chem. 272, 24646–24656. 48. Thompson, J.D., Higgins, D.G. & Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. 49. Scha ¨ gger, H. & von Jagow, G. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368–379. 3586 S. Studer et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . A critical motif for oligomerization and chaperone activity of bacterial a- heat shock proteins Sonja Studer, Markus Obrist, Nicolas Lentze and Franz Narberhaus Institute of Microbiology, Eidgeno ¨ ssische. studies have mainly been focused on the mammalian representatives aA- and aB-crystallin. These proteins proved remarkably resistant against mutational alterations. For example, a wide variety of truncations. C-terminal regions and analyzed them by gel filtration, citrate synthase assay and coaffinity purification. Truncation of more than the initial few amino acids of the N-terminal region led to the formation of distinct