F. Narberhaus, Institute of Microbiology, ETH-Zentrum, Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland. Fax: + 41 1632 1148, Tel.: + 41 1632 2586, E-mail: firstname.lastname@example.org
Oligomerization into multimeric complexes is a prerequisite for the chaperone function of almost all α-crystallin type heat shock proteins (α-Hsp), but the molecular details of complex assembly are poorly understood. The α-Hsp proteins 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 α-Hsp functionality. This I-X-I motif is one of the characteristic consensus motifs of the α-Hsp family, and here we provide experimental evidence of its structural and functional importance. α-Hsp proteins lacking the C-terminal extension were inactive, but still able to form dimers. Here, we demonstrate that the central α-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.
Heat shock or other forms of stress induce the expression of α-heat shock proteins (α-Hsp proteins) in a broad range of prokaryotic and eukaryotic organisms [1–5]. α-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 α-Hsp proteins examined display chaperone activity in vitro, measured by their ability to protect model substrates from thermally or chemically induced aggregation [8–13].
α-Hsp proteins are named after their most prominent representative, α-crystallin, which prevents protein precipitation in the vertebrate eye lens. They are mostly referred to as small heat shock proteins (sHsp), because their monomeric 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 α-Hsp proteins. Moreover, native α-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 α-Hsp proteins is highly dynamic, which is often reflected by pronounced size heterogeneity or rapid subunit exchange . Mammalian members of the α-Hsp family are remarkably polydisperse [20,21], and some bacterial proteins such as Escherichia coli IbpB also display pronounced size heterogeneity . 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].
α-Hsp proteins are widely distributed but poorly conserved. One major characteristic of this protein family is the presence of a central α-crystallin domain, flanked by a N-terminal region and a C-terminal extension . The highest degree of amino-acid similarity is found within theα-crystallin domain, while the N-terminal region and the C-terminal extension are variable in length and sequence. Naturally occurring α-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 α-Hsp structure–function studies have mainly been focused on the mammalian representatives αA- and αB-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 α-Hsp proteins were reported to affect chaperone activity  or complex formation [26,32].
The C-terminal extension, being located on the outer surface of the α-Hsp oligomers, is generally assumed to contribute to complex solubility , but its further functional implication remains unclear. The recently resolved crystal structures of a plant and an archaeal α-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 striking feature of the poorly conserved C-terminal extension is the presence of an isoleucine-X-isoleucine (I-X-I) motif in the majority of α-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 α-Hsp family . 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 α-crystallin and other vertebrate α-Hsp proteins [27,31,33,35], but only in the case of one plant α-Hsp were C-terminal truncations observed to reduce complex size .
Altogether, the oligomerization principles of α-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 α-Hsp proteins [4,13,37]. It contains at least 10 α-Hsp proteins, which are highly induced upon heat shock . The sequences of seven B. japonicum α-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 . 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 α-Hsp proteins of the same class .
The present study demonstrates that chaperone activity of B. japonicumα-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 α-Hsp proteins, and that the isolated α-crystallin domain is unable to dimerize.
Plasmids for the expression of B. japonicum hspF (pRJ5306) and hspH (pRJ5307) provided with a C-terminal His6 tag have been described previously . 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 BsaI on hspF and SfiI on hspH. PCR products were digested with either BsaI or SfiI and NdeI (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 α-crystallin domain or the α-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™ 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 α-Hsp versions carrying a His6 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 NdeI and XhoI and ligated into a pET24b vector digested with the same endonucleases. The correct nucleotide 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 °C to D600 = 0.6, induced by addition of isopropyl thio-β-d-galactoside to a final concentration of 0.5 mm, and grown for a further 2–3 h. After harvesting, cells were resuspended in binding buffer (500 mm KCl, 20 mm Tris/HCl, 5 mm imidazole, pH 7.9) containing 1 mm phenylmethanesulfonyl fluoride and 10 µg·mL−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 . The column was pre-equilibrated with binding buffer and then washed with washing buffer (500 mm KCl, 20 mm Tris/HCl, pH 7.9) containing increasing imidazole concentrations (5–50 mm). The imidazole concentration was finally raised to 250 mm in order to elute bound proteins. If protein purity was not satisfactory, the eluate was diluted to an imidazole concentration below 50 mm and applied to a second column, either Ni-nitrilotriacetic acid/agarose (Qiagen) 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. α-Hsp proteins were preincubated at 43 °C in 1 mL of 50 mm sodium phosphate, pH 6.8, for at least 15 min. The assay was started by addition of CS to a final concentration of 600 nm. 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 mm Tris/HCl, 1 mm EDTA, pH 8.0). For each protein, chaperone activity assays were performed at least twice with preparations from independent purifications.
Analytical size exclusion chromatography of purified proteins 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 mm KCl, 20 mm Tris/HCl, 250 mm imidazole, pH 7.9), 200-µL protein samples were injected and separated at a flow rate of 0.6 mL·min−1. Absorbance was recorded at 280 nm. The following standards were used 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.
Copurification of untagged α-Hsp proteins with His6-tagged variants by Ni-nitrilotriacetic acid affinity chromatography was used to investigate protein–protein interactions of certain truncated α-Hsp proteins. The applied denaturation–renaturation protocol has been described previously .
Construction of HspH and HspF derivatives for structure-function studies
In order to study the principles of α-Hsp assembly and the relation between complex formation and chaperone activity, we constructed a series of truncated and point-mutated α-Hsp variants. A total of 19 α-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 α-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 (Δ3N), HspH(Δ9N), HspH(Δ15N), HspH(Δ20N), and HspF(Δ5N), HspF(Δ30N), HspF(Δ40N). In HspH(Δ20N) and HspF(Δ40N), approximately half of the N-terminal region was eliminated, including a proline and an arginine residue conserved in all seven B. japonicumα-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. coliα-Hsp proteins. To investigate its role in oligomerization, we constructed two α-Hsp derivatives lacking one [HspF(Δ5C)] or both [HspH(Δ20C)] isoleucine residues. Two further constructs, HspH(Δ5C) and HspH(Δ15C), 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, consisting of the α-crystallin domain alone [HspH(α)], the α-crystallin domain plus the N-terminal region [HspH(Nα)], or the α-crystallin domain followed by the C-terminal extension [HspH(αC)], were analyzed.
Alterations in the N-terminal region affect chaperone activity and oligomerization
As demonstrated previously , full-length class A and class B α-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 (; 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(Δ3N) and HspF(Δ5N) could not be distinguished 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 α-Hsp complexes.
Removal of additional amino acids, however, drastically altered the characteristics of either protein. HspH(Δ9N), HspH(Δ15N) and HspH(Δ20N) 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(Δ3N)], a considerable portion of the protein eluted after 12–13 min in the void volume of the column, reflecting the tendency of all B. japonicumα-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(Δ30N) and HspF(Δ40N) suggested that both proteins were only dimers (Fig. 3F,G), whereas HspH derivatives devoid of the first half of the N-terminal region [HspH(Δ20N)] appeared as octameric complexes. In either case, it is evident that a largely intact N-terminal region isstrictly required for the formation of active α-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).
Two conserved isoleucines are essential for α-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(Δ5C) and HspH(Δ15C), 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 assembled into large complexes (Fig. 5A,B). But while HspH(Δ5C) appeared similar to full-length HspH in terms of oligomer formation, HspH(Δ15C) formed larger aggregates with an apparent molecular mass exceeding 2 MDa. All C-terminally truncated α-Hsp derivatives exhibited an increased tendency to precipitate.
Elimination of the entire I-X-I motif in HspH(Δ20C) 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(Δ5C) was sufficient to completely abolish chaperone activity (Fig. 4D) and severely impaired oligomer formation (Fig. 5D). The apparent molecular mass of 44–48 kDa probably represents 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 α-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 α-Hsp region responsible for dimerization, we initially attempted to purify the isolated α-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 His6-tagged HspH variants, i.e. HspH(α)–His6, HspH(Nα)–His6, HspH(αC)–His6 and HspH(HΔ20N)–His6 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 His6-tagged species was indicative of protein–protein interactions, whereas proteins that did not interact with the His6-tagged species were not retained on the column .
Untagged HspH did not coelute with the His6-tagged α-crystallin domain (Fig. 6A), suggesting that the isolated α-crystallin domain was unable to dimerize. The α-crystallin domain carrying the C-terminal extension appeared to enable weak interactions with native HspH, because after copurification of HspH(αC)–His6 with HspH, a faint band corresponding to HspH was visible on SDS gels. In contrast, HspH(Nα)–His6, an HspH derivative consisting of the α-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(Δ20N)–His6. This suggests that the α–Hsp interaction is in part mediated by a portion of the N-terminal region in vicinity of the α-crystallin domain.
To ensure that efficient dimerization requires solely α-crystallin domain and N-terminal region and not the C-terminal extension, HspH(Nα)–His6 was also subjected to a copurification assay with untagged HspH(Nα). The latter protein alone did not bind to the Ni-nitrilotriacetic acid resin (Fig. 6B, lane 5). However, it co-eluted with HspH(Nα)–His6 (Fig. 6B, lane 4). Size exclusion chromatography confirmed that HspH(Nα) was present as small, presumably dimeric species, which were, as expected, devoid of chaperone 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.
Up to now, systematic α-Hsp structure–function studies have almost exclusively focused on α-crystallins from higher organisms. Introduction of mutations often had little impact on oligomerization and chaperone activity, as mammalian α-Hsp proteins are very resistant to mutational changes owing to their intrinsic plasticity [20,26–30,39]. In comparison with mammalian α-crystallins, the oligomerization principles of bacterial α-Hsp proteins have received little attention. Available data indicate that α-Hsp proteins from prokaryotes and plants may differ significantly from their mammalian counterparts in structure and function [40,41]. Some prokaryotic α-Hsp proteins possess remarkably rigid structures [11,14] that render them more susceptible to point mutations and truncations. We therefore chose two distantly-related bacterial α-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  and confirmed with purified proteins in the present study. Transmission electron microscopical analysis of both proteins revealed roughly spherical structures (data not shown) similar to those observed for other members of the α-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. Interestingly, 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 α-Hsp complexes. Even complexes that exceed the size of the wild-type protein, e.g. HspHΔ15C, retained chaperone activity. Much like our α-Hsp variants, naturally occurring multimerization-incompetent α-Hsp proteins have poor or lacking chaperone activity [24,25,42]. The chaperone activity of mammalian α-crystallins, on the other hand, appears to be much more resistant against structural modifications. Human αB-crystallin does not require a multimeric α-Hsp complex for chaperone activity . The functional, substrate-binding entity might actually be an α-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 α-crystallin domain. Crystallographic data from M. jannaschii Hsp16.5  and wheat Hsp16.9 , as well as spin labeling studies with αA-crystallin and Hsp27  indicate that the dimeric building block of many α-Hsp proteins is formed by interacting β strands in the α-crystallin domain.
Although this may also apply to the B. japonicumα-Hsp proteins, the isolated α-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 α-crystallin domain to dimerize. It is conceivable that the α-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 β strand overlaps the junction of N-terminal region and α-crystallin domain of M. jannaschii Hsp16.5 . However, even the addition of four N-terminal amino acids to the deduced α-crystallin domain of HspH (Fig. 1) in order to avoid disruption of a predicted β strand was not sufficient for dimer formation. This argues that more residues towards the N-terminus are involved in dimerization.
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 more drastically with oligomerization than comparable alterations in HspH (compare Fig. 3D,F). In line with clearly deviating N-terminal sequences (Fig. 1), oligomerization of HspH and HspF might be mediated by different interacting regions. Such distinctions most likely explain why α-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 . In α-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 α-Hsp proteins may differ from their counterparts in prokaryotes and plants. The crystal structure of wheat Hsp16.9 demonstrates that the N-terminal region of this protein is involved in numerous subunit contacts . It is puzzling that only every other N-terminal region in the Hsp16.9 complex butall N-termini in M. jannaschii Hsp16.5 are highly disordered [14,34]. Possibly, the N-terminal regions of B. japonicumα-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. Nevertheless, 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 β strand in the α-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 α-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, alterations outside of the I/V-X-I/V motif did not impair oligomer formation of vertebrate α-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 . In αA-crystallin, C-terminal truncations touching the I/V-X-I/V motif did not inhibit multimerization, but reduced chaperone activity . Only in one particular plant α-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 . 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 substantiate that oligomerization of prokaryotic α-Hsp proteins is a complicated multistep process that differs from the assembly of eukaryotic α-Hsp proteins and merits further investigation.
We thank Hauke Hennecke for support and encouragement. This study was supported by a grant from the Swiss National Foundation.