INTRODUCTION

Molecular chaperones belong to a class of proteins whose function is to interact with and stabilize proteins that are partially or totally unfolded, as is the case when proteins are in the process of being synthesized, translocated across a membrane, or damaged by conditions of cellular stress. Many chaperones are expressed at higher levels during biological stresses, and are members of heat shock protein (HSP)¹ families (1-4). Whereas some chaperones (HSP70, HSP40, and HSP60) are involved in protein folding under normal conditions in vivo (5-7), others such as HSP104 (8-10), inducible HSP70s (11), and small HSPs (12-15) are known to play important roles in protecting organisms from stress.

The small HSPs (smHSPs) form a structurally divergent protein family with members present in Archaea, Bacteria, and Eukarya (16, 17). The presence of an evolutionarily conserved -crystallin domain distinguishes all smHSPs and -crystallins (18-20). This domain is preceded by an N-terminal domain, which is highly variable in size and sequence, and is followed by a short, poorly conserved C-terminal extension. Some smHSP genes contain an intron which delineates the N-terminal and -crystallin domains (21, 22), and structural studies support a two-domain structure (20, 23, 24) consisting mostly of -sheets (25, 26) for smHSPs. The C-terminal extensions of smHSPs appear relatively unstructured (27, 28) and are known to undergo numerous modifications, including truncations (reviewed in Ref. 29).

A common feature of smHSPs is their formation of large oligomeric complexes. The simplest and best characterized smHSP quaternary structure is the 150-kDa trimer of trimers formed by Mycobacterium tuberculosis HSP16.3 (30). Plant smHSPs assemble into complexes of 200-300 kDa (31), and the typical oligomeric size of -crystallins and smHSPs from yeast and mammals is between 400 and 800 kDa (29, 32). Several contrasting models have been proposed to account for the arrangement of subunits within the smHSP aggregate. The review by Groenen et al. (29) addresses this issue in detail, and suggests that although there is as yet no single consistent model for smHSP quaternary structure, it is likely that the smHSP N-terminal domain is buried in the aggregate whereas the C-terminal domain is exposed at the surface, and that all subunits are in equivalent positions within a roughly spherical aggregate.

Numerous in vitro studies have documented the ability of smHSPs to prevent the aggregation of various test proteins at elevated temperatures (26, 33-35). In the cell, cytoskeletal elements, which are particularly sensitive to cellular stresses (36), appear to be substrates for smHSPs. Specific binding of smHSPs to actin and the intermediate filaments desmin, vimentin, and glial fibrillary acidic protein have been demonstrated (37-40). HSP27 has been implicated in regulating the dynamics of actin filaments (41, 42), and -crystallin participates in intermediate filament assembly (40). Furthermore, HSP27 and -crystallin can enhance the survival of cells subjected to heat shock or oxidative stresses by conferring increased stability to actin fibers (13, 43-45).

Although many smHSPs are present during development under physiological conditions, smHSPs are among the most highly inducible HSPs during heat shock or other stresses (15). Four of over 15 smHSP genes present in the nematode Caenorhabditis elegans (hsp16-1, hsp16-2, hsp16-41, and hsp16-48) have been shown to be expressed only under stress conditions, in a tissue general manner (46-49). More significantly, their expression correlates specifically with the presence of agents which induce protein damage (50).

In an effort to understand the oligomeric assembly and chaperone activity of smHSPs in general, studies on the structure and function of the 16-kDa C. elegans HSP16-2 wild-type protein and various derivatives were undertaken. We show that binding to unfolded protein and chaperone activity is strictly correlated with the ability of the smHSP to form multimers. The nonconserved N-terminal region is buried within the smHSP complex and is required for subunit assembly, whereas most of the C-terminal extension is not required for multimerization or chaperone activity, but is possibly involved in maintaining the solubility of the complex. Investigations on the binding of wild-type HSP16-2 and derivatives to the cytoskeletal elements actin and tubulin show that smHSPs exhibit little or no substrate specificity and have a high affinity for unfolded, but not for aggregated or native polypeptides. The conclusions from our in vitro mutagenesis studies on HSP16-2 are supported by data from an unusually diminutive (12.6 kDa), naturally-occurring smHSP from C. elegans (51).

EXPERIMENTAL PROCEDURES

Porcine heart citrate synthase (CS), bovine serum albumin (BSA), bovine IgG, PIPES, EGTA, and 5-[(4,6-dichlorotriazin-2-yl)amino]-fluorescein (DTAF) were from Sigma. ATP, ADP, GTP, and 1,4-dithiothreitol (DTT) were from Boehringer. MES was purchased from Calbiochem. Pyrene iodoacetamide and 7-chloro-4-nitrobenzene-2-oxa-1,3-diazole (NBD) came from Molecular Probes. Bis(sulfosuccinimidyl)suberate (BS³) was purchased from Pierce. Urea and guanidine hydrochloride were obtained from Aldrich Biochemicals. L-[³⁵S]Methionine was purchased from Amersham. All other chemicals used were analytical grade.

The hsp16-2 coding region had previously been subcloned into the pRSET A expression vector (Invitrogen) at the BamHI-EcoRI site (50) for producing H₆HSP16-2 (i.e. wild-type HSP16-2 fused to an N-terminal 4-kDa polyhistidine-containing tag). A construct designed to express wild-type HSP16-2 was prepared by excising the NdeI-BamHI fragment containing the polyhistidine tag from this plasmid, blunting the ends, and religating. To prepare the vector encoding H₆1-15 HSP16-2 (a tagged HSP16-2 lacking the first 15 amino acids), the hsp16-2-containing clone was amplified using polymerase chain reaction and primers 5-GACTCGAGGTGATCTTATGAG and 5-CGATGAATTCGTTATTCAGCAGATTTCTCTTCGAC (XhoI and EcoRI restriction sites underlined); the XhoI-EcoRI-restricted polymerase chain reaction product was subcloned into pRSET B (XhoI-EcoRI). The vector encoding H₆130-145 HSP16-2 (like H₆HSP16-2 but missing the last 16 amino acids) was generated by subcloning the polymerase chain reaction product amplified with primers 5-GCTGAATTCTTATCCTTGAACCGCTTCTTTC and 5-GCGTGGATCCATGTCACTTTACCACTATTTC into pRSET A (BamHI-EcoRI). The constructs designed to encode H₆1-32 and H₆1-44 HSP16-2 were prepared by subcloning the PstI-EcoRI fragment of the pH₆HSP16-2 vector into pRSET B (PstI-EcoRI), and the HpaI-HindIII fragment of the same vector into pRSET B (PvuII-HindIII), respectively. The hsp12.6 coding region was amplified from first-strand cDNA and subcloned into the BglII-HindIII site of a modified pRSET A lacking the polyhistidine-containing NdeI-BamHI fragment (51).

The expression constructs were transformed into the BL21(DE3) Escherichia coli strain, grown to an OD₆₀₀ of 0.8-1.0, induced with 1 mM isopropyl-1-thio--D-galactopyranoside, and harvested 3 h later (52). All HSP16-2 proteins formed insoluble inclusion bodies which were solubilized in TEND buffer (50 mM Tris-HCl, 0.1 mM EDTA, 50 mM NaCl, 1 mM DTT, pH 7.5) supplemented with 8 M urea. HSP16-2 was first purified by size-exclusion chromatography over Sephacryl S-100 in TEND, 4 M urea. The peak fractions were then dialyzed against TEND buffer, and the native complex was purified by separation on Sephacryl S300-HR in TEND buffer. The polyhistidine-containing proteins were purified by Ni²⁺-chelate affinity chromatography in 8 M urea as described by the manufacturer (Qiagen), and then dialyzed in TEND buffer. HSP12.6 was produced in E. coli as a soluble protein. Cells resuspended in TEND buffer supplemented with protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml pepstatin A, 10 µg/ml leupeptin, 2 mM EDTA) were broken in a French press, and the clarified supernatant was chromatographed on Sephacryl S200-HR in TEND buffer. Peak fractions containing HSP12.6 were further purified by chromatography on a hydroxylapatite support. Purification steps were carried out on ice or at 4 °C. All smHSPs were stored at 70 °C after purification (and dialysis) except for H₆130-145 HSP16-2 which was only dialyzed before use because of its insolubility following freeze-thawing.

To estimate the size of the native smHSP complexes, approximately 5 mg of purified protein was subjected to SEC over Sephacryl S-300HR. The column was first calibrated with the following molecular mass standards: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), and RNase A (14 kDa). The proteins were chromatographed on the column in TEND buffer at room temperature. Samples were separated on SDS gels and the elution volume of each was used to estimate the molecular weight.

Sedimentation velocity experiments were carried out with a Beckman Optima XL-A analytical ultracentrifuge equipped with an AN 60Ti four-hole rotor and cells with two-channel 12-mm path length centerpieces. Sample volumes of 400 µl were centrifuged at 60,000 rpm. Radial scans of absorbance at 278 nm were taken at 10-min intervals. Data were analyzed to provide the apparent distribution of sedimentation coefficients using the programs DCDT (53) and SVEDBERG (54). The partial specific volumes, , at 20 °C, were calculated from the amino acid compositions and the solvent density was 1.00 g/cm³. The degrees of hydration of the totally unfolded proteins was estimated based on the amino acid compositions by the method of Kuntz (55) according to Laue et al. (56). The degrees of hydration used for all calculations, 0.316, 0.314, and 0.337 g of H₂O per g of protein, for wild-type HSP16-2, H₆130-145 HSP16-2, and H₆1-44 HSP16-2, respectively, were the result of correcting the calculated degrees of hydration by a factor 0.7 obtained by comparing degrees of hydration for several proteins in their folded state to that based on their amino acid composition (57).

Cross-linking reactions containing 1.5 µM smHSP monomers, 15 µM BSA, and 2 mM freshly prepared BS³ were carried out in cross-linking buffer (25 mM MES, pH 7.5, 25 mM NaCl, 0.5 mM DTT) for 30 min at room temperature and analyzed by Western blotting using an antibody against HSP16-2 (50).

H₆HSP16-2 was purified and dialyzed against NP buffer (50 mM NaCl, 50 mM NaPO₄, pH 8.0). Two samples containing 175 µg of H₆HSP16-2 were prepared, one in NP buffer (sample N for "native") and the other in NP buffer containing 6 M urea (sample D for "denatured"). A small amount of Ni²⁺-chelate affinity resin (30 µl) was added to samples N and D. The samples were mixed for 15 min, then washed twice with NP buffer (without urea for sample N and with 6 M urea for sample D, both at pH 6.3), and any bound protein was eluted with 100 mM EDTA. Aliquots of the pH 6.3 and EDTA washes of sample N and the EDTA washes of sample D were analyzed on a 12% SDS gel.

The thermally-induced aggregation of a 900-µl citrate synthase solution (150 nM monomers), with smHSPs or control proteins, was measured by light scattering at 320 nm in a Cary 210 Varian spectrophotometer equipped with a thermostatted cell compartment preheated to 45 °C. The effect of HSP16-2 on the chemically-induced aggregation of CS was measured in an SPF-500C^TM stirred-cell spectrofluorometer with excitation and emission wavelengths set to 500 nm and a band pass of 2 nm. CS was denatured in 6 M guanidinium chloride for at least 45 min and diluted 100-fold in a total volume of 2 ml to give a final monomeric concentration of 200 nM. All aggregation experiments were carried out in 50 mM NaPO₄ buffer, pH 7.5, and normalized to control experiments in which aggregation with CS alone was defined as being 100% of total CS aggregation.

The kinetics of HSP16-2·actin and HSP16-2·tubulin binary complex formation were measured by diluting ³⁵S-labeled 7.5 M urea-denatured -actin or -tubulin (prepared as described in Ref. 58) 100-fold (final actin and tubulin concentrations were approximately 225 nM) into a solution containing 1.6 µM HSP16-2 complex and mixing quickly. Appropriate dilutions of HSP16-2 were prepared using "refolding buffer" (25 mM MES, pH 7.5, 25 mM NaCl, and 0.5 mM DTT). At each time point, 20 µl of the reaction mixture was mixed with 5 µl of glycerol and frozen immediately in an ethanol/dry ice bath. Samples were then separated on 4.5% nondenaturing gels (58). To measure the binding of HSP16-2 actin and tubulin intermediates formed along the aggregation pathway, ³⁵S-labeled denatured actin or tubulin was first diluted into refolding buffer and incubated for various lengths of time. At each time point, an aliquot was added to a tube containing HSP16-2 such that the concentration of HSP16-2 oligomers was 1.6 µM and that of actin/tubulin approximately 225 nM. The binding was allowed to proceed for 20 min at room temperature, and the reactions were analyzed on nondenaturing polyacrylamide gels. The yield of binary complex was quantitated using a PhosphorImager.

Actin was purified from rabbit muscle acetone powder (59, 60) and isolated as CaATP-globular (G)-actin by chromatography through Sephadex G-200 (61) at 4 °C equilibrated in 5 mM Tris-HCl, pH 7.8, 0.2 mM DTT, 0.2 mM ATP, 0.1 mM CaCl₂, 0.01% NaN₃. G-actin (50 µM) was stored on ice and used within 2 weeks. Pyrenyl-labeled actin and NBD-labeled actin were prepared according to Kouyama and Mihashi (62) and Detmers et al. (63), respectively. Pure tubulin was prepared from fresh pig brain by three assembly-disassembly cycles (64) followed by phosphocellulose (Whatman P11) chromatography (65). Tubulin (100-150 µM) in 0.1 M PIPES, pH 6.9, 1 mM EGTA, 0.5 mM MgCl₂ and 1 mM GTP was flash frozen in liquid nitrogen and stored at 80 °C. DTAF-labeled tubulin was prepared as described by Mejillano and Himes (66). Native pyrenyl- or NBD-actin and DTAF-tubulin were denatured by addition of urea to a final concentration of 7.5 M.

All measurements were made at 20 °C in a Spex fluorolog 2 spectrofluorometer. The excitation monochromator was set at 345, 468, and 492 nm and the emission recorded at 386, 535, and 517 nm for pyrenyl-actin, NBD-actin, and DTAF-tubulin, respectively.

SmHSP protein concentrations were determined using the Bio-Rad protein assay kit and bovine IgG as standard. The concentration of CS was determined by absorbance at 280 nm using an extinction coefficient of 1.55 × 10⁵ M¹ cm¹ (67). The concentration of HSP16-2 and H₆130-145 complexes were calculated according to the average of the two molecular masses obtained by sedimentation velocity measurements (317 and 421 kDa, respectively). All other smHSP concentrations were based on the calculated molecular weights. Actin and tubulin protein concentrations were determined by the Lowry method (68). Molecular weights of 42,000 and 100,000 were employed for actin (69) and tubulin (70), respectively.

RESULTS

Production of HSP16-2 Variants for Structure-Function Studies

Although the sizes of smHSPs vary considerably, a detailed sequence comparison of known members suggests that the minimal functional unit consists of a core region of about 85 amino acids (the -crystallin domain) which is flanked by an N-terminal region of at least 39 residues (in E. coli IbpA), and a C-terminal extension of at least 12 residues (in Neurospora crassa HSP30). Accordingly, the most compact smHSP characterized to date is E. coli IbpA, which has a very short C terminus of 14 residues and totals 137 amino acids (15.8 kDa). A novel family of 12.2-12.6 kDa smHSPs from C. elegans represents the only known exception to the apparent minimum size restriction of smHSPs; interestingly, one of these proteins, HSP12.6, was shown to lack the typical quaternary structural and functional characteristics of smHSPs (51). C. elegans HSP16-2 (145 amino acids) is only slightly larger than IbpA, and is therefore a good model smHSP for delineating the structural requirements for smHSP oligomerization and chaperone activity in these proteins. Expression constructs harboring wild-type HSP16-2 and six derivatives were created (Fig. 1A). All derivatives have a 4-kDa N-terminal tag which contains a polyhistidine sequence (H₆). H₆HSP16-2 represents wild-type HSP16-2 with an N-terminal H₆ fusion. H₆1-15, H₆1-32, and H₆1-44 HSP16-2 derivatives have approximately one-third, two-thirds, and all of the N-terminal domain of HSP16-2 deleted, respectively. Lastly, H₆130-145 HSP16-2 is equivalent to H₆HSP16-2 with the last 16 C-terminal residues removed. All smHSPs were produced in E. coli and purified to 90% homogeneity (Fig. 1B). An alignment of C. elegans HSP16-2 and HSP12.6, as well as other protein sequences from various organisms (Fig. 1C), illustrates the three distinct regions found in smHSPs, and depicts the regions of the HSP16-2 derivatives which are truncated relative to wild-type HSP16-2, HSP12.6, and other smHSPs.

HSP16-2 Quaternary Structure and Subunit Orientation

We first estimated the size of recombinant wild-type HSP16-2 by SEC on a calibrated Sephacryl S-300HR column. The majority of HSP16-2 elutes as a single peak between the molecular weight markers thyroglobulin (669 kDa) and ferritin (440 kDa), and has an apparent molecular mass of 550 kDa (Fig. 2A). The estimated size of the complex is essentially identical to that of HSP16-2 isolated from a heat-shocked nematode extract (71). Interestingly, the H₆HSP16-2 protein also assembles into large oligomeric complexes of somewhat larger size (680 kDa), as judged by SEC (Fig. 2B).

Sedimentation velocity measurements confirm the oligomeric nature of HSP16-2. Raw sedimentation data were fitted by nonlinear least-squares procedures as described by Philo (54). The data fitted relatively well to a two-component system involving a 10.5 S and a 14.7 S species with proportions of about 58 and 42%, respectively, at an HSP16-2 concentration of 1.0 mg/ml. Using the relation (s₁/s₂)³ = (M₁/M₂)² and bovine serum albumin as a reference (57), we obtained apparent M_r values of 239,470 and 394,920 for the two species, consistent with the behavior of wild-type HSP16-2 as two populations of oligomers consisting of approximately 14 and 24 subunits. The frictional ratio values (f/f₀) suggest that HSP16-2 is slightly asymmetrical. The above sedimentation velocity data are summarized in Table I.

Table I. Hydrodynamic parameters of wild-type HSP16-2 and two truncated forms The conformational parameters were calculated as described under "Materials and Methods," using the molecular mass and the partial specific volume () values determined from the amino acid composition. Experimental points representing the distribution of each molecular form were decomposed into Gaussian curves. The surface of each Gaussian is proportional to the concentration of the corresponding oligomeric form. Numerical values for the n-mer of each species were obtained by dividing the experimental molecular mass of each species by the corresponding theoretical molecular mass. s0 20,w is the sedimentation coefficient; f and f0 are the frictional coefficients and Rs the Stokes radius. Molecular mass   s0 20,w f/f0 Rs n-mer Relative abundance Da cm3 g1 S m % Theoretical   Wild type HSP16-2 16,232 0.735 Experimental   Wild type HSP16-2     Form 1 239,470 0.735 10.48 1.10 4.82  × 109 14-15 58     Form 2 394,920 0.735 14.47 1.08 5.65  × 109 24-25 42 Theoretical   H6130-145   HSP16-2 18,580 0.723 Experimental   H6130-145   HSP16-2     Form 1 322,395 0.723 12.8 1.16 5.29  × 109 17-18 49     Form 2 520,385 0.723 17.6 1.16 6.20  × 109 28 51 Theoretical   H61-44   HSP16-2 15,227 0.723 Experimental   H61-44   HSP16-2     Form 1 14,990 0.723 1.66 1.14 1.92  × 109 1 95     Form 2 56,235 0.723 4.0 1.15 2.99  × 109 3-4 5

Since the H₆HSP16-2 derivative also forms an aggregate, we determined whether its polyhistidine-containing tag was exposed. Aliquots of native or urea-denatured H₆HSP16-2 were incubated with nickel-chelate affinity resin, and after washing the resin to remove nonspecifically adsorbed proteins, specifically bound protein was eluted with 100 mM EDTA. No detectable binding of the native H₆HSP16-2 to the nickel resin was seen, whereas the unfolded H₆HSP16-2, with its exposed H₆ tag, bound efficiently (Fig. 3). This suggests that the N-termi-nal region of wild-type HSP16-2 is sequestered within the interior of the native complex.

HSP16-2 Interacts with Unfolded Citrate Synthase and Prevents Its Aggregation

To confirm that HSP16-2 possesses the chaperone activity of typical smHSPs, its effect on the aggregation of CS was studied. The denaturation and renaturation of CS has been well studied (72), and CS has been used in chaperone studies with HSP90 and associated proteins (73-75), GroEL (76), and smHSPs (30, 34, 35). When CS is incubated alone at 40-45 °C it aggregates irreversibly, as detected by light scattering at 320 nm, and loses activity. Incubation of CS with increasing amounts of HSP16-2 results in a concomitant decrease in CS aggregation (Fig. 4A). At molar ratios of HSP16-2 complex to CS monomer of 1:1 or higher, aggregation of CS is almost completely inhibited (summarized in Fig. 4B). The H₆HSP16-2 variant is also equally effective in preventing CS aggregation (51). When HSP16-2 was added to a solution of CS which had aggregated for 15 min, further aggregation was prevented but existing CS aggregates were not re-solubilized (not shown). This behavior of HSP16-2 parallels that of bovine -crystallin and M. tuberculosis HSP16.3 (30, 77).

The aggregation of proteins at high temperatures may involve unfolded intermediates which are different from those generated when the proteins are diluted into buffer from chaotropes such as urea or guanidine hydrochloride. We therefore examined the effect of HSP16-2 on the aggregation of 6 M guanidine hydrochloride-denatured CS which occurs upon 100-fold dilution of the chaotrope. It was found that HSP16-2 interacts with and stabilizes CS diluted from denaturant essentially as efficiently as it prevents the thermally-induced aggregation of CS (Fig. 4C).

HSP16-2 Forms Stable Binary Complexes with Denatured Actin and Tubulin

Although smHSPs have been shown to bind to and stabilize actin filaments (38, 41, 45), detailed studies on the nature of the interaction between smHSPs and actin have not been reported. To further explore this property of smHSPs, we first investigated the formation of binary complexes between HSP16-2 and unfolded -actin using a gel mobility assay. Fig. 5A shows reactions in which urea-denatured ³⁵S-labeled actin was diluted 100-fold into a solution with (lanes 1-4) or without (lanes 5 and 6) HSP16-2 or with HSP16-2 and a 2-fold molar excess of native BSA (lane 7), and separated on a nondenaturing gel. The denatured actin in reaction 4 was first incubated for 150 min before it was mixed with HSP16-2. The top panel of Fig. 5A shows the Coomassie Blue-stained native gel (with the positions of the HSP16-2 complex and BSA indicated), and the bottom panel represents an autoradiogram of the same gel. In lanes 1-3, the binary (HSP16-2·actin) complex is visible, whereas if the actin is allowed to aggregate for 150 min before HSP16-2 is added, essentially no binary complex is detectable (lane 4). Reactions in which actin was incubated in the absence of HSP16-2 show only a trace of actin aggregates (lanes 5 and 6). The presence of a 2-fold molar excess of BSA has no effect on binary complex formation between HSP16-2 and denatured actin (lane 7), indicating that HSP16-2 but not BSA binds to the unfolded polypeptide.

Binary complex formation between HSP16-2 and unfolded -actin and -tubulin.

A time course of binary complex formation between HSP16-2 and unfolded actin (Fig. 5B) reveals that approximately 70% of the binary complexes are formed within the first minute of incubation. Interestingly, identical kinetic experiments performed with HSP16-2 and labeled, denatured -tubulin gave very similar results (Fig. 5C). This demonstrates that HSP16-2 has similar affinities for unfolded actin and tubulin, despite the fact that these proteins share no sequence homology. Although the binding of HSP16-2 to denatured actin is initially very rapid, it then proceeds with observable kinetics until a plateau is reached after about 20 min. The slower phase of binary complex formation may indicate that the actin intermediates formed at later times are less well recognized by the smHSP or that the available binding sites become progressively saturated.

In light of the above results, the ability of HSP16-2 to interact with actin and tubulin intermediates formed along the aggregation pathway was investigated. These intermediates are distinct from the molten-globule intermediates formed by non-cytoskeletal proteins in that they form relatively slowly (58). A constant amount of HSP16-2 was added to a reaction mixture containing actin which had been incubated alone in buffer for varying amounts of time. The binding reactions were allowed to proceed for 20 min, and then were analyzed on a nondenaturing gel (Fig. 5D). Under these conditions, the yield of binary complex formed decreases to almost zero over time. After a brief 2-min preincubation of the actin alone, a significant proportion (about 25%) of the actin fails to form a complex with HSP16-2, and after a 5-min preincubation, only approximately 50% of the actin is found in a binary complex, relative to the amount of actin bound to HSP16-2 in the absence of a preincubation. The same experiment with HSP16-2 and -tubulin gave almost identical results (Fig. 5E). It is noteworthy that the chaperonins HSP60 and CCT also have a decreasing ability to recognize actin intermediates which form during aggregation over the same time period (58). Overall, these studies suggest that HSP16-2 has a distinctly greater affinity for unfolded actin and tubulin intermediates which occur early on the aggregation pathway, but displays little protein specificity.

The affinity of HSP16-2 for denatured and native forms of actin and tubulin was further examined using fluorescently-labeled derivatives. Addition of increasing amounts of HSP16-2 to denatured pyrenyl-actin and DTAF-tubulin resulted in a quenching of the fluorescence of these proteins (Fig. 6, A and B). These data are well fitted on the simplifying assumption that HSP16-2 binds to labeled, denatured actin and tubulin with a single association constant of 2.8 × 10⁶ M¹. The stoichiometry of binding between the unfolded protein and the HSP16-2 complex is 1:1. In contrast, addition of up to a 5 M excess of HSP16-2 complex to the native pyrenyl- or NBD-labeled actin and DTAF-tubulin did not result in a quenching of the fluorescence of the labeled proteins. This further demonstrates that HSP16-2 has an equally high affinity for unfolded actin and tubulin, and also reveals that HSP16-2 has little or no affinity for the same native proteins.

In Vitro Mutagenesis of HSP16-2

The C-terminal extensions of smHSPs are variable in size and sequence, but often contain a region of limited similarity, having the motif (R/K)X(I/V)X(I/V) (Fig. 1C). We therefore sought to determine whether the C-terminal extension of HSP16-2 (including the conserved motif RSIPI) plays a structural or functional role by studying H₆130-145 HSP16-2. Analysis of H₆130-145 HSP16-2 by SEC reveals that like H₆HSP16-2, the N-terminally-tagged variant forms a somewhat larger aggregate than wild-type HSP16-2, with an estimated M_r of 675,000 (Fig. 2B). This was confirmed by sedimentation velocity measurements (Table I), which as is the case for the wild-type protein, show that H₆130-145 HSP16-2 exists as two high M_r species. The M_r values are 322,395 and 520,385, consistent with oligomeric complexes of approximately 17 and 28 subunits.

We then tested the ability of H₆130-145 HSP16-2 to protect CS from aggregating at an elevated temperature (Fig. 7). Surprisingly, removal of the C-terminal extension has little or no influence on the chaperone function of HSP16-2: H₆130-145 HSP16-2 is as effective as the wild-type smHSP in suppressing CS aggregation (compare with Fig. 4B). Confirmation that this derivative interacts with unfolded proteins was obtained from fluorescence quenching experiments on pyrenyl-labeled actin (Fig. 6C) and NBD-actin and DTAF-tubulin (not shown).

Interestingly, over 90% of H₆130-145 HSP16-2 precipitates from solution after freeze-thawing, while wild-type HSP16-2 and H6HSP16-2 remain completely soluble following the same treatment (not shown). This suggests that the solubility or perhaps stability of the HSP16-2 complex is compromised in the C-terminal truncation mutant.

Perhaps the most conspicuous feature of smHSPs is the presence of a nonconserved N-terminal domain which is highly variable in size. Three N-terminal truncations of HSP16-2, 1-15, 1-32, and 1-44 were used to probe the function of this domain. To examine the oligomeric nature of these N-terminal truncations as well as of other HSP16-2 derivatives, the purified proteins were subjected to chemical cross-linking using the homobifunctional cross-linker BS³ (78). To minimize nonspecific intermolecular cross-linking of non-oligomerized smHSP subunits, cross-linking reactions were carried out in the presence of low protein (1.5 µM monomer) and BS³ (2 mM) concentrations, and a 10-fold molar excess of BSA (15 µM). Reactions were analyzed by Western blotting with an anti-HSP16-2 polyclonal antibody (Fig. 8). All of the proteins yielded discernible monomeric, dimeric, and trimeric species. From the large number of products present in the high molecular mass range (above 100 kDa and at the origin of the separating gel), it can be concluded that the HSP16-2, H₆HSP16-2, and H₆130-145 proteins also form higher-order oligomers which are too large to be well resolved on the 12% SDS gel. In contrast, the H₆1-15, H₆1-32, and H₆1-44 HSP16-2 proteins yielded essentially no high M_r cross-linked products but could be cross-linked to trimers (and possibly tetramers), suggesting that they exist as small aggregates. The presence of the N-terminal H₆ tag in each of these truncated proteins is unlikely to have resulted in disruption of the multimers since both H₆HSP16-2 and H₆130-145 HSP16-2 are able to form large oligomeric complexes.

The sizes of the N-terminally-truncated smHSPs were estimated by SEC on a Sephacryl S-300HR column. These smHSP derivatives elute as proteins of 45-115 kDa (Fig. 2B), and therefore appear to form small aggregates of trimers-heptamers. These results resemble those obtained on the N-terminally-deleted A-, B-crystallin, and HSP25 (79, 80). To obtain more accurate data on the subunit stoichiometry of one of the truncated smHSPs, sedimentation velocity measurements were performed on H₆1-44 HSP16-2. These yielded molecular weights of 14,990 and 56,235, consistent with the behavior of H₆1-44 HSP16-2 as a monomer (95%) with a small proportion (5%) of tetramers (Table I).

Since the N-terminally-truncated smHSPs did not form native-like oligomeric complexes, it was of interest to determine whether they still possessed molecular chaperone activity. The three derivatives were therefore tested for an ability to prevent thermally- or chemically-induced aggregation of CS. None of these smHSPs was effective in suppressing CS aggregation, even at a molar excess of 65 molecules of smHSP to 1 CS monomer (not shown). Furthermore, H₆1-44 HSP16-2 did not quench the fluorescence of denatured forms of pyrenyl-actin (Fig. 6D) or NBD-actin and DTAF-tubulin, and had no effect on the fluorescence of the native labeled proteins (not shown).

The Properties of a 12.6-kDa SmHSP Support the HSP 16-2 Structure-Function Analyses

The C. elegans genome contains at least four highly conserved members of a novel class of smHSPs, of which one member (HSP12.6) is shown aligned to other smHSPs in Fig. 1C. HSP12.6 possesses a well conserved -crystallin domain, but in comparison to other smHSPs, it has both the smallest N-terminal domain at 25 amino acids, and the shortest C-terminal extension, with only 2 amino acids. The present studies on wild-type HSP16-2 and derivatives would predict that HSP12.6 should be unable to multimerize due to its short N-terminal domain, and as a result should lack molecular chaperone activity. Indeed, our recent characterization of HSP12.6 revealed that unlike other smHSPs, HSP12.6 is monomeric and is unable to prevent the thermally-induced aggregation of citrate synthase (51).

We investigated whether HSP12.6 could interact with unfolded proteins even though it is incapable of preventing their aggregation. As with the mostly monomeric H₆1-44 HSP16-2, HSP12.6 did not interact with unfolded, pyrenyl-labeled actin (Fig. 6D). Similarly, no quenching of fluorescence was obtained with native actin, or with native or unfolded tubulin (not shown). The structural and functional data on HSP12.6 therefore support the notion that the distal end of the smHSP N-terminal domain is required for multimerization, and that chaperone activity is only possible in the context of an oligomeric assembly.

DISCUSSION

The HSP16s are among the smallest members of the -crystallin family of HSPs, and we have shown here that they possess the multimeric structure and chaperone activities typical of these proteins. Very few mutational studies have been carried out on smHSPs, and these have concentrated mainly on mammalian -crystallins (79-83). To our knowledge, the present studies on HSP16-2 represent the most extensive structure-function analysis of a stress-inducible smHSP reported to date. The small size of the HSP16s is an advantage in such studies, and the combination of in vitro mutagenesis with functional chaperone assays, substrate binding measurements, and sedimentation studies reported here provide a number of important insights into the structure and function of smHSPs.

Sedimentation velocity measurements reveal that recombinant wild-type HSP16-2 forms heterogeneous mixtures of two particles of 238 and 412 kDa, suggesting that the number of subunits per complex is approximately 14 and 24, respectively. In contrast, recombinant and natural HSP16-2 elutes as an ~550-kDa oligomer by SEC. The reason for the discrepancy between the two sizing techniques is unclear; it is possible that the HSP16-2 aggregates are loosely packed, or that they are non-globular as suggested by the frictional ratio values obtained (Table I). Examination of HSP16-2 by electron microscopy reveals globular but relatively heterogeneous particles with diameters ranging between 10 and 15 nm (not shown). Interestingly, a similar apparent dichotomy exists for mammalian HSP27, which by SEC behaves as an ~800-kDa oligomer, whereas by native gel electrophoresis migrates as a doublet of approximately 200 and 250 kDa (43). In sucrose gradients, HSP27 sediments as two distinct forms, one of 300-400 kDa and another of ~65 kDa (84).

The H₆HSP16-2 variant, which forms fully active oligomeric complexes that are slightly larger than those of wild-type HSP16-2, was used to determine the orientation of the HSP16-2 monomers within the native complex. It was found that the 4-kDa N-terminal polyhistidine-containing tag was inaccessible for binding to a Ni²⁺-chelate affinity matrix. Thus either the tag is collaterally positioned in the complex and masked by the HSP16-2 protein and neighboring subunits (which would very likely have a detrimental effect on the assembly of the complex), or, more likely, the tag is accommodated within a central cavity in the complex. Furthermore, the fact that HSP16-2 can accommodate an additional 4 kDa (36 residues) at its N terminus, making this domain larger than those of -crystallins and Drosophila HSP27, and only 8 residues shorter than that of murine HSP25 (refer to the alignment in Fig. 1C), suggests that the arrangement of subunits within smHSP complexes provides considerable freedom for N-terminal regions of different lengths and amino acid sequence. Indeed, the alternatively-spliced A^ins-crystallin variant which contains a 23-residue insertion near the N-terminal/-crystallin domain boundary also assembles into oligomers (85). Interestingly, the increased length of the N-terminal region in the A^ins-crystallin leads to an increase in the size of the native complex, a situation analogous to that seen with H₆HSP16-2.

The present work shows that deletion of only approximately one-third (15 residues) of the N-terminal domain of HSP16-2 results in a dramatic reduction in the size of the complex as judged by SEC and cross-linking analysis, suggesting that the region involved in multimerization may be distally located in the N-terminal domain. It is therefore conceivable that the HSP16-2 monomers have an extended conformation with the N-terminal regions oriented toward the interior of the complex and providing the necessary contacts for subunit aggregation. In support of this hypothesis, HSP12.6, which has 16 fewer residues than HSP16-2 in the N-terminal region, is monomeric and predicted to be asymmetrical by sedimentation velocity analysis (51).

The studies by Merck and co-workers (79, 80) suggest the existence of at least two major sites of interactions between smHSP monomers: one within the N-terminal domain, which permits the assembly of the complex, and the other within the C-terminal domain, which appears to have an inherent ability to form smaller oligomers. Similarly, Chang et al. (30) found that mycobacterial HSP16.3 nonamers could be disassembled into trimers by treatment with 4 M urea or 1 M guanidine hydrochloride, suggesting that two different types of subunit interactions exist: one involved in trimer formation and another which participates in the oligomerization of trimers. While our SEC and cross-linking experiments on N-terminally-truncated HSP16-2 mutants also suggest that smHSP complexes may assemble from small oligomers, sedimentation velocity measurements of H₆1-44 HSP16-2 and HSP12.6, which possess complete -crystallin domains, show these proteins to be largely monomeric. While it is therefore possible that N-terminally-truncated smHSPs exist as monomers that are in slow equilibrium with higher-order oligomeric species, early cross-linking experiments using a series of cross-linkers with different spacer lengths suggest that -crystallin aggregates are not likely to be built up of smaller clusters (86), and a recent study provides evidence that the minimal cooperative unit of -crystallin is the monomer (87).

The removal of the last 16 residues of HSP16-2 has no effect on multimerization, demonstrating that this region does not participate in subunit assembly. Interestingly, the C-terminal truncation affects the solubility of the oligomer, perhaps by destabilizing the native protein structure. A role for the C-terminal extension in the solubilization of -crystallin particles has recently been suggested (82). It has also been shown by ¹H NMR studies that the last 8-10 and 18 C-terminal residues of -crystallins and HSP25, respectively, form flexible extensions on the surface of the oligomers (27, 28). Taken together, these data suggest that the C-terminal extensions of HSP16-2 and other smHSPs are on the surface (exposed to solvent), where they impart increased solubility and perhaps stability to the complexes.

Under stress conditions, heat shock proteins such as HSP60, HSP70, HSP90, and smHSPs likely prevent partially unfolded proteins from aggregating and becoming insoluble (33, 34, 74, 88, 89). Whereas most HSPs perform necessary functions in non-stressed cells and are also induced following cellular insults (1, 10, 15), the C. elegans HSP16-class smHSPs are produced exclusively when nematodes are exposed to a heat shock or to proteotoxic agents (48, 50). Our studies on HSP16-2 confirm the ability of this specialized class of chaperone to interact with and stabilize proteins which become unfolded following heat or chemical treatment.

It is noteworthy that in eukaryotes, cytoskeletal proteins are one of the most sensitive targets of cellular stresses, and there is increasing evidence that smHSPs specifically interact with cytoskeletal elements, modulating their polymerization, and protecting them during stress conditions (13, 36-38, 40, 41, 43). We have shown that the affinity of HSP16-2 for native actin and tubulin is negligible, whereas it is very high for the same thermally- or chemically-unfolded proteins. The interaction of cardiac B-crystallin with actin and desmin filaments is enhanced during ischemic conditions, where a decrease in the pH of the cytosol is known to affect the stability of cytoskeletal elements (38). HSP16-2 has nearly identical affinities for actin and tubulin, and can also prevent the aggregation of citrate synthase, suggesting that this chaperone has a general affinity for unfolded proteins. Other smHSPs also display little protein specificity, so whether smHSP chaperones have specialized roles in protecting the cellular cytoskeleton network during stresses remains to be seen.

Recent data suggest that -crystallin preferentially interacts with thermally-generated early unfolding intermediates which have exposed hydrophobic regions and are committed to the aggregation pathway (90-92). The present study demonstrates that HSP16-2 is equally capable of preventing the aggregation of thermally- and chemically-unfolded citrate synthase. Furthermore, we show that HSP16-2 has a considerably greater affinity for actin and tubulin unfolded intermediates which occur early on the aggregation pathway, and has little or no affinity for native or aggregated proteins.

The mechanism by which smHSPs interact with and prevent unfolded polypeptides from aggregating is unknown. Although it is generally agreed that hydrophobic regions in smHSPs are important for polypeptide binding (83, 93), hydrophilic interactions may be necessary as well (81, 94). Interestingly, Lee et al. (95) recently found that a hydrophobic reporter molecule, bis-ANS, could be photoincorporated into specific regions of pea HSP18.1 which may be important for substrate binding. One site of incorporation was localized to the sequence ADLPGLKKEEVKV (homologous regions found 13 residues into the -crystallin domains of other smHSPs; see Fig. 1C). This region lies within the first 30% or so of the -crystallin domain, and is well conserved in HSP16-2 and other smHSPs (Fig. 1C). This region was not altered in our deletion studies. The fact that the inactive, low-M_r HSP16-2 N-terminal deletion mutants and the naturally monomeric HSP12.6 both retain this sequence but lack chaperone activity lends support to the idea that multimerization is essential for substrate binding.

Since most of the C-terminal extension of HSP16-2 is dispensable for polypeptide binding and chaperone activity, and is likely to be on the surface of the oligomeric complex, the interaction with unfolded polypeptides presumably involves other regions of the smHSP, such as the clefts formed between subunits. Such clefts can only exist in the context of an oligomeric assembly, which we have shown is necessary for polypeptide binding. Moreover, studies with unstructured spin-labeled peptides suggested that these were not bound on the surface or clustered in an interior cavity of the -crystallin complex, but rather were strongly immobilized at least 25 Å apart in polar environments (94). A detailed computer-generated model of the interaction between partially unfolded -crystallin and -crystallin also predicts the site of polypeptide interaction to be within clefts in the -crystallin complex (96).

Molecular chaperones such as those belonging to the HSP60 and HSP104 families are active as homo- or heteroligomers, requiring multiple subunit contacts with the unfolded polypeptide for activity (5, 97). SmHSPs also form large oligomeric assemblies of one or two types of subunits, but their quaternary structures are much more variable, ranging from toroids (98) to roughly globular particles of various sizes, shapes, subunit stoichiometries, and compositions (29, 30, 35). Several plausible models for smHSP quaternary structures have been proposed (reviewed in Ref. 29), but the lack of detailed structural data do not yet allow a definitive choice to be made among these. However, the micellar-like model first proposed by Augusteyn and Koretz (99) seems best able to accommodate the variability in subunit stoichiometry and particle morphology seen in many smHSPs, as well as the findings reported here, i.e. that a heterologous protein sequence, appended to a distal N-terminal region essential for multimerization, can be sequestered within the particles. Furthermore, our data support a model in which multimerization is a prerequisite both for the interaction of the chaperone with unfolded protein and for chaperone activity. A model consistent with our findings and summarizing some structural and functional characteristics of smHSPs is presented in Fig. 9.

The function of HSP16-2 in vivo may be to interact with and stabilize a large variety of unfolding proteins, effectively buffering the cell against the deleterious effects caused by misfolded and aggregated proteins. Linder et al. (100) reported on a novel 18-kDa C. elegans protein most similar (32-38% overall amino acid sequence identity) to the C. elegans HSP16 smHSPs and named SEC-1 (for small embryonic Chaperone-1) (see Fig. 1C for alignment for sequence). SEC-1 is detectable only during early embryogenesis, where it performs an essential but unknown function. Interestingly, SEC-1 confers thermotolerance to E. coli, even though its expression is not augmented by environmental stresses. Therefore, although the physiological functions of this smHSP are not redundant with those of the stress-inducible HSP16s, it is likely that both possess similar structural and functional characteristics as molecular chaperones.

Despite considerable effort, conditions were not found under which HSP16-2 could refold denatured test proteins in vitro. The fate(s) of the unfolded proteins bound to smHSPs is poorly understood. It is now established that many molecular chaperones cooperate in refolding denatured proteins (6, 89, 101, 102), and are involved in targeting proteins for degradation (Ref. 103, also reviewed in Ref. 104). Recently, HSP25 and HSP70 were shown to act in concert in vitro in the refolding of citrate synthase (105), and Lee et al. (95) demonstrated that denatured firefly luciferase bound to HSP18.1 could be refolded in the presence of rabbit reticulocyte lysate (which contains the chaperones necessary for refolding denatured luciferase) and ATP. It is therefore likely that in vivo, the smHSP-bound unfolded proteins are folded by molecular chaperones such as those involved in de novo protein folding (HSP40/HSP70, HSP60; Ref. 5), or under certain conditions are degraded by the cellular proteolytic system.