Structural diversity in the small heat shock protein superfamily: control of aggregation by the N-terminal region

John C. Salerno1, Cheryl L. Eifert, Kathleen M. Salerno and Jane F. Koretz

Center for Biophysics and Department of Biology, Rensselaer Polytechnic Institute, Science Center, Troy, NY 12180-3590, USA

1 To whom correspondence should be addressed. e-mail: salerj{at}rpi.edu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Acknowledgment
 References
 
The small heat shock protein superfamily, extending over all kingdoms, is characterized by a common core domain with variable N- and C-terminal extensions. The relatively hydrophobic N-terminus plays a critical role in promoting and controlling high-order aggregation, accounting for the high degree of structural variability within the superfamily. The effects of N-terminal volume on aggregation were studied using chimeric and truncated proteins. Proteins lacking the N-terminal region did not aggregate above the tetramers, whereas larger N-termini resulted in large aggregates, consistent with the N-termini packing inside the aggregates. Variation in an extended internal loop differentiates typical prokaryotic and plant superfamily members from their animal counterparts; this implies different geometry in the dimeric building block of high-order aggregates.

Keywords: aggregation/{alpha}-crystallin/heat shock protein/molecular chaperone/structure


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Acknowledgment
 References
 
The small heat shock protein (sHSP) superfamily is ancient and diverse, including representatives from eubacteria, archaea, plants and animals (Van Der Ouderaa et al., 1974Go; Ingolia and Craig, 1982Go; Jones et al., 1986Go; Hay and Petrach, 1987Go; Lauzon et al., 1990Go; Tseng et al., 1992Go; Gaestel et al., 1993Go; Heidelbach et al., 1993Go; Merck et al., 1993Go; Sutton et al., 1996Go; de Jong et al., 1998Go; Koretz et al., 1998Go). Prokaryotic members of the superfamily are often in the 12–18 kDa range, but many eukaryotic homologs are significantly larger; sHSPs range over 12–40 kDa. The core structural domain of all these proteins is similar in size and has been designated the ‘crystallin domain’ after the {alpha}-crystallins, a group of homologous sHSP superfamily members most heavily expressed in mammalian lens. The N- and C-terminal sequences outside this common domain vary in length and amino acid composition. A major function of heat shock proteins is to act as molecular chaperones to prevent misfolding or the aggregation of partially folded proteins (Horwitz, 1992Go; Jakob et al., 1993Go).

The structure of the thermophilic bacterial heat shock protein HSP16.5 from Methanococcus jannaschii has been solved by Kim and co-workers (Kim et al., 1998Go), revealing a core region in which two ß-sheets form a ‘butter sandwich’ around an interior composed almost exclusively of hydrophobic side chains. HSP16.5 is of interest because of its thermophilic origin, solved structure and ability to stabilize a wide variety of constitutive proteins when it is expressed in Escherichia coli. Other sHSPs, while sharing the common core domain, differ in aggregation, chaperone-like activity and stability. Recently the structure of a second member of the superfamily was solved (van Montfort et al., 2001Go), revealing strong similarities and a few interesting differences. Here we report initial studies using molecular chimeras within the sHSP superfamily to investigate the origins of these differences.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Acknowledgment
 References
 
Sequence alignments and homology modeling

Initial sequence alignments were generated using multiple alignment (CLUSTAL W) and pairwise alignment (ALINORM) programs. The results were adjusted manually. Structural modeling based on the alignments and the atomic coordinates of HSP16.5 (Kim et al., 1998Go) and HSP 16.9 from wheat (van Montfort et al., 2001Go) was carried out using the InsightII/homology modeling package from Molecular Simulations. Alternative alignments were examined using steric and energetic criteria. Data from spin label studies (Berengian et al., 1999Go) were considered in the choice of alignments (e.g. selection of ß-strand start positions). Crude models were refined using Discover. Refinement included splice point repairs to produce favorable bond geometries and energy minimization carried out on all atoms except backbone atoms in conserved secondary structural elements.

PCR amplification

Oligonucleotide sequences were designed to anneal specifically to the {alpha}A-crystallin gene (bovine), such that the 5' oligonucleotide would begin amplification at residue 51 in order to eliminate the N-terminal region. The 3' oligonucleotide incorporates the {alpha}A-crystallin stop codon and introduces an XhoI site. After endonuclease digestion with XhoI, the length of the predicted {alpha}A-crystallin gene product is 124 residues. The oligonucleotide sequences used were upstream 5'-TCCCTCTTCCGCACCGTGCTGG-3' and downstream 5'-GCTTTGTTAGCAGCTCGAGCCTTAGGACGAG-3'. Addi tionally, a 15-residue N-terminal region, containing a start codon and preceded by an NdeI site, was attached 5' to the N-terminally deleted {alpha}A gene discussed above (using overlap extension amplification). The sequences of the serine/glycine N-terminal oligonucleotides were upstream 5'-CATATGG ACGTCACCACCGGAACCGGAACCACCGGAACCACCGCTAGC-3' and downstream 5'-CCAGCACGGTGCGGAA GAGGGAGCTAGCGGTGGTTCCGGT-3'.

The total length of the {alpha}A-crystallin{Delta}51+ construct is 139 residues. The sequence of the {alpha}A-crystallin{Delta}51+ gene was verified using an ABI 373 sequencer. The T7 promoter primer (upstream) and the T7 terminator primer (downstream) anneal to the pet20b vector.

Chimeras were designed to place the N-terminal extension of HSP16.5 on {alpha}-crystallin and to place the N-terminal extension of {alpha}-crystallin on HSP16.5. Fragments were obtained by PCR by methods closely analogous to those described above and chimeric genes formed by ligation. Chimeric products included the first 32 residues of HSP16.5 and the first 50 residues of {alpha}-crystallin as N-terminal regions.

Protein expression and purification

The {alpha}A-crystallin{Delta}51+ and chimeric genes were ligated into the pet20b vector (Novagen) and subsequently transformed into the E.coli expression strain BL21 (DE3) pLysS. Cell lysis and supernatant preparation were performed according to Horwitz et al. (Horwitz et al., 1998Go). Protein supernatant was applied, at ~2.0 ml/min, to a Hiprep 16/10 Q XL column (Amersham-Pharmacia) that had been equilibrated with 20 mM Tris–100 mM NaCl. The {Delta}51+ constructed protein eluted in 350 mM NaCl and these fractions were applied to a ~100 ml bed volume column packed with Sephacryl S-400 gel filtration material. The column was equilibrated with 20 mM Tris–250 mM NaCl and elution was carried out at ~1.0 ml/min.

Aggregate size

The size of the {alpha}A-crystallin{Delta}51+ protein was determined using a Superose 12 HR 10/30 gel exclusion column (Amersham-Pharmacia) run at 0.5 ml/min in 20 mM Tris (pH 8.0)–200 mM NaCl. Standards were ß-amylase 200 000, bovine serum albumin 66 000, carbonic anhydrase 29 000 and cytochrome c 12 400. The size of other chimeric aggregates was determined under similar conditions, but using a Superose 6 HR gel exclusion column and high molecular weight standards.


    Results and discussion
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Acknowledgment
 References
 
Homology modeling

Homology within the sHSP superfamily extends over many families and crosses kingdom boundaries (Merck et al., 1993Go; de Jong et al., 1998Go; Koretz et al., 1998Go). Figure 1 presents a representative sample (Van Der Ouderaa et al., 1974Go; Ingolia and Craig, 1982Go; Jones et al., 1986Go; Hay and Petrach, 1987Go; Lauzon et al., 1990Go; Tseng et al., 1992Go; Gaestel et al., 1993Go; Heidelbach et al., 1993Go; Sutton et al., 1996Go) selected from an extensive multiple alignment containing hundreds of sequences. The smallest members of the superfamily are clearly single-domain structures dominated by ß-sheet motifs. Larger sHSPs and {alpha}-crystallins are structurally similar, with additional insertions and a significant N-terminal extension. Most larger members have molecular weights of 25–27 kDa; the 2-fold size difference between these and the smallest HSPs reflects N- and C-terminal extensions too small to be domains, combined with internal insertions corresponding to extended loops connecting units of conserved secondary structure. Almost all members of the sHSP superfamily are single-domain proteins; a few ~40 kDa heat shock proteins contain two repeats.



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Fig. 1. Multiple sequence alignment of representative members of the small heat shock protein superfamily (Van Der Ouderaa et al., 1974Go; Ingolia and Craig, 1982Go; Jones et al., 1986Go; Hay and Petrach, 1987Go; Lauzon et al., 1990Go; Tseng et al., 1992Go; Gaestel et al., 1993Go; Heidelbach et al., 1993Go; Sutton et al., 1996Go). Sequences correspond to accession numbers O2495337, Q06823, P06582, P02510, P02470, P24622, P27777, P19243 and P14602; the genetically modified wheat HSP sequence was taken from the structure described by van Montfort et al. (van Montfort et al., 2001Go). The putative disordered N-terminal region shows little homology between families, whereas the region corresponding to the ß-sheet domain of sHSP16.5 is much more highly conserved. Regions of similarity among eukaryotic proteins are present in the N-terminal region, while similarity in the swapped strand region is higher between plant and bacterial sequences.

 
Kim et al. reported the first sHSP structure (MjHSP16.5), crystallized as a spherical 24-subunit aggregate (Kim et al., 1998Go). The building block for the aggregate is a dimer; each monomer consists of two antiparallel ß-sheets and contributes a ß-strand, labeled ‘swapped’ in Figure 1, to the N-terminal edge of the other monomer. The authors assigned secondary structure for eight sHSPs and constructed a crystallin molecular model (Salerno and Koretz, 1999Go; Koretz and Salerno, 2000Go). Although sHSP structures superficially resemble immunoglobin (Mornon et al., 1998Go), the ß-sheet topologies are completely different (Koretz et al., 1998Go). Large-scale multiple alignments suggest alignment uncertainty in the first few ß-strands arising from difficulty in reconciling physical data with homology. Berengian et al. used spin labeling data to deduce structural details in {alpha}-crystallin (Berengian et al., 1999Go). The absence of interactions expected for an HSP16.5 ß1 cognate led them to conclude that this strand is absent; this is likely to be correct. Most of their conclusions are consistent with homology, but their alignment forces an insertion into the cognate of HSP16.5 ß2 for the homologous rodent sequences.

The alignment shown in Figure 1 indicates the conserved ß-strands of the core structure with boxes labeled ß1–ß7. These strands are present in both available structures; the first strand in this nomenclature is also the first strand in the wheat structure, which corresponds to ß2 in wheat HSP16.9. Additional features not included in the conserved core are indicated by dotted boxes. In some regions, notably the N-terminal region preceding the core, existing crystal structures are unrelated and the dotted lines indicate different features.

Figure 2 shows a homology model of the {alpha}A-crystallin core, using the solved structures as a basis set; conserved secondary structural elements were based primarily on wheat HSP16.9 (van Montfort et al., 2001Go). This improves our earlier model using HSP16.5 coordinates (Koretz and Salerno, 2000Go), but retains its overall features. Relaxation removed all significant unfavorable contacts, generating a free energy of –672 kcal/mol using van der Waals and electrostatic terms. The core is composed of two sheets, formed by alternating sequence elements and enclosing a hydrophobic core. The surface of this brick-like structure is largely hydrophilic, but contains hydrophobic patches that almost certainly function in aggregation.



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Fig. 2. Ribbon representation of backbone topology of {alpha}A-crystallin, based on homology modeling to HSP16.9 and HSP16.5. Residues are color-coded for hydrophobicity, with red the most hydrophobic and blue the most hydrophilic. Only the extended core region of {alpha}A-crystallin (residues 59–145) is presented here. The line represents the {alpha} trace of half of a superimposed HSP16.9 dimer, indicating the position of the other subunit and the difficulty of forming a swapped strand cognate in {alpha}-crystallin. The HSP16.9 backbone is yellow in helical regions and brick red in strands.

 
Most crystallin mutants are similar to wild-type protein (Muchowski et al., 1999Go) because hydrophobic interactions are relatively non-specific and a large number of interactions are involved. An exception is R120G in {alpha}B-crystallin (or R116G in {alpha}A-crystallin) (Bova et al., 1999Go), which greatly decreases the stability of the native structure. R116/R120 is unusual in that it is a hydrophilic residue directed into the core. The function of the R116/R120 cognate in HSP16.5 is to form an H-bond to the backbone of the loop between the first and second ß-strands. The bulkier side chains in crystallin make it difficult to model a corresponding element.

The R116 cognate in wheat HSP16.9, R108, is not directed towards an acceptor in the ß1–ß2 loop. Because the aromatic side chain of F41 blocks access to this region, R108 forms an intermonomer H-bond with the E100 side chain in the ß4–5 loop, which is involved in strand swapping interactions in the dimer. The N-terminal region of HSP16.9 consists of a large open loop including two {alpha}-helices; towards the N-terminus a short ß-cap covers part of the strand covered by ß1 in HSP16.5. The ß-cap runs parallel to the interior strand, whereas ß1 of HSP16.5 is antiparallel. The N-terminus of {alpha}-crystallin is not obviously homologous to HSP16.9, but contains a similar motif that could serve as a cap.

The highly conserved PK motif following from the core domain in sHSPs is a strong helix initiator that forms an N-terminal cap on the short second helix of HSP16.5. Its presence in other sHSPs and {alpha}-crystallin is suggestive of a short helix. The HSP16.9 cognate is not exactly helical, but suggests a helical ancestor; this variability is often seen in the helical returns of families of parallel ß-sheet proteins. It is followed in HSP16.5 by a terminal ß-strand, which mediates the formation of higher order aggregates by inserting two hydrophobic residues into the interior of a neighboring dimer. The distance between the PK and the hydrophobic region is variable, suggesting a different aggregation geometry. In HSP16.9, C-terminal extensions are oriented differently within a dimer. The pattern suggests that the N- and C-termini may interact.

The loop containing the swapped strand is critical to HSP16.5 dimerization. Large-scale alignments with hundreds of homologues reveal that most prokaryotic and plant sHSPs contain a 20–23-residue cognate and a similar strand-swapping interaction is present in HSP16.9. This loop is much shorter in animal sHSPs and {alpha}-crystallin (~14 vs 23 residues); modeling shows that it is too small to form the same dimer-promoting structure (see Figure 2). It is still the longest internal loop, however, and may play a role in forming a dimer with altered properties (e.g. geometry, flexibility, stability).

The absence of a cognate in crystallins and animal sHSPs is significant for the structure of the N-terminal edge of the core. Rodent crystallins have a major insertion in the initial ß-strand covered by the swapped strand and such a large insertion is unlikely in an internal ß-strand. The shorter loop corresponding to the region may leave the initial strand exposed in animal sHSPs; in this case the insertion in the rodent crystallins could correspond structurally to a ß-blowout. If this is true, the structural model of Berengian et al. (Berengian et al., 1999Go) could be correct for some crystallins, but the region would not be structurally conserved.

Chimera expression and structural properties

The sequence homology and structural relationships in the sHSP superfamily suggest that differences in the N- and C-terminal regions, and also the variable swapped strand region, are responsible for great differences in aggregation between proteins sharing a nearly identical common core. Existing crystal structures point to the importance of the swapped and C-terminal regions in the formation of dimers and tetramers, respectively; N-terminal sequences may be the key in further oligomer formation. The greatest variability within the superfamily is seen in the extent and conformation of the hydrophobic N-terminal tail preceding the onset of the common domain. Calculations indicate that the N-terminal regions of many large sHSPs are too large to pack inside the compact aggregates of smaller homologues. This suggests that N-terminal volume is a major controlling factor of aggregation in the sHSP superfamily (Haley et al., 2000Go). Because of the large amount of information available on HSP16.5 and {alpha}-crystallin, we chose these as examples of sHSPs with short and long N-termini.

Figure 3 shows the constructs designed to test the effect of the N-terminal sequence on sHSP aggregation. {alpha}-Crystallin{Delta}51+ is a truncated construct in which the material corresponding to the disordered 32 N-terminal residues of HSP16.5 has been replaced by a short hydrophilic leader sequence. This construct removes the N-terminal region’s influence on aggregation. If hydrophobic residues in this region drive aggregation according to the micelle hypothesis, this construct would be expected to be soluble, forming dimers and tetramers because of the intact swapped strand and C-terminal regions respectively.



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Fig. 3. Schematic showing truncated constructs and chimeras designed to test the role of the N-terminal region in aggregation.

 
The {alpha}-crystallin-HSP16.5 chimera has the longer hydrophobic N-terminus of {alpha}-crystallin attached to the core structural domain of HSP16.5 (Eifert et al., 2000Go). Since the larger N-terminal region should not be able to pack in the interior of the HSP16.5 particle, this chimera is designed to force HSP16.5 into {alpha}-crystallin-like behavior in that it must form large, disordered aggregates.

The HSP16.5-{alpha}-crystallin construct represents the ‘reverse chimera’; the shorter 32-residue HSP16.5 N-terminus is attached to the {alpha}-crystallin core structural domain. The construct is designed to determine if the relaxation of N-terminal steric constraints is sufficient to allow {alpha}-crystallin dimers to pack into small ordered structures comparable to HSP16.5. The {alpha}-crystallin dimer structure must be different from the dimer structure of HSP16.5, because of the drastic shortening of the swapped strand loop. Large differences in the dimer or tetramer structure would mitigate against packing into an HSP16.5-type aggregate.

Table I summarizes the aggregation size data obtained for {alpha}-crystallins, HSP16.5 and the series of constructs, all of which were expressed at high levels (~20 mg from a 1 l culture) in E.coli. Analytical FPLC on Superose 12HR of {alpha}-crystallin{Delta}51+ (Figure 4) indicates that it exists in solution primarily in a dimer–tetramer equilibrium; the majority species is the tetramer at ~60 kDa. Native recombinant {alpha}-crystallin aggregates are an order of magnitude larger; because of the larger aggregates, chromatography was done on Superose 6HR, producing results of similar quality. Since sequence regions corresponding to those involved in dimer formation and dimer–dimer interaction in HSP16.5 are intact in {alpha}-crystallin{Delta}51+, this result argues strongly that the major role of the N-terminus is in higher order aggregation. The lack of large aggregates strongly suggests that the driving force for their formation is hydrophobic interaction between the N-termini, confirming the central idea of the micelle hypothesis (Augusteyn and Koretz, 1987Go; Radlick and Koretz, 1992Go) and identifying the extent of the hydrophobic component, which is much smaller than previously believed. The result demonstrates the feasibility of engineering the aggregation properties of sHSPs by manipulating the N-terminus, in this case producing both soluble, highly expressed {alpha}-crystallin constructs and HSP16.5 derivatives with altered aggregation properties.


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Table I. Aggregate size of sHSP proteins and constructs
 


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Fig. 4. Sepharose 12HR chromatography results for the truncated {alpha}A-crystallin{Delta}51+ construct, showing the major peak at 33 min corresponding to the 60 kDa tetramer. The shoulder corresponds to the dimer at approximately 30 kDa. Standards were cytochrome c, 12.4 kDa; carbonic anhydrase, 24 kDa; bovine serum albumin, 66 kDa; and ß-amylase, 200 kDa.

 
The {alpha}-crystallin-HSP16.5 chimera forms large aggregates identical in size with native {alpha}-crystallin to within our ability to measure them. This indicates that, as postulated from modeling, N-terminal packing limits the ability of sHSPs to form small aggregates. It also shows that N-termini of sHSPs can drive the aggregation of distant sHSP superfamily members and that the interactions can be relatively non-specific, supporting the proposal that hydrophobic interactions play a major role. The ‘reverse’ HSP16.5–{alpha}-crystallin chimera can be expressed at high levels, but replacement of the {alpha}-crystallin N-terminus with the HSP16.5 N-terminus is insufficient to convert {alpha}-crystallin to HSP16.5-like aggregation behavior. As indicated by chromatographic data, the construct exists in a variety of aggregation states, with none as small as HSP16.5. The N-terminus of HSP16.5 is sufficient to drive aggregation, but the dimer and/or tetramer configuation of {alpha}-crystallin, determined by the core structural domain and C-terminal tail, appears to be inconsistent with HSP16.5-like high-order aggregates. We plan further modifications to improve our understanding of the interaction between different levels of structure. The N-terminal region of {alpha}-crystallin is significantly larger than the corresponding region in HSP16.5. Good evidence suggests that the disordered 32 N-terminal residues of HSP16.5 are packed inside the ‘hollow’ sphere formed by the 24-subunit aggregate (Kim et al., 1998Go). The interior ‘empty’ space, about 140 000 Å3, is just large enough to accommodate these residues, which are significantly more hydrophobic than those found on the outside of the sphere, leaving enough space for the packing of at most one additional domain (~20 000 Å3). If the N-terminal extension of {alpha}-crystallin is packed within the aggregate, it must prevent the formation of an ordered structure such as the 24-subunit spheroid of HSP16.5, because the larger hydrophobic region will not fit into such a small central core. As indicated by the altered properties of the {alpha}-crystallin{Delta}51+ construct, removal of these residues is sufficient to produce soluble tetrameric {alpha}-crystallin. Although it is likely that the corresponding N-terminal regions of other sHSPs pack inside their aggregates, homology between these regions does not extend throughout the superfamily. The ordered N-terminal region of HSP16.9 leads to the formation of a characteristic double disk-shaped aggregate unlike {alpha}-crystallin or HSP16.5 aggregates. Half of the HSP16.9 monomers have N-terminal regions visible in the crystal structure and packing inside one disk; the other half are differently oriented and do not diffract. None of the 32 N-terminal residues of HSP16.5 diffract and all appear to pack inside the spherical aggregate.

The protein micelle model of {alpha}-crystallin aggregation has been successful in rationalizing many features of {alpha}-crystallin’s behavior (Augusteyn and Koretz, 1987Go; Radlick and Koretz, 1992: Haley et al., 1998Go). It is instructive to consider briefly the characteristics of micelles formed by smaller amphipathic molecules; these characteristics are strongly affected by the relative sizes of the hydrophilic and hydrophobic regions. Amphipaths with small hydrophobic volumes and large hydrophilic cross-sections form small aggregates because the hydrophilic region can tile the surface of a small sphere in which the hydrophobic volumes can pack. Amphipaths with larger hydrophobic volumes relative to the hydrophilic cross-section form larger aggregates so that the spherical surface tiled by the hydrophilic region contains a larger volume per subunit. For very large hydrophobic volumes or special geometric constraints, other structures can be favored, ranging from non-spherical micelles to the familiar bi-lamellar structures of phospholipid membranes. Our results suggest that the N-terminal region corresponds to the hydrophobic volume, whereas the hydrophilic cross-section is provided by the common core domain.

Given the apparent packing of the N-terminal 32 residues of HSP16.5 in the interior of the aggregate, it is likely that the size and properties of the aggregates formed by members of the sHSP superfamily are in part controlled by the volume of the N-terminal extension. We propose that a major reason for N-terminal variability within the superfamily is to control aggregate size, order and geometry. This does not rule out the possibility that parts of this region are involved in more specific interactions with other monomers or that the C-terminal extension may also have a role in interprotein interactions. Scattering studies of a construct of {alpha}B-crystallin (Feil et al., 2001Go), similar to tetrameric {alpha}-crystallin{Delta}51+ but dimeric owing to the removal of 18 residues from the C-terminus as well, lend support to this hypothesis.


    Acknowledgment
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Acknowledgment
 References
 
The authors are grateful for the support of NIH grant EY10011.


    References
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Acknowledgment
 References
 
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Received March 15, 2003; revised May 20, 2003; accepted September 4, 2003.