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
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Abstract |
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Keywords: aggregation/-crystallin/heat shock protein/molecular chaperone/structure
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Introduction |
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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., 1998), 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., 2001
), 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.
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Methods |
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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., 1998) and HSP 16.9 from wheat (van Montfort et al., 2001
) 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., 1999
) 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 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
A-crystallin stop codon and introduces an XhoI site. After endonuclease digestion with XhoI, the length of the predicted
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
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 A-crystallin
51+ construct is 139 residues. The sequence of the
A-crystallin
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 -crystallin and to place the N-terminal extension of
-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
-crystallin as N-terminal regions.
Protein expression and purification
The A-crystallin
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., 1998
). 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 Tris100 mM NaCl. The
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 Tris250 mM NaCl and elution was carried out at
1.0 ml/min.
Aggregate size
The size of the A-crystallin
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.
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Results and discussion |
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Homology within the sHSP superfamily extends over many families and crosses kingdom boundaries (Merck et al., 1993; de Jong et al., 1998
; Koretz et al., 1998
). Figure 1 presents a representative sample (Van Der Ouderaa et al., 1974
; Ingolia and Craig, 1982
; Jones et al., 1986
; Hay and Petrach, 1987
; Lauzon et al., 1990
; Tseng et al., 1992
; Gaestel et al., 1993
; Heidelbach et al., 1993
; Sutton et al., 1996
) 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
-crystallins are structurally similar, with additional insertions and a significant N-terminal extension. Most larger members have molecular weights of 2527 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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The alignment shown in Figure 1 indicates the conserved ß-strands of the core structure with boxes labeled ß1ß7. 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 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., 2001
). This improves our earlier model using HSP16.5 coordinates (Koretz and Salerno, 2000
), 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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The R116 cognate in wheat HSP16.9, R108, is not directed towards an acceptor in the ß1ß2 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 ß45 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 -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
-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 -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 2023-residue cognate and a similar strand-swapping interaction is present in HSP16.9. This loop is much shorter in animal sHSPs and -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., 1999) 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., 2000). Because of the large amount of information available on HSP16.5 and
-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. -Crystallin
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 regions 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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The HSP16.5--crystallin construct represents the reverse chimera; the shorter 32-residue HSP16.5 N-terminus is attached to the
-crystallin core structural domain. The construct is designed to determine if the relaxation of N-terminal steric constraints is sufficient to allow
-crystallin dimers to pack into small ordered structures comparable to HSP16.5. The
-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 -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
-crystallin
51+ (Figure 4) indicates that it exists in solution primarily in a dimertetramer equilibrium; the majority species is the tetramer at
60 kDa. Native recombinant
-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 dimerdimer interaction in HSP16.5 are intact in
-crystallin
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, 1987
; Radlick and Koretz, 1992
) 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
-crystallin constructs and HSP16.5 derivatives with altered aggregation properties.
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The protein micelle model of -crystallin aggregation has been successful in rationalizing many features of
-crystallins behavior (Augusteyn and Koretz, 1987
; Radlick and Koretz, 1992: Haley et al., 1998
). 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 B-crystallin (Feil et al., 2001
), similar to tetrameric
-crystallin
51+ but dimeric owing to the removal of 18 residues from the C-terminus as well, lend support to this hypothesis.
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Acknowledgment |
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Received March 15, 2003; revised May 20, 2003; accepted September 4, 2003.