Báo cáo khoa học: Glutamic acid residues in the C-terminal extension of small heat shock protein 25 are critical for structural and functional integrity

Glutamic acid residues in the C-terminal extension of small heat shock protein 25 are critical for structural and functional integrity Amie M. Morris1, Teresa M. Treweek2, J. A. Aquilina1, John A. Carver3 and Mark J. Walker1

1 School of Biological Sciences, University of Wollongong, Australia 2 Graduate School of Medicine, University of Wollongong, Australia 3 School of Chemistry & Physics, The University of Adelaide, Australia

Keywords C-terminal extension; Hsp25; molecular chaperone; protein aggregation; small heat shock protein

Correspondence M. J. Walker, School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia Fax: +61 2 4221 4135 Tel: +61 2 4221 3439 E-mail: mwalker@uow.edu.au J. A. Carver, School of Chemistry & Physics, The University of Adelaide, Adelaide, SA 5005, Australia Fax: +61 8 8303 4380 Tel: +61 8 8303 3110 E-mail: john.carver@adelaide.edu.au

(Received 19 February 2008, revised 14 September 2008, accepted 29 September 2008)

doi:10.1111/j.1742-4658.2008.06719.x

Small heat shock proteins (sHsps) are intracellular molecular chaperones that prevent the aggregation and precipitation of partially folded and destabilized proteins. sHsps comprise an evolutionarily conserved region of 80–100 amino acids, denoted the a-crystallin domain, which is flanked by regions of variable sequence and length: the N-terminal domain and the C-terminal extension. Although the two domains are known to be involved in the organization of the quaternary structure of sHsps and interaction with their target proteins, the role of the C-terminal extension is enigmatic. Despite the lack of sequence similarity, the C-terminal extension of mam- malian sHsps is typically a short, polar segment which is unstructured and highly flexible and protrudes from the oligomeric structure. Both the polar- ity and flexibility of the C-terminal extension are important for the mainte- nance of sHsp solubility and for complexation with its target protein. In this study, mutants of murine Hsp25 were prepared in which the glutamic acid residues in the C-terminal extension at positions 190, 199 and 204 were each replaced with alanine. The mutants were found to be structurally altered and functionally impaired. Although there were no significant dif- ferences in the environment of tryptophan residues in the N-terminal domain or in the overall secondary structure, an increase in exposed hydro- phobicity was observed for the mutants compared with wild-type Hsp25. The average molecular masses of the E199A and E204A mutants were comparable with that of the wild-type protein, whereas the E190A mutant was marginally smaller. All mutants displayed markedly reduced thermo- stability and chaperone activity compared with the wild-type. It is con- cluded that each of the glutamic acid residues in the C-terminal extension is important for Hsp25 to act as an effective molecular chaperone.

Small heat shock proteins (sHsps) are a family of intracellular molecular chaperones defined by the pres- ence of an evolutionarily conserved region of 80–100 amino acid residues, denoted the a-crystallin domain [1]. Despite having a relatively small monomeric size (12–43 kDa) [2], sHsps exist under physiological condi-

tions as large oligomers of up to 50 subunits and 1.2 MDa in mass [3,4]. sHsps are found in most cell types in most organisms, and their expression is upreg- ulated under a range of stress conditions, such as heat, oxidative conditions, pH changes, infection and in many disease states characterized by the formation of

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Abbreviations ADH, alcohol dehydrogenase; ANS, 8-anilinonaphthalene-1-sulfonate; sHsp, small heat shock protein.

A. M. Morris et al. Glutamic acid mutants of Hsp25

insoluble amyloid plaques, e.g. Alzheimer’s, Creutz- feldt–Jakob and Parkinson’s diseases [5–8]. Increased levels of sHsps, in particular aB-crystallin and Hsp27, are observed in the brains of sufferers of these diseases [9,10]. Hsp25 is the murine homologue of Hsp27.

The alteration of the properties of the C-terminal extension also leads to significant changes to the struc- ture and function of sHsps. The chaperone activity of Hsp30C is impaired when the polarity of the C-termi- nal extension is reduced [25], and introduction of hydrophobicity into the C-terminal extension of aA-crystallin results in immobilization of the C-termi- nal extension and reduced chaperone activity [21]. Conversely, an increase in the charge of the extension of aA-crystallin results in no significant changes in chaperone activity relative to wild-type aA-crystallin [26,27], highlighting the importance of the polar resi- dues in the C-terminal extension of sHsps.

Stress conditions can promote the partial unfolding of proteins, which subsequently leads to the exposure of hydrophobic residues [11]. This increase in surface- exposed hydrophobicity encourages partially folded proteins to mutually associate and potentially precipi- tate [12]. sHsps prevent the aggregation of such pro- teins by interacting with them and sequestering them into a large complex. The recognition of target pro- teins by sHsps occurs through exposed hydrophobic regions, and the resultant complex is stabilized through electrostatic interactions [13]. Target proteins are held in a folding-competent conformation until conditions are permissive for their refolding or degradation, with the former requiring the input of another chaperone protein, e.g. Hsp70 [14].

equivalents

The thermostability of proteins from thermophilic organisms is related to electrostatic interactions through the presence of polar and charged groups, as well as hydrophobic and packing effects [28]. These proteins typically have a higher proportion of polar and charged residues, primarily glutamic acid and lysine, than their mesophilic Interactions between [29]. aB-crystallin subunits can be inhibited by the replace- ment of glutamic acid residues in the a-crystallin domain with other residues, possibly through decreased electro- static interactions and increased electrostatic repulsion [30]. Similarly, it is likely that the glutamic acid residues in the C-terminal extension of Hsp25 are important for electrostatic interactions with the solvent and potentially other regions of sHsp.

sHsps comprise three structural regions: the con- served a-crystallin domain is flanked by an N-terminal domain and a C-terminal extension, both of which are of variable length and sequence. Overall homology amongst sHsps is therefore low [15]. Although exten- sive work has been undertaken to elucidate the func- tions of the N-terminal domain and a-crystallin domain, the role of the C-terminal extension is less clear. Despite its variability, the C-terminal extension is a short region which is polar, highly flexible and unstructured, and extends freely from the sHsp oligo- mer [16]. These general properties are essential for the correct functioning of sHsps as molecular chaperones. Removal of the C-terminal extension inhibits the chap- erone activity of aA-crystallin and Xenopus Hsp30C [17,18], and also leads to a decrease in the solubility of Hsp25, aA-crystallin and Caenorhabditis Hsp16-2 [17,19,20].

Although some studies have examined the role of residues in the C-terminal extensions of other sHsps, notably aA- and aB-crystallin, the flexible regions of these proteins are unique and distinct from that of Hsp25. The a-crystallins contain negatively charged residues only near the anchor point of the flexible region to the domain core, whereas Hsp25 has glu- tamic acid residues spaced along its flexible extension. Investigation into the function of these uniquely positioned residues in Hsp25 has not been performed previously.

The C-terminal extension acts as a solubilizer to coun- teract the hydrophobicity associated with target protein sequestration [21]. The flexibility of the C-terminal extensions of Hsp25 and aA-crystallin is maintained in the final sHsp–target protein complex, with the exten- sions remaining solvent exposed. Under heat stress, the extension of aB-crystallin has been shown to exhibit reduced flexibility on sHsp–target complex formation, implying that the extension may be involved in target protein capture and have functions in addition to acting as a solubilizer [22]. The oligomeric sizes of aA-crystal- lin, bacterial Hsp16.3 and bacterial HspH are affected by C-terminal extension removal [23,24], indicating that the C-terminal extension is involved in the quaternary structural arrangement of sHsps.

Site-directed mutagenesis has been used in this study to produce Hsp25 alanine substitution mutants of the glutamic acid residues in the C-terminal extension, i.e. E190A, E199A and E204A. An additional mutant, Q194A, was also prepared and included as a control. The role of the C-terminal extension and each of the mutated residues was investigated by comparison of the structure and function of these mutants with those of wild-type Hsp25. The glutamic acid residue mutants displayed altered structure and impaired thermostabil- ity and chaperone activity compared with the wild-type protein, highlighting the importance of the negatively charged glutamic acid residues in the C-terminal exten- sion of Hsp25.

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Table 1) and in seven of the corresponding murine sHsps (not shown), and is a predominant residue in the flexible regions of both human and murine sHsps. The majority of residues present in the flexible regions of the extensions (71% and 74% for human and mur- ine, respectively) are those that have been shown to promote disorder (Table 1) [32]. Although the C-termi- nal extension of human Hsp27 contains an aspartic acid residue, that of murine Hsp25 does not (Fig. 1). Thus, apart from the C-terminal carboxyl group, the three glutamic acid residues are the only source of negative charge in the flexible extension of Hsp25.

Expression and purification of wild-type and mutant Hsp25 proteins

Fig. 1. C-terminal extension sequences of murine Hsp25 and the 10 human sHsps aligned at their IXI motifs [67,68]. The IXI motifs are shown in italics and the known flexible regions of various sHsps, as determined by NMR spectroscopy [26,31,76], are in bold. Residues used in the tally for Table 1 are underlined. Accession numbers were: Hsp27 (P04792), MKBP (Q16082), aA-crystallin (P02489), aB-crystallin (P02511), Hsp20 (O14558), HspB7 (Q9UBY9), Hsp22 (Q9UJY1), HspB9 (Q9BQS6) and ODFP (Q14990).

Hsp25 mutants were designed to investigate the impor- tance of the negatively charged residues in the C-termi- nal extension. Wild-type Hsp25 and the Q194A and glutamic acid residue mutants were purified success- fully, as confirmed by the observation of the correct masses by ESI-MS (not shown).

Results

CD spectroscopy of wild-type and mutant Hsp25

Sequence analysis of the C-terminal extensions of mammalian sHsps

The C-terminal extensions of mammalian sHsps are highly variable in length and sequence, yet they share the characteristics of being polar, flexible and unstruc- tured, suggesting that the types of residue present in the C-terminal extension, rather than their sequence, are important. This was investigated by analysing the amino acid residues corresponding to the known flexi- ble regions of aA- and aB-crystallin and Hsp25 [16,26,31] (Fig. 1). Proline is present in six of the eight human sHsps that contain a flexible region (Fig. 1,

Far-UV CD spectroscopy was performed to determine whether substitution of the glutamine or glutamic acid residues resulted in any alteration to the overall sec- ondary structure of Hsp25. A broad minimum at 217 nm was observed for all spectra (Fig. 2, Table 2), indicative of the predominance of b-sheet structure [33]. The estimation of secondary structure content obtained by deconvolution of the spectra was consis- tent with previous measurements [20], with wild-type Hsp25 having secondary structure contents of 38% b-sheet and 6% a-helix at 25 (cid:2)C. A slight increase in

Table 1. Frequency of amino acid residues in the flexible region of the C-terminal extensions of human and murine sHsps. Residues present in the flexible region of the C-terminal extension of each of the eight human sHsps that contain a flexible region (underlined residues in Fig. 1) are tallied. Only the totals are given for murine sHsps. Residues that promote disorder are in bold and those that promote order are in italic [32]. Residues are denoted as charged (+ or )), polar (p) or nonpolar (n).

Residue T C L D V N Y I M F W H P A E K S R Q G

) ) p n p n n n n n + p 1 + 2 p 2 + 1 p 1 n 2 n 1 1 1 1 1 1 3 1 1 1 n 5 3 1 3 3 6 2 2 n 1 5 2 2 3 1 3 1 1 1 1 1 1 2 1

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Charge Hsp27 HspB2 aA-crystallin aB-crystallin Hsp20 Hsp22 HspB9 ODFP Human total Murine total 6 1 4 5 6 1 1 4 3 1 3 2 3 4 1 1 2 3 2 2 1 1 2 3 1 1 1 1 1 1 0 0 0 0 4 17 18 16 13 14 14 1 8 9 2 7 10 1 2 4 4 3 4 1 3 5

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) and E204A ( ), Q194A ( ), E190A ( ), E199A (

Fig. 2. Far-UV CD spectra of wild-type ( ) Hsp25 at 25, 37 and 55 (cid:2)C in 10 mM sodium phosphate buffer (pH 7.5). No significant differences in secondary structure between wild-type and mutant proteins were observed at any of the three temperatures.

Table 2. Summary of changes in structure and function of C-terminal Hsp25 mutants. Comparisons are made with wild-type Hsp25. Qualita- tive comparisons are given for exposed clustered hydrophobicity, thermostability and chaperone activity (DC18, Hsp25 truncation mutant lacking the C-terminal 18 residues; ND, not determined).

Hsp25 mutant Charge Secondary structure Exposed clustered hydrophobicity Average molecular mass Thermostability Chaperone activity Reference

No change +1 +1 +1 No change 27% increase 46% increase 54% increase 65% increase 31% decrease No change 53 kDa smaller No change No change No change No change Poor Poor Poor ND No change Decreased Decreased Decreased Decreased This study This study This study This study [20] Q194A E190A E199A E204A DC18 No change No change No change No change Increased a-helix

[36]. Fluorescence spectroscopy was performed on wild-type and mutant Hsp25 to detect any changes in the environment of the tryptophan residues resulting from the mutations. The tryptophan residues of Hsp25 are located in the N-terminal domain at positions 16, 22, 43, 46 and 52, and in the a-crystallin domain at position 99. A fluorescence maximum (Fmax) of approximately 4700 arbitrary units with a wavelength at maximum fluorescence (kmax) of 340.2 nm was observed for wild-type Hsp25 (not shown). No shift in kmax was observed for the mutants. A shift in kmax is indicative of a change in the polarity of the tryptophan environment [37]. Therefore, the overall tryptophan environment was not significantly affected by substitu- tion of the glutamic acid residues in the C-terminal extension, which are all distant in primary structure from the tryptophan residues.

negative ellipticity and flattening of the spectra were observed for wild-type and mutant Hsp25 samples with increasing temperature from 25 to 55 (cid:2)C. The increase in negative ellipticity at around 210 nm implies an increase in a-helical content [33]. However, following deconvolution of the spectra, changes to each of the structural element proportions were less than 5% between all spectra, and were deemed to be insignifi- cant [34]. The overall increase in negative ellipticity at higher temperatures is consistent with a slight increase in or stabilization of secondary structure [35]. In com- paring the mutants with wild-type Hsp25, the consis- tency of the deconvolution data suggests that it is unlikely that the secondary structure is altered signifi- cantly as a result of the mutations, i.e. there was little difference in overall secondary structure between the wild-type and mutant proteins.

Tryptophan fluorescence spectroscopy of wild-type and mutant Hsp25

8-Anilinonaphthalene-1-sulfonate (ANS) binding fluorescence spectroscopy of wild-type and mutant Hsp25

The binding of ANS and other hydrophobic probes to a protein enables the comparative determination of the

Tryptophan fluorescence depends strongly on the local environment of the amino acid and is a sensitive probe of conformation in the vicinity of tryptophan residues

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), E190A ( ), E199A ( ), E199A ( ), E190A ( ), Q194A ( ) Hsp25 and buffer (

Fig. 3. ANS binding fluorescence emission spectra of wild-type ( ) and E204A ( ). Experiments were performed at 25 (cid:2)C with 85 lM ANS and an excitation wavelength of 387 nm. Samples were prepared to a final concentration of 5 lM in 50 mM sodium phosphate buffer containing 0.02% NaN3. (pH 7.3) Increases in maximum fluorescence of 27, 45, 53 and 63% were observed for the Q194A, E190A, E199A and E204A mutants, respectively, in comparison with wild-type Hsp25.

), Fig. 4. Size-exclusion chromatography FPLC of wild-type ( Q194A ( ) ) and E204A ( Hsp25. Samples were prepared to a final concentration of 30 lM in 50 mM sodium phosphate buffer (pH 7.3) containing 0.02% NaN3, with 100 lL being loaded onto the column. The peak positions of the elution of molecular standards are indicated at the top of the graph. The elution of wild-type Hsp25 corresponds to an average molecular mass of 613 kDa. No significant differences in mass were observed for the Q194A, E199A and E204A mutants. The E190A mutant eluted at a volume corresponding to an average molecular mass of 560 kDa.

the E190A mutant,

exposed clustered hydrophobicity of the protein and, if altered, indicates a perturbation in tertiary structure [38]. Such probes bind noncovalently to regions on pro- teins that contain exposed clusters of hydrophobic ami- noacyl residues, resulting in an increase in fluorescence [39]. ANS binding fluorescence of wild-type and mutant Hsp25 reached a maximum at a final concentration of 85 lm ANS, and the fluorescence values presented (Fig. 3, Table 2) are the means of the plateau region of the ANS binding curves (75–95 lm) (not shown). Wild- type Hsp25 resulted in an ANS binding fluorescence of approximately 570 arbitrary units. The Q194A, E190A, E199A and E204A mutants exhibited increases in ANS binding fluorescence of approximately 27%, 45%, 53% and 63%, respectively, compared with the wild-type protein, indicating that all of the mutants have greater clustered hydrophobicity exposed to the solvent, and thus an altered tertiary structure.

the Q194A, 26–27 subunits. The peak maxima of E199A and E204A mutants eluted at volumes almost identical to that of the wild-type, indicating very simi- lar average oligomeric sizes to the wild-type (Table 2). The small extra peak in the elution profile of E199A represents a protein of less than 250 kDa in mass. The oligomeric species of wild-type Hsp25 is in equilibrium with a tetrameric form [40], and so the smaller species may be a tetramer. Elution of the E190A mutant was delayed slightly compared with elution of the wild-type protein, with the elution peak corresponding to a calculated average molecular mass of approximately 53 kDa smaller than wild-type Hsp25, and to an aver- age oligomer of 24–25 subunits. Thus, with the excep- tion of the oligomeric size of Hsp25 was not affected by glutamine or glutamic acid residue mutations.

Oligomer formation by wild-type and mutant Hsp25

Thermostability studies of wild-type and mutant Hsp25

aggregates,

Size-exclusion chromatography was performed in order to determine whether the glutamic acid substitutions affected the oligomeric size of Hsp25. Wild-type Hsp25 eluted between the molecular weight markers thyro- globulin (mass of 669 kDa) and apoferritin (mass of 443 kDa) (Fig. 4), with an average molecular mass of 613 ± 185 kDa, as calculated from the standard curve (not shown), corresponding to an average oligomer of

The thermostability of wild-type and mutant Hsp25 was investigated by monitoring the increase in light scattering at 360 nm as a result of the formation of large followed by precipitation with increasing temperature. Wild-type Hsp25 was very heat stable and remained in solution up to temperatures of 100 (cid:2)C (Fig. 5, Table 2). No precipitate was observed

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of precipitation of yeast ADH for monitoring precipi- tation was found to be 55 (cid:2)C (not shown), and the inactivation and precipitation of yeast ADH at this temperature have been well characterized [44]. This temperature was also well below the onset of aggrega- tion and precipitation for wild-type and mutant Hsp25 proteins, as shown by thermostability studies. Any precipitation observed was therefore not attributable to Hsp25 instability.

), Q194A ( ), E199A ( ) and E204A (

Complete suppression [45] of yeast ADH precipita- tion was observed for wild-type Hsp25 at a molar ratio of 1.4 : 1.0 Hsp25 : ADH (Fig. 6). A decrease in sup- pression of ADH precipitation was observed at this ratio for all of the glutamic acid residue mutants. At all other ratios, the E199A mutant showed minor reductions in suppression of ADH precipitation com- pared with wild-type Hsp25, whereas the E190A and E204A mutants showed markedly reduced suppression. The Q194A mutant showed similar levels of suppres- sion of aggregation to the wild-type protein at all ratios.

), Fig. 5. Thermostability profiles of wild-type ( ) Hsp25. Samples E190A ( were prepared to a final concentration of 0.2 mgÆmL)1 in 50 mM sodium phosphate buffer (pH 7.3). The temperature was increased at a rate of 1 (cid:2)CÆmin)1. Wild-type Hsp25 and Q194A remained in solution up to temperatures of 100 (cid:2)C. By contrast, the E190A, E199A and E204A mutants precipitated out of solution within 2 (cid:2)C of the onset of precipitation at 68 (cid:2)C for E190A and 70 (cid:2)C for E199A and E204A.

the lower

and the small increase in light scattering at tempera- tures above approximately 70 (cid:2)C is consistent with an increase in aggregate size [41]. The Q194A mutant showed a light scattering profile comparable with that of the wild-type protein. In marked contrast, the glu- tamic acid residue mutants all precipitated out of solu- i.e. at tion within 2 (cid:2)C of the onset of aggregation, approximately 68 (cid:2)C for E190A and 70 (cid:2)C for E199A and E204A. Decreased light scattering after maximum precipitation had been reached resulted from the pre- cipitate sinking to the bottom of the cuvette and there- fore not obscuring the light path [42]. Thus, the Q194A mutant showed thermostability similar to that of wild- type Hsp25, whereas the glutamic acid residue mutants exhibited significantly decreased thermostability.

Functional chaperone activity assays of wild-type and mutant Hsp25

Precipitation of insulin can be initiated by the addi- tion of a reducing agent, such as dithiothreitol, which cleaves the disulfide bonds between the A and B chains of insulin, resulting in the aggregation and precipita- tion of the B chain. Reduction stress assays are advan- tageous over thermal stress assays as they can be performed at physiological temperatures, i.e. 37 (cid:2)C. The precipitation of insulin under reduction stress was completely suppressed by wild-type Hsp25 at a molar ratio of 0.5 : 1.0 Hsp25 : insulin (Fig. 7), with all mutants showing comparable suppression of insulin precipitation at this ratio. All of the glutamic acid in particular the E204A mutant, residue mutants, displayed reduced suppression at ratio (0.05 : 1.0), and the E190A mutant showed reduced suppression at 0.25 : 1.0. At all ratios, the Q194A mutant exhibited very similar levels of suppression of insulin B chain precipitation to the wild-type protein. the thermal and reduction stress Taken together, assays demonstrate that each of the glutamic acid mutants, in particular E190A and E204A, are signifi- than is wild-type cantly less effective chaperones Hsp25 (Table 2).

Discussion

The chaperone activity of wild-type and mutant Hsp25 was assessed by determining the ability of these proteins to prevent the amorphous aggregation and precipitation of target proteins under stress conditions. Assays were performed with alcohol dehydrogenase (ADH) under heat stress and insulin under reduction stress in the pres- ence of varying concentrations of Hsp25.

the extremity of

Many proteins contain intrinsically disordered regions that are necessary for their function [46]. Accordingly, these regions have a higher frequency of disorder-pro- moting residues [32,47]. Such is the case for the flexible regions located at the C-terminal extensions of human and murine sHsps. Despite the

Yeast ADH is a tetramer of four equal subunits with a total molecular mass of 141 kDa [43]. Thermal stress assays using this enzyme are commonly per- formed at temperatures of 48–60 (cid:2)C. The optimal rate

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Fig. 6. Chaperone activity of wild-type and mutant Hsp25, as measured by the suppression of precipitation of ADH under thermal stress. Ratios represent the molar concentration of Hsp25 monomers to ADH subunits. Assays were performed at 55 (cid:2)C in 50 mM sodium phos- phate buffer (pH 7.3) containing 0.02% NaN3. Traces are the average of duplicates. The precipitation of ADH was completely suppressed at an Hsp25 : ADH ratio of 1.4 : 1.0. The Q194A mutant showed comparable chaperone activity with the wild-type protein. Each of the glutamic acid residue mutants displayed a decrease in chaperone activity compared with wild-type Hsp25.

low sequence similarity, these regions are abundant in disorder-promoting residues, such as proline [48]. The hydrophilicity, lack of structure and associated flexibil- ity are essential for the solubilizing role of the exten- sion in the sHsp and complexation of the sHsp with target proteins. The unstructured and highly dynamic nature of this flexible region also ensures that it does not interfere with or block the preceding conserved IXI motif, which is important in subunit–subunit inter- actions [49].

Despite the low sequence similarity throughout the sHsp family, Hsp25 has a very similar, predominantly b-sheet, secondary structure to that of other sHsps, including mammalian aA- and aB-crystallin and bacte- rial IpbB [50,51]. The glutamic acid residue substitu- tions did not affect the secondary structure of Hsp25, indicating that these residues, which are part of an unstructured region, are not important for the determi-

nation of this level of structure. In support of this con- clusion, mutants of aA- and aB-crystallin, in which the C-terminal extensions were swapped, have been shown to have secondary structures similar to each other and to their wild-type counterparts [52]. Similarly, the removal of the C-terminal extension of aA-crystallin, aB-crystallin and bacterial Hsp16.3 produces proteins with secondary structure comparable with that of the respective wild-type proteins [17,53,54] The secondary structure of wild-type and mutant Hsp25 did not change significantly from 25 to 55 (cid:2)C, consistent with previous findings that Hsp25, a-crystallin and IpbB resist changes to secondary structure with increasing temperatures up to approximately 60 (cid:2)C [50,55,56]. The secondary structure of Hsp25 has also been shown to be stable under mildly denaturing conditions [40], and temperatures of at least 60 (cid:2)C are required for a loss of b-sheet structure [40].

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Fig. 7. Chaperone activity of wild-type and mutant Hsp25, as measured by the suppression of precipitation of insulin under reduction stress. Ratios represent the molar concentration of Hsp25 monomers to insulin molecules. Assays were performed at 37 (cid:2)C in 50 mM sodium phos- phate buffer (pH 7.3) containing 0.02% NaN3. Traces are the average of triplicates. The precipitation of insulin was completely suppressed at an Hsp25 : insulin ratio of 0.5 : 1.0. The Q194A mutant showed comparable chaperone activity with the wild-type protein. Each of the glutamic acid residue mutants displayed a decrease in chaperone activity compared with wild-type Hsp25.

in exposed hydrophobicity, as

suggesting that

least

Although the secondary structure of Hsp25 was not altered as a result of the mutations, significant differ- indicated by ences ANS binding, were observed, the same elements of secondary structure were adopted, but that the subunits were arranged differently from that of the wild-type protein. Because five of the six in Hsp25 are located in the tryptophan residues N-terminal domain, trypto- the comparable overall phan exposure in the mutants compared with the wild-type protein indicates that the structure of the N-terminal domain is maintained, at in the vicinity of the tryptophan residues. The increase in exposed clustered hydrophobicity observed for the mutants is therefore likely to arise from rearrange- ments of secondary structural elements in the a-crys- tallin domain.

The slightly reduced oligomeric size of the E190A mutant compared with the wild-type suggests that the E190 residue is important for the correct formation of the quaternary structure of Hsp25. Examination of the crystal structures of Hsp16.9 and Hsp16.5, which do not have flexible C-terminal extensions, shows that the conserved IXI motif (residues 185–187 in Hsp25) forms hydrophobic contacts with a groove between b-strands in the a-crystallin domain of another monomer, and that this interaction is essential for the oligomerization of sHsps [57]. Truncation from the C-terminus of aA-crystallin to remove the IXI motif renders the pro- tein unable to form oligomers [58], indicating that interactions involving the IXI motif are also essential for the oligomerization of mammalian sHsps contain- ing a flexible C-terminal extension. Because of the proximity between the E190 residue of Hsp25 and the

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IXI motif, it is possible that substitution of this residue disrupts the interaction between the IXI motif and the hydrophobic groove, resulting in an altered oligomeric structure. The formation of large sHsp oligomers is also dependent on interactions between N-terminal domains [23], which are not affected by mutations in the C-terminal extension. The comparability of oligo- meric sizes between the mutants and wild-type Hsp25 clearly demonstrates this.

the protein [15,26]. Removal of

poorly at lower ratios towards insulin under reduction stress. These functional differences were not a result of the lack of solubilization of the chaperone, as both wild- type Hsp25 and the glutamic acid residue mutants of Hsp25 were stable in solution at the temperatures at which these assays were performed. Recognition of and interaction with target proteins by sHsps is largely hydrophobic in nature [13]. On this basis, it would be expected that the Hsp25 mutants, with increased surface hydrophobicity, would display enhanced chaperone ability compared with the wild-type protein [51]. However, the structural changes associated with the mutations appear to have a greater influence on the chaperone activity than simply the degree of exposed hydrophobicity [62].

[17–20]. The

In mammalian sHsps, the presence of a flexible, sol- vent-exposed C-terminal extension helps to counteract the large amount of hydrophobicity exposed by the the remainder of C-terminal extension results in a decrease in thermo- stability of Hsp25, aA-crystallin, Xenopus Hsp30C and Caenorhabditis Hsp16-2 drastically reduced thermostability of the glutamic acid residue mutants demonstrates the importance of each of the negatively charged residues in the C-terminal extension in maintaining the solubility of the Hsp25 oligomer. Similarly, the introduction of hydrophobicity into the in a C-terminal extension of aA-crystallin results decrease in the thermostability of this sHsp [21]. These data suggest that relatively modest alterations to the C-terminal extensions of sHsps, resulting in a decrease in polarity, are sufficient to disrupt the ability of the C-terminal extensions to efficiently act as solubilizers.

chaperone binding sites,

temperature-induced increase

Although conclusive identification of the chaperone binding sites of sHsps remains elusive, there is evidence that the binding of target proteins occurs in the groove between monomers, and involves a b-sheet region located at the beginning of the a-crystallin domain cor- responding to residues 70–88 in aA-crystallin [63,64]. The changes in tertiary structure observed for the mutants, as evidenced by the alteration in exposed hydrophobicity, could result in the disruption of bind- ing sites, and thus hindered recognition and sequestra- tion of target proteins. These changes may also inhibit stabilization by electrostatic interactions, resulting in less effective target protein sequestration. The decrease in polarity of the C-terminal extension may also facili- tate interaction between the extension and hydro- phobic resulting in the binding sites being less accessible to the target protein, leading to a decrease in target protein binding [26].

At temperatures above 60 (cid:2)C, Hsp25 and the a-crys- tallins undergo changes in their tertiary structure that result in the exposure of hydrophobic regions [56,59]. The onset of precipitation of the glutamic acid residue mutants of Hsp25 corresponds approximately to this temperature. The in hydrophobic exposure did not induce the precipitation of wild-type Hsp25, although a small increase in light scattering implies the formation of larger aggregates [41]. Thermostable proteins display more effective bur- ial of hydrophobic regions than do less thermostable proteins [28]. The increase in exposed hydrophobicity coupled with the associated with the mutations, temperature-induced increase in hydrophobicity, is consistent with the poor thermostability observed for the glutamic acid residue mutants.

The glutamic acid residue mutants showed reduced chaperone activity compared with wild-type Hsp25 towards target proteins under different assay conditions, a property that has also been observed recently for wild- type and mutant forms of aB-crystallin [60,61]. The E190A and E204A mutants performed poorly compared with the wild-type protein in both assays, most notably at the lower Hsp25 : target protein ratios used. The E199A mutant showed somewhat decreased chaperone activity towards ADH under heat stress, but performed

In summary, the three negatively charged glutamic acid residues in the C-terminal extension of Hsp25 (E190, E199 and E204) are essential for the correct structure and function of this sHsp. These residues contribute to the polarity of the extension and pro- mote its disorder, ensuring that the C-terminal exten- sion remains unstructured and solvent exposed, and therefore able to perform its solubilizing role in the sHsp and in the complexes formed with target proteins during chaperone action. Indeed, the presence of sig- nificant regions of structural disorder is a common characteristic of molecular chaperones, and is integral to their effective chaperone action [65]. Despite an the Q194A alteration in exposed hydrophobicity, mutant showed comparable oligomeric size and func- tional properties to those of wild-type Hsp25. Thus, residues in the flexible region of the C-terminal exten- sion are not equally important for Hsp25 to perform its role as a molecular chaperone, emphasizing the importance of the glutamic acid residues.

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Experimental procedures

Sequence analysis of the C-terminal extension of mammalian sHsps

The C-terminal extensions of the 10 human sHsps [66] were aligned according to their IXI motifs, when present. When absent, the alignments were based on those of Fontaine et al. [67] and Franck et al. [68]. Residues that aligned with the known flexible regions of aA- and aB-crystallin [16] were tallied. The C-terminal extensions of the equivalent murine sHsps were similarly analysed.

Site-directed mutagenesis of pAK3038-Hsp25

was induced with 0.4 mm isopropyl thio-b-d-galactoside. Cells were harvested by centrifugation and lysed as described. After ultracentrifugation, dithiothreitol, polyeth- yleneimine and EDTA were added to the supernatant to final concentrations of 10 mm, 0.12% (v ⁄ v) and 1 mm, respectively, and the lysate was incubated and centrifuged as described. The final supernatant was filtered through a 0.22 lm Minisart filter (Sartorius, Epsom, UK) before (Sigma-Aldrich, being loaded onto a DEAE-Sephacel St Louis, MO, USA) column with a volume of approxi- mately 90 mL. Recombinant Hsp25 was eluted with 100 mm NaCl in 20 mm Tris ⁄ HCl buffer (pH 8.5) contain- ing 1 mm EDTA and 0.02% (w ⁄ v) NaN3. Fractions con- taining Hsp25 were concentrated to approximately 5 mL and dithiothreitol was added to a final concentration of 50 mm. The sample was incubated at room temperature for 30 min before being loaded onto a Sephacryl S-300HR (Pharmacia, Uppsala, Sweden) column with a volume of approximately 470 mL. Recombinant Hsp25 eluted in the first peak with 50 mm Tris ⁄ HCl buffer (pH 8.0) containing 1 mm EDTA and 0.02% (w ⁄ v) NaN3. Fractions containing Hsp25 were concentrated, dialysed exhaustively against, or exchanged into, MilliQ water and lyophilized. Both chromatographic steps were performed at 4 (cid:2)C. The purity of recombinant proteins was confirmed by nanoscale ESI-MS.

Far-UV CD spectroscopy

secondary

structure

of

the primers

Site-directed mutagenesis was performed using the Quik- Change(cid:3) system (Stratagene, La Jolla, CA, USA), accord- ing to the manufacturer’s instructions, except that 14 cycles were used (Cooled-Palm 96, Corbett Research, Mortlake, NSW, Australia). All primers were synthesized by Sigma Genosys (Castle Hill, NSW, Australia). The primer pairs for site-directed mutagenesis were as follows: 5¢-TTCGA GGCCCGCGCCGCAATTGGGGGCCCAGAA-3¢ and 5¢- TTCTGGGCCCCCAATTGCGGCGCGGGCCTCGAA-3¢ for E190A, 5¢-ATTCCGGTTACTTTCGCGGCCCGCGC CCAAATT-3¢ and 5¢-AATTTGGGCGCGGGCCGCGA AAGTAACCGGAAT-3¢ for E190A, 5¢-CAAATTGGGGG CCCAGCAGCTGGGAAGTCTGAA-3¢ and 5¢-TTCAGA CTTCCCAGCTGCTGGGCCCCCAATTTG-3¢ for E199A, and 5¢-GAAGCTGGGAAGTCTGCACAGTCTGGAGCC AAG-3¢ and 5¢-CTTGGCTCCAGACTGTGCAGACTTCC CAGCTTC-3¢ for E204A. Mutated codons are shown in italic type. Dimethylsulfoxide was added to a final concen- tration of 5% (v ⁄ v) to reactions in which strong secondary interactions were likely, as advised by the supplier. Success- ful mutagenesis was confirmed by DNA sequence analysis of the forward and reverse strands with BigDye(cid:4) Termina- tor Ready Reaction Mix (Applied Biosystems, Foster City, CA, USA) on a Prism 377 DNA sequencer (Applied 5¢-TCTCGGAGATCC Biosystems) using GACAGA-3¢ and 5¢-CTTTCGGGCTTTGTTAGCAG-3¢, respectively.

CD spectra were acquired on a J-810 spectropolarimeter (Jasco, Tokyo, Japan) with an attached Peltier temperature- controlled water circulator. Samples were prepared in 10 mm phosphate buffer (pH 7.5) to a final concentration of 10–15 lm and filtered through a 0.22 lm Minisart filter. Spectra were recorded at 25, 37 and 55 (cid:2)C, and are accu- mulations of 16 scans recorded from 190 to 250 nm with a path length of 1 mm. The sample concentration was deter- mined using a bicinchoninic acid assay (Sigma-Aldrich). An composition was estimation performed using the cdsstr program [70–72] in the DICHROWEB Online Circular Dichroism Analysis suite [73,74].

Expression and purification of wild-type and mutant Hsp25

Intrinsic tryptophan fluorescence and ANS binding fluorescence spectroscopy

water

circulator

pAK3038-Hsp25 was a gift from M. Gaestel (Institute of Biochemistry, Hannover, Germany). DNA was transformed into electrically competent BL21(DE3) Escherichia coli before expression. Expression and purification of murine Hsp25 and mutants were performed according to the method described by Horwitz et al. [69] with minor changes. Transformed cells were grown in Luria–Bertani medium containing 0.4% (w ⁄ v) glucose and 100 lgÆmL)1 ampicillin to select for pAK3038-Hsp25. Protein expression

All fluorescence studies were performed at 25 (cid:2)C using an F-4500 fluorescence spectrophotometer (Hitachi High-Tech- nologies, Tokyo, Japan) with a Thermomix temperature- controlled (B. Braun, Melsungen, Germany). Samples were prepared in 50 mm phosphate buffer (pH 7.3) containing 0.02% (w ⁄ v) NaN3 to a final concentration of 5 lm, as calculated from A280 values of the samples, an extinction coefficient of 1.87 for a 1 mgÆmL)1 solution of Hsp25 [75] and molecular mass. An

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tration of 20 mm and monitored by light scattering at 360 nm. Assays were performed in triplicate.

A. M. Morris et al. Glutamic acid mutants of Hsp25

Acknowledgements

excitation wavelength of 295 nm was used for intrinsic fluorescence, with emission spectra recorded from 300 to 450 nm. ANS binding fluorescence was performed with an excitation wavelength of 387 nm. Sequential aliquots of freshly prepared ANS were added to the samples and mixed thoroughly until the emission fluorescence at 479 nm reached a maximum. Emission spectra were then recorded from 400 to 650 nm.

We are grateful to Professor Matthias Gaestel for pro- viding the plasmid pAK3038. This work was supported by grants to JAC from the National Health and Medi- cal Research Council of Australia and the Australian Research Council.

Size-exclusion chromatography

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