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 pptx

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Glutamic acid residues in the C-terminal extensionof small heat shock protein 25 are critical for structuraland functional integrityAmie M. Morris1, Teresa M. Treweek2, J. A. Aquilina1, John A. Carver3and Mark J. Walker11 School of Biological Sciences, University of Wollongong, Australia2 Graduate School of Medicine, University of Wollongong, Australia3 School of Chemistry & Physics, The University of Adelaide, AustraliaSmall heat shock proteins (sHsps) are a family ofintracellular molecular chaperones defined by the pres-ence of an evolutionarily conserved region of 80–100amino 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 and1.2 MDa in mass [3,4]. sHsps are found in most celltypes in most organisms, and their expression is upreg-ulated under a range of stress conditions, such as heat,oxidative conditions, pH changes, infection and inmany disease states characterized by the formation ofKeywordsC-terminal extension; Hsp25; molecularchaperone; protein aggregation; small heatshock proteinCorrespondenceM. J. Walker, School of Biological Sciences,University of Wollongong, Wollongong,NSW 2522, AustraliaFax: +61 2 4221 4135Tel: +61 2 4221 3439E-mail: mwalker@uow.edu.auJ. A. Carver, School of Chemistry &Physics, The University of Adelaide,Adelaide, SA 5005, AustraliaFax: +61 8 8303 4380Tel: +61 8 8303 3110E-mail: john.carver@adelaide.edu.au(Received 19 February 2008, revised 14September 2008, accepted 29 September2008)doi:10.1111/j.1742-4658.2008.06719.xSmall heat shock proteins (sHsps) are intracellular molecular chaperonesthat prevent the aggregation and precipitation of partially folded anddestabilized proteins. sHsps comprise an evolutionarily conserved region of80–100 amino acids, denoted the a-crystallin domain, which is flanked byregions of variable sequence and length: the N-terminal domain and theC-terminal extension. Although the two domains are known to be involvedin the organization of the quaternary structure of sHsps and interactionwith 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 andhighly 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. Inthis study, mutants of murine Hsp25 were prepared in which the glutamicacid residues in the C-terminal extension at positions 190, 199 and 204were each replaced with alanine. The mutants were found to be structurallyaltered and functionally impaired. Although there were no significant dif-ferences in the environment of tryptophan residues in the N-terminaldomain 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 werecomparable with that of the wild-type protein, whereas the E190A mutantwas 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 extensionis important for Hsp25 to act as an effective molecular chaperone.AbbreviationsADH, alcohol dehydrogenase; ANS, 8-anilinonaphthalene-1-sulfonate; sHsp, small heat shock protein.FEBS Journal 275 (2008) 5885–5898 ª 2008 The Authors Journal compilation ª 2008 FEBS 5885insoluble amyloid plaques, e.g. Alzheimer’s, Creutz-feldt–Jakob and Parkinson’s diseases [5–8]. Increasedlevels 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.Stress conditions can promote the partial unfoldingof proteins, which subsequently leads to the exposureof hydrophobic residues [11]. This increase in surface-exposed hydrophobicity encourages partially foldedproteins to mutually associate and potentially precipi-tate [12]. sHsps prevent the aggregation of such pro-teins by interacting with them and sequestering theminto a large complex. The recognition of target pro-teins by sHsps occurs through exposed hydrophobicregions, and the resultant complex is stabilized throughelectrostatic interactions [13]. Target proteins are heldin a folding-competent conformation until conditionsare permissive for their refolding or degradation, withthe former requiring the input of another chaperoneprotein, e.g. Hsp70 [14].sHsps comprise three structural regions: the con-served a-crystallin domain is flanked by an N-terminaldomain and a C-terminal extension, both of which areof variable length and sequence. Overall homologyamongst sHsps is therefore low [15]. Although exten-sive work has been undertaken to elucidate the func-tions of the N-terminal domain and a-crystallindomain, the role of the C-terminal extension is lessclear. Despite its variability, the C-terminal extensionis a short region which is polar, highly flexible andunstructured, and extends freely from the sHsp oligo-mer [16]. These general properties are essential for thecorrect 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 ofHsp25, aA-crystallin and Caenorhabditis Hsp16-2[17,19,20].The C-terminal extension acts as a solubilizer to coun-teract the hydrophobicity associated with target proteinsequestration [21]. The flexibility of the C-terminalextensions of Hsp25 and aA-crystallin is maintained inthe final sHsp–target protein complex, with the exten-sions remaining solvent exposed. Under heat stress, theextension of aB-crystallin has been shown to exhibitreduced flexibility on sHsp–target complex formation,implying that the extension may be involved in targetprotein capture and have functions in addition to actingas a solubilizer [22]. The oligomeric sizes of aA-crystal-lin, bacterial Hsp16.3 and bacterial HspH are affectedby C-terminal extension removal [23,24], indicating thatthe C-terminal extension is involved in the quaternarystructural arrangement of sHsps.The alteration of the properties of the C-terminalextension also leads to significant changes to the struc-ture and function of sHsps. The chaperone activity ofHsp30C is impaired when the polarity of the C-termi-nal extension is reduced [25], and introduction ofhydrophobicity into the C-terminal extension ofaA-crystallin results in immobilization of the C-termi-nal extension and reduced chaperone activity [21].Conversely, an increase in the charge of the extensionof aA-crystallin results in no significant changes inchaperone activity relative to wild-type aA-crystallin[26,27], highlighting the importance of the polar resi-dues in the C-terminal extension of sHsps.The thermostability of proteins from thermophilicorganisms is related to electrostatic interactions throughthe presence of polar and charged groups, as well ashydrophobic and packing effects [28]. These proteinstypically have a higher proportion of polar and chargedresidues, primarily glutamic acid and lysine, than theirmesophilic equivalents [29]. Interactions betweenaB-crystallin subunits can be inhibited by the replace-ment of glutamic acid residues in the a-crystallin domainwith other residues, possibly through decreased electro-static interactions and increased electrostatic repulsion[30]. Similarly, it is likely that the glutamic acid residuesin the C-terminal extension of Hsp25 are important forelectrostatic interactions with the solvent and potentiallyother regions of sHsp.Although some studies have examined the role ofresidues in the C-terminal extensions of other sHsps,notably aA- and aB-crystallin, the flexible regions ofthese proteins are unique and distinct from that ofHsp25. The a-crystallins contain negatively chargedresidues only near the anchor point of the flexibleregion to the domain core, whereas Hsp25 has glu-tamic acid residues spaced along its flexible extension.Investigation into the function of these uniquelypositioned residues in Hsp25 has not been performedpreviously.Site-directed mutagenesis has been used in this studyto produce Hsp25 alanine substitution mutants of theglutamic 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 themutated residues was investigated by comparison ofthe structure and function of these mutants with thoseof wild-type Hsp25. The glutamic acid residue mutantsdisplayed altered structure and impaired thermostabil-ity and chaperone activity compared with the wild-typeprotein, highlighting the importance of the negativelycharged glutamic acid residues in the C-terminal exten-sion of Hsp25.Glutamic acid mutants of Hsp25 A. M. Morris et al.5886 FEBS Journal 275 (2008) 5885–5898 ª 2008 The Authors Journal compilation ª 2008 FEBSResultsSequence analysis of the C-terminal extensionsof mammalian sHspsThe C-terminal extensions of mammalian sHsps arehighly variable in length and sequence, yet they sharethe characteristics of being polar, flexible and unstruc-tured, suggesting that the types of residue present inthe C-terminal extension, rather than their sequence,are important. This was investigated by analysing theamino 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 eighthuman sHsps that contain a flexible region (Fig. 1,Table 1) and in seven of the corresponding murinesHsps (not shown), and is a predominant residue inthe flexible regions of both human and murine sHsps.The majority of residues present in the flexible regionsof the extensions (71% and 74% for human and mur-ine, respectively) are those that have been shown topromote disorder (Table 1) [32]. Although the C-termi-nal extension of human Hsp27 contains an asparticacid residue, that of murine Hsp25 does not (Fig. 1).Thus, apart from the C-terminal carboxyl group, thethree glutamic acid residues are the only source ofnegative charge in the flexible extension of Hsp25.Expression and purification of wild-type andmutant Hsp25 proteinsHsp25 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 andglutamic acid residue mutants were purified success-fully, as confirmed by the observation of the correctmasses by ESI-MS (not shown).CD spectroscopy of wild-type and mutant Hsp25Far-UV CD spectroscopy was performed to determinewhether substitution of the glutamine or glutamic acidresidues resulted in any alteration to the overall sec-ondary structure of Hsp25. A broad minimum at217 nm was observed for all spectra (Fig. 2, Table 2),indicative of the predominance of b-sheet structure[33]. The estimation of secondary structure contentobtained by deconvolution of the spectra was consis-tent with previous measurements [20], with wild-typeHsp25 having secondary structure contents of 38%b-sheet and 6% a-helix at 25 °C. A slight increase inFig. 1. C-terminal extension sequences of murine Hsp25 and the 10human sHsps aligned at their IXI motifs [67,68]. The IXI motifs areshown in italics and the known flexible regions of various sHsps, asdetermined by NMR spectroscopy [26,31,76], are in bold. Residuesused in the tally for Table 1 are underlined. Accession numberswere: Hsp27 (P04792), MKBP (Q16082), aA-crystallin (P02489),aB-crystallin (P02511), Hsp20 (O14558), HspB7 (Q9UBY9), Hsp22(Q9UJY1), HspB9 (Q9BQS6) and ODFP (Q14990).Table 1. Frequency of amino acid residues in the flexible region of the C-terminal extensions of human and murine sHsps. Residues presentin the flexible region of the C-terminal extension of each of the eight human sHsps that contain a flexible region (underlined residues inFig. 1) are tallied. Only the totals are given for murine sHsps. Residues that promote disorder are in bold and those that promote order arein italic [32]. Residues are denoted as charged (+ or )), polar (p) or nonpolar (n).Residue PAEKST C R L D QGVNYI M FWHCharge nn) +p p p + n ) pnnpnnn nn +Hsp27 153221 1 1 1 12HspB2 536 1 1 11aA-crystallin 212131aB-crystallin 2323 1 1Hsp20 31 1Hsp22 1 2 1 1 1HspB9 1 1 11ODFP 4124 2 1 1 1 1211 1Human total 17 16 14 8 7 6 5 4 4 3 3332221 10 0Murine total 18 13 14 9 10 1 6 4 3 2 4543131 10 0A. M. Morris et al.Glutamic acid mutants of Hsp25FEBS Journal 275 (2008) 5885–5898 ª 2008 The Authors Journal compilation ª 2008 FEBS 5887negative ellipticity and flattening of the spectra wereobserved for wild-type and mutant Hsp25 samples withincreasing temperature from 25 to 55 °C. The increasein negative ellipticity at around 210 nm implies anincrease in a-helical content [33]. However, followingdeconvolution of the spectra, changes to each of thestructural element proportions were less than 5%between all spectra, and were deemed to be insignifi-cant [34]. The overall increase in negative ellipticity athigher temperatures is consistent with a slight increasein 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 isunlikely that the secondary structure is altered signifi-cantly as a result of the mutations, i.e. there was littledifference in overall secondary structure between thewild-type and mutant proteins.Tryptophan fluorescence spectroscopy ofwild-type and mutant Hsp25Tryptophan fluorescence depends strongly on the localenvironment of the amino acid and is a sensitive probeof conformation in the vicinity of tryptophan residues[36]. Fluorescence spectroscopy was performed onwild-type and mutant Hsp25 to detect any changes inthe environment of the tryptophan residues resultingfrom the mutations. The tryptophan residues of Hsp25are located in the N-terminal domain at positions 16,22, 43, 46 and 52, and in the a-crystallin domain atposition 99. A fluorescence maximum (Fmax)ofapproximately 4700 arbitrary units with a wavelengthat maximum fluorescence (kmax) of 340.2 nm wasobserved for wild-type Hsp25 (not shown). No shift inkmaxwas observed for the mutants. A shift in kmaxisindicative of a change in the polarity of the tryptophanenvironment [37]. Therefore, the overall tryptophanenvironment was not significantly affected by substitu-tion of the glutamic acid residues in the C-terminalextension, which are all distant in primary structurefrom the tryptophan residues.8-Anilinonaphthalene-1-sulfonate (ANS) bindingfluorescence spectroscopy of wild-type andmutant Hsp25The binding of ANS and other hydrophobic probes to aprotein enables the comparative determination of theTable 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 mutantlacking the C-terminal 18 residues; ND, not determined).Hsp25 mutant ChargeSecondarystructureExposed clusteredhydrophobicityAverage molecularmass ThermostabilityChaperoneactivity ReferenceQ194A No change No change 27% increase No change No change No change This studyE190A +1 No change 46% increase 53 kDa smaller Poor Decreased This studyE199A +1 No change 54% increase No change Poor Decreased This studyE204A +1 No change 65% increase No change Poor Decreased This studyDC18 No change Increased a-helix 31% decrease No change ND Decreased [20]Fig. 2. Far-UV CD spectra of wild-type ( ), Q194A ( ), E190A ( ), E199A ( ) and E204A ( ) Hsp25 at 25, 37 and 55 °Cin10 mM sodium phosphate buffer (pH 7.5). No significant differences in secondary structure between wild-type and mutant proteins wereobserved at any of the three temperatures.Glutamic acid mutants of Hsp25 A. M. Morris et al.5888 FEBS Journal 275 (2008) 5885–5898 ª 2008 The Authors Journal compilation ª 2008 FEBSexposed clustered hydrophobicity of the protein and, ifaltered, 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 mutantHsp25 reached a maximum at a final concentration of85 lm ANS, and the fluorescence values presented(Fig. 3, Table 2) are the means of the plateau region ofthe ANS binding curves (75–95 lm) (not shown). Wild-type Hsp25 resulted in an ANS binding fluorescence ofapproximately 570 arbitrary units. The Q194A, E190A,E199A and E204A mutants exhibited increases in ANSbinding fluorescence of approximately 27%, 45%, 53%and 63%, respectively, compared with the wild-typeprotein, indicating that all of the mutants have greaterclustered hydrophobicity exposed to the solvent, andthus an altered tertiary structure.Oligomer formation by wild-type andmutant Hsp25Size-exclusion chromatography was performed in orderto determine whether the glutamic acid substitutionsaffected the oligomeric size of Hsp25. Wild-type Hsp25eluted between the molecular weight markers thyro-globulin (mass of 669 kDa) and apoferritin (mass of443 kDa) (Fig. 4), with an average molecular mass of613 ± 185 kDa, as calculated from the standard curve(not shown), corresponding to an average oligomer of26–27 subunits. The peak maxima of the Q194A,E199A and E204A mutants eluted at volumes almostidentical 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 E199Arepresents a protein of less than 250 kDa in mass. Theoligomeric species of wild-type Hsp25 is in equilibriumwith a tetrameric form [40], and so the smaller speciesmay be a tetramer. Elution of the E190A mutant wasdelayed slightly compared with elution of the wild-typeprotein, with the elution peak corresponding to acalculated average molecular mass of approximately53 kDa smaller than wild-type Hsp25, and to an aver-age oligomer of 24–25 subunits. Thus, with the excep-tion of the E190A mutant, the oligomeric size ofHsp25 was not affected by glutamine or glutamic acidresidue mutations.Thermostability studies of wild-type andmutant Hsp25The thermostability of wild-type and mutant Hsp25was investigated by monitoring the increase in lightscattering at 360 nm as a result of the formation oflarge aggregates, followed by precipitation withincreasing temperature. Wild-type Hsp25 was very heatstable and remained in solution up to temperatures of100 °C (Fig. 5, Table 2). No precipitate was observedFig. 3. ANS binding fluorescence emission spectra of wild-type(), Q194A ( ), E190A ( ), E199A ( ) and E204A() Hsp25 and buffer ( ). Experiments were performed at25 °C with 85 lM ANS and an excitation wavelength of 387 nm.Samples were prepared to a final concentration of 5 lM in 50 mMsodium phosphate buffer (pH 7.3) containing 0.02% NaN3.Increases in maximum fluorescence of 27, 45, 53 and 63% wereobserved 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 (), E190A ( ), E199A ( ) and E204A ( )Hsp25. Samples were prepared to a final concentration of 30 lM in50 mM sodium phosphate buffer (pH 7.3) containing 0.02% NaN3,with 100 lL being loaded onto the column. The peak positions ofthe elution of molecular standards are indicated at the top of thegraph. The elution of wild-type Hsp25 corresponds to an averagemolecular mass of 613 kDa. No significant differences in masswere observed for the Q194A, E199A and E204A mutants. TheE190A mutant eluted at a volume corresponding to an averagemolecular mass of 560 kDa.A. M. Morris et al. Glutamic acid mutants of Hsp25FEBS Journal 275 (2008) 5885–5898 ª 2008 The Authors Journal compilation ª 2008 FEBS 5889and the small increase in light scattering at tempera-tures above approximately 70 °C is consistent with anincrease in aggregate size [41]. The Q194A mutantshowed a light scattering profile comparable with thatof the wild-type protein. In marked contrast, the glu-tamic acid residue mutants all precipitated out of solu-tion within 2 °C of the onset of aggregation, i.e. atapproximately 68 °C for E190A and 70 °C for E199Aand E204A. Decreased light scattering after maximumprecipitation 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 Q194Amutant showed thermostability similar to that of wild-type Hsp25, whereas the glutamic acid residue mutantsexhibited significantly decreased thermostability.Functional chaperone activity assays of wild-typeand mutant Hsp25The chaperone activity of wild-type and mutant Hsp25was assessed by determining the ability of these proteinsto prevent the amorphous aggregation and precipitationof target proteins under stress conditions. Assays wereperformed with alcohol dehydrogenase (ADH) underheat stress and insulin under reduction stress in the pres-ence of varying concentrations of Hsp25.Yeast ADH is a tetramer of four equal subunitswith a total molecular mass of 141 kDa [43]. Thermalstress assays using this enzyme are commonly per-formed at temperatures of 48–60 °C. The optimal rateof precipitation of yeast ADH for monitoring precipi-tation was found to be 55 °C (not shown), and theinactivation and precipitation of yeast ADH at thistemperature have been well characterized [44]. Thistemperature was also well below the onset of aggrega-tion and precipitation for wild-type and mutant Hsp25proteins, as shown by thermostability studies. Anyprecipitation observed was therefore not attributableto Hsp25 instability.Complete suppression [45] of yeast ADH precipita-tion was observed for wild-type Hsp25 at a molar ratioof 1.4 : 1.0 Hsp25 : ADH (Fig. 6). A decrease in sup-pression of ADH precipitation was observed at thisratio for all of the glutamic acid residue mutants. Atall other ratios, the E199A mutant showed minorreductions in suppression of ADH precipitation com-pared with wild-type Hsp25, whereas the E190A andE204A mutants showed markedly reduced suppression.The Q194A mutant showed similar levels of suppres-sion of aggregation to the wild-type protein at allratios.Precipitation of insulin can be initiated by the addi-tion of a reducing agent, such as dithiothreitol, whichcleaves the disulfide bonds between the A and B chainsof 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 beperformed at physiological temperatures, i.e. 37 °C.The precipitation of insulin under reduction stress wascompletely suppressed by wild-type Hsp25 at a molarratio of 0.5 : 1.0 Hsp25 : insulin (Fig. 7), with allmutants showing comparable suppression of insulinprecipitation at this ratio. All of the glutamic acidresidue mutants, in particular the E204A mutant,displayed reduced suppression at the lower ratio(0.05 : 1.0), and the E190A mutant showed reducedsuppression at 0.25 : 1.0. At all ratios, the Q194Amutant exhibited very similar levels of suppression ofinsulin B chain precipitation to the wild-type protein.Taken together, the thermal and reduction stressassays demonstrate that each of the glutamic acidmutants, in particular E190A and E204A, are signifi-cantly less effective chaperones than is wild-typeHsp25 (Table 2).DiscussionMany proteins contain intrinsically disordered regionsthat 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 flexibleregions located at the extremity of the C-terminalextensions of human and murine sHsps. Despite theFig. 5. Thermostability profiles of wild-type ( ), Q194A ( ),E190A (), E199A ( ) and E204A ( ) Hsp25. Sampleswere prepared to a final concentration of 0.2 mgÆmL)1in 50 mMsodium phosphate buffer (pH 7.3). The temperature was increasedat a rate of 1 °CÆmin)1. Wild-type Hsp25 and Q194A remained insolution up to temperatures of 100 °C. By contrast, the E190A,E199A and E204A mutants precipitated out of solution within 2 °Cof the onset of precipitation at 68 °C for E190A and 70 °C forE199A and E204A.Glutamic acid mutants of Hsp25 A. M. Morris et al.5890 FEBS Journal 275 (2008) 5885–5898 ª 2008 The Authors Journal compilation ª 2008 FEBSlow sequence similarity, these regions are abundant indisorder-promoting residues, such as proline [48]. Thehydrophilicity, 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 withtarget proteins. The unstructured and highly dynamicnature of this flexible region also ensures that it doesnot interfere with or block the preceding conservedIXI motif, which is important in subunit–subunit inter-actions [49].Despite the low sequence similarity throughout thesHsp family, Hsp25 has a very similar, predominantlyb-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 anunstructured 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 theC-terminal extensions were swapped, have been shownto have secondary structures similar to each other andto their wild-type counterparts [52]. Similarly, theremoval of the C-terminal extension of aA-crystallin,aB-crystallin and bacterial Hsp16.3 produces proteinswith secondary structure comparable with that of therespective wild-type proteins [17,53,54] The secondarystructure of wild-type and mutant Hsp25 did notchange significantly from 25 to 55 °C, consistent withprevious findings that Hsp25, a-crystallin and IpbBresist changes to secondary structure with increasingtemperatures up to approximately 60 °C [50,55,56].The secondary structure of Hsp25 has also been shownto be stable under mildly denaturing conditions [40],and temperatures of at least 60 °C are required for aloss of b-sheet structure [40].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 °Cin50mM sodium phos-phate buffer (pH 7.3) containing 0.02% NaN3. Traces are the average of duplicates. The precipitation of ADH was completely suppressed atan Hsp25 : ADH ratio of 1.4 : 1.0. The Q194A mutant showed comparable chaperone activity with the wild-type protein. Each of theglutamic acid residue mutants displayed a decrease in chaperone activity compared with wild-type Hsp25.A. M. Morris et al. Glutamic acid mutants of Hsp25FEBS Journal 275 (2008) 5885–5898 ª 2008 The Authors Journal compilation ª 2008 FEBS 5891Although the secondary structure of Hsp25 was notaltered as a result of the mutations, significant differ-ences in exposed hydrophobicity, as indicated byANS binding, were observed, suggesting that thesame elements of secondary structure were adopted,but that the subunits were arranged differently fromthat of the wild-type protein. Because five of the sixtryptophan residues in Hsp25 are located in theN-terminal domain, the comparable overall trypto-phan exposure in the mutants compared with thewild-type protein indicates that the structure of theN-terminal domain is maintained, at least in thevicinity of the tryptophan residues. The increase inexposed clustered hydrophobicity observed for themutants 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 E190Amutant compared with the wild-type suggests that theE190 residue is important for the correct formation ofthe quaternary structure of Hsp25. Examination of thecrystal structures of Hsp16.9 and Hsp16.5, which donot have flexible C-terminal extensions, shows that theconserved IXI motif (residues 185–187 in Hsp25) formshydrophobic contacts with a groove between b-strandsin the a-crystallin domain of another monomer, andthat this interaction is essential for the oligomerizationof sHsps [57]. Truncation from the C-terminus ofaA-crystallin to remove the IXI motif renders the pro-tein unable to form oligomers [58], indicating thatinteractions involving the IXI motif are also essentialfor the oligomerization of mammalian sHsps contain-ing a flexible C-terminal extension. Because of theproximity between the E190 residue of Hsp25 and theFig. 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 °Cin50mM sodium phos-phate buffer (pH 7.3) containing 0.02% NaN3. Traces are the average of triplicates. The precipitation of insulin was completely suppressedat an Hsp25 : insulin ratio of 0.5 : 1.0. The Q194A mutant showed comparable chaperone activity with the wild-type protein. Each of theglutamic acid residue mutants displayed a decrease in chaperone activity compared with wild-type Hsp25.Glutamic acid mutants of Hsp25 A. M. Morris et al.5892 FEBS Journal 275 (2008) 5885–5898 ª 2008 The Authors Journal compilation ª 2008 FEBSIXI motif, it is possible that substitution of this residuedisrupts the interaction between the IXI motif and thehydrophobic groove, resulting in an altered oligomericstructure. The formation of large sHsp oligomers isalso dependent on interactions between N-terminaldomains [23], which are not affected by mutations inthe C-terminal extension. The comparability of oligo-meric sizes between the mutants and wild-type Hsp25clearly demonstrates this.In mammalian sHsps, the presence of a flexible, sol-vent-exposed C-terminal extension helps to counteractthe large amount of hydrophobicity exposed by theremainder of the protein [15,26]. Removal of theC-terminal extension results in a decrease in thermo-stability of Hsp25, aA-crystallin, Xenopus Hsp30C andCaenorhabditis Hsp16-2 [17–20]. The drasticallyreduced thermostability of the glutamic acid residuemutants demonstrates the importance of each of thenegatively charged residues in the C-terminal extensionin maintaining the solubility of the Hsp25 oligomer.Similarly, the introduction of hydrophobicity into theC-terminal extension of aA-crystallin results in adecrease in the thermostability of this sHsp [21]. Thesedata suggest that relatively modest alterations to theC-terminal extensions of sHsps, resulting in a decreasein polarity, are sufficient to disrupt the ability of theC-terminal extensions to efficiently act as solubilizers.At temperatures above 60 °C, Hsp25 and the a-crys-tallins undergo changes in their tertiary structure thatresult in the exposure of hydrophobic regions [56,59].The onset of precipitation of the glutamic acid residuemutants of Hsp25 corresponds approximately to thistemperature. The temperature-induced increase inhydrophobic exposure did not induce the precipitationof wild-type Hsp25, although a small increase in lightscattering implies the formation of larger aggregates[41]. Thermostable proteins display more effective bur-ial of hydrophobic regions than do less thermostableproteins [28]. The increase in exposed hydrophobicityassociated with the mutations, coupled with thetemperature-induced increase in hydrophobicity, isconsistent with the poor thermostability observed forthe glutamic acid residue mutants.The glutamic acid residue mutants showed reducedchaperone activity compared with wild-type Hsp25towards 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]. TheE190A and E204A mutants performed poorly comparedwith the wild-type protein in both assays, most notablyat the lower Hsp25 : target protein ratios used. TheE199A mutant showed somewhat decreased chaperoneactivity towards ADH under heat stress, but performedpoorly at lower ratios towards insulin under reductionstress. These functional differences were not a result ofthe lack of solubilization of the chaperone, as both wild-type Hsp25 and the glutamic acid residue mutants ofHsp25 were stable in solution at the temperatures atwhich these assays were performed. Recognition of andinteraction with target proteins by sHsps is largelyhydrophobic in nature [13]. On this basis, it would beexpected that the Hsp25 mutants, with increased surfacehydrophobicity, would display enhanced chaperoneability compared with the wild-type protein [51].However, the structural changes associated with themutations appear to have a greater influence on thechaperone activity than simply the degree of exposedhydrophobicity [62].Although conclusive identification of the chaperonebinding sites of sHsps remains elusive, there is evidencethat the binding of target proteins occurs in the groovebetween monomers, and involves a b-sheet regionlocated 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 themutants, as evidenced by the alteration in exposedhydrophobicity, could result in the disruption of bind-ing sites, and thus hindered recognition and sequestra-tion of target proteins. These changes may also inhibitstabilization by electrostatic interactions, resulting inless effective target protein sequestration. The decreasein polarity of the C-terminal extension may also facili-tate interaction between the extension and hydro-phobic chaperone binding sites, resulting in thebinding sites being less accessible to the target protein,leading to a decrease in target protein binding [26].In summary, the three negatively charged glutamicacid residues in the C-terminal extension of Hsp25(E190, E199 and E204) are essential for the correctstructure and function of this sHsp. These residuescontribute to the polarity of the extension and pro-mote its disorder, ensuring that the C-terminal exten-sion remains unstructured and solvent exposed, andtherefore able to perform its solubilizing role in thesHsp and in the complexes formed with target proteinsduring chaperone action. Indeed, the presence of sig-nificant regions of structural disorder is a commoncharacteristic of molecular chaperones, and is integralto their effective chaperone action [65]. Despite analteration in exposed hydrophobicity, the Q194Amutant 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 performits role as a molecular chaperone, emphasizing theimportance of the glutamic acid residues.A. M. Morris et al. Glutamic acid mutants of Hsp25FEBS Journal 275 (2008) 5885–5898 ª 2008 The Authors Journal compilation ª 2008 FEBS 5893Experimental proceduresSequence analysis of the C-terminal extension ofmammalian sHspsThe C-terminal extensions of the 10 human sHsps [66] werealigned according to their IXI motifs, when present. Whenabsent, the alignments were based on those of Fontaineet al. [67] and Franck et al. [68]. Residues that aligned withthe known flexible regions of aA- and aB-crystallin [16]were tallied. The C-terminal extensions of the equivalentmurine sHsps were similarly analysed.Site-directed mutagenesis of pAK3038-Hsp25Site-directed mutagenesis was performed using the Quik-ChangeÒsystem (Stratagene, La Jolla, CA, USA), accord-ing to the manufacturer’s instructions, except that 14 cycleswere used (Cooled-Palm 96, Corbett Research, Mortlake,NSW, Australia). All primers were synthesized by SigmaGenosys (Castle Hill, NSW, Australia). The primer pairsfor site-directed mutagenesis were as follows: 5¢-TTCGAGGCCCGCGCCGCAATTGGGGGCCCAGAA-3¢ and 5¢-TTCTGGGCCCCCAATTGCGGCGCGGGCCTCGAA-3¢for E190A, 5¢-ATTCCGGTTACTTTCGCGGCCCGCGCCCAAATT-3¢ and 5¢-AATTTGGGCGCGGGCCGCGAAAGTAACCGGAAT-3¢ for E190A, 5¢-CAAATTGGGGGCCCAGCAGCTGGGAAGTCTGAA-3¢ and 5¢-TTCAGACTTCCCAGCTGCTGGGCCCCCAATTTG-3¢ for E199A,and 5¢-GAAGCTGGGAAGTCTGCACAGTCTGGAGCCAAG-3¢ and 5¢-CTTGGCTCCAGACTGTGCAGACTTCCCAGCTTC-3¢ for E204A. Mutated codons are shown initalic type. Dimethylsulfoxide was added to a final concen-tration of 5% (v ⁄ v) to reactions in which strong secondaryinteractions were likely, as advised by the supplier. Success-ful mutagenesis was confirmed by DNA sequence analysisof the forward and reverse strands with BigDyeÔ Termina-tor Ready Reaction Mix (Applied Biosystems, Foster City,CA, USA) on a Prism 377 DNA sequencer (AppliedBiosystems) using the primers 5¢-TCTCGGAGATCCGACAGA-3¢ and 5¢-CTTTCGGGCTTTGTTAGCAG-3¢,respectively.Expression and purification of wild-type andmutant Hsp25pAK3038-Hsp25 was a gift from M. Gaestel (Institute ofBiochemistry, Hannover, Germany). DNA was transformedinto electrically competent BL21(DE3) Escherichia colibefore expression. Expression and purification of murineHsp25 and mutants were performed according to themethod described by Horwitz et al. [69] with minorchanges. Transformed cells were grown in Luria–Bertanimedium containing 0.4% (w ⁄ v) glucose and 100 lgÆmL)1ampicillin to select for pAK3038-Hsp25. Protein expressionwas induced with 0.4 mm isopropyl thio-b-d-galactoside.Cells were harvested by centrifugation and lysed asdescribed. After ultracentrifugation, dithiothreitol, polyeth-yleneimine and EDTA were added to the supernatant tofinal concentrations of 10 mm, 0.12% (v ⁄ v) and 1 mm,respectively, and the lysate was incubated and centrifugedas described. The final supernatant was filtered through a0.22 lm Minisart filter (Sartorius, Epsom, UK) beforebeing loaded onto a DEAE-Sephacel (Sigma-Aldrich,St Louis, MO, USA) column with a volume of approxi-mately 90 mL. Recombinant Hsp25 was eluted with100 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 mLand dithiothreitol was added to a final concentration of50 mm. The sample was incubated at room temperature for30 min before being loaded onto a Sephacryl S-300HR(Pharmacia, Uppsala, Sweden) column with a volume ofapproximately 470 mL. Recombinant Hsp25 eluted in thefirst peak with 50 mm Tris ⁄ HCl buffer (pH 8.0) containing1mm EDTA and 0.02% (w ⁄ v) NaN3. Fractions containingHsp25 were concentrated, dialysed exhaustively against,or exchanged into, MilliQ water and lyophilized. Bothchromatographic steps were performed at 4 °C. The purityof recombinant proteins was confirmed by nanoscaleESI-MS.Far-UV CD spectroscopyCD spectra were acquired on a J-810 spectropolarimeter(Jasco, Tokyo, Japan) with an attached Peltier temperature-controlled water circulator. Samples were prepared in10 mm phosphate buffer (pH 7.5) to a final concentrationof 10–15 lm and filtered through a 0.22 lm Minisart filter.Spectra were recorded at 25, 37 and 55 °C, and are accu-mulations of 16 scans recorded from 190 to 250 nm with apath length of 1 mm. The sample concentration was deter-mined using a bicinchoninic acid assay (Sigma-Aldrich). Anestimation of secondary structure composition wasperformed using the cdsstr program [70–72] in theDICHROWEB Online Circular Dichroism Analysis suite[73,74].Intrinsic tryptophan fluorescence and ANSbinding fluorescence spectroscopyAll fluorescence studies were performed at 25 °C using anF-4500 fluorescence spectrophotometer (Hitachi High-Tech-nologies, Tokyo, Japan) with a Thermomix temperature-controlled water circulator (B. Braun, Melsungen,Germany). Samples were prepared in 50 mm phosphatebuffer (pH 7.3) containing 0.02% (w ⁄ v) NaN3to a finalconcentration of 5 lm, as calculated from A280values ofthe samples, an extinction coefficient of 1.87 for a1mgÆmL)1solution of Hsp25 [75] and molecular mass. AnGlutamic acid mutants of Hsp25 A. M. Morris et al.5894 FEBS Journal 275 (2008) 5885–5898 ª 2008 The Authors Journal compilation ª 2008 FEBS[...]... 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Glutamic acid residues in the C-terminal extension of small heat shock protein 25 are critical for structural and functional integrity Amie. 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
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