Báo cáo khoa học: Structural and functional roles for b-strand 7 in the a-crystallin domain of p26, a polydisperse small heat shock protein from Artemia franciscana pdf

Structural and functional roles for b-strand in the a-crystallin domain of p26, a polydisperse small heat shock protein from Artemia franciscana Yu Sun, Svetla Bojikova-Fournier and Thomas H MacRae Department of Biology, Dalhousie University, Halifax, NS, Canada Keywords a-crystallin domain; Artemia franciscana; molecular chaperone; p26 structure ⁄ function; small heat shock protein Correspondence T H MacRae, Department of Biology, Dalhousie University, Halifax, NS, Canada B3H 4J1 Fax: +1 902 4943736 Tel: +1 902 4946525 E-mail: tmacrae@dal.ca (Received July 2005, revised 26 December 2005, accepted January 2006) doi:10.1111/j.1742-4658.2006.05129.x Oviparous development in the extremophile crustacean, Artemia franciscana, generates encysted embryos which enter a profound state of dormancy, termed diapause Encystment is marked by the synthesis of p26, a polydisperse small heat shock protein thought to protect embryos from stress In order to elucidate structural ⁄ functional relationships within p26 and other polydisperse small heat shock proteins, and to better define the protein’s role during diapause, amino acid substitutions R110G, F112R, R114A and Y116D were generated within the p26 a-crystallin domain by site-directed mutagenesis These residues were chosen because they are highly conserved across species boundaries, and molecular modelling indicates that they are part of a key structural interface between dimers The F112R mutation, which had the greatest impact on oligomerization, placed two charged residues at the p26 dimer–dimer interface, demonstrating the importance of b-strand in tetramer formation All mutated versions of p26 were less able than wild-type p26 to confer thermotolerance on transformed bacteria and they exhibited diminished chaperone action in three in vitro assays; however, all variants retained protective activity This apparent stability of p26 may, by prolonging effective chaperone life in vivo, enhance embryo stress resistance All substitutions modified p26 intrinsic fluorescence, surface hydrophobicity and secondary structure, and the pronounced changes in variant R114A, as indicated by these physical measurements, correlated with the greatest loss of function Although mutation R114A had the greatest effect on p26 chaperoning, it had the least on oligomerization These results demonstrate that in contrast to many other small heat shock proteins, p26 effectiveness as a chaperone is independent of oligomerization The results also reinforce the idea, occasioned by modelling, that R114 is removed slightly from dimer–dimer interfaces Moreover, b-strand is shown to have an important role in oligomerization of p26, a function first proposed for this structural element upon crystallization of wheat Hsp16.9, a small heat shock protein with different quaternary structure Protein folding and maintenance of an appropriate 3D structure occur with the assistance of molecular chaperones, including Hsp60 (chaperonins), Hsp70, Hsp90, Hsp104 ⁄ ClpB, Hsp110 and the small heat shock proteins (sHSPs) [1–6] Several chaperones are actively involved in protein folding, whereas others, and in particular the sHSPs, protect proteins during stresses such as heat shock, oxidation and hypoxia ⁄ anoxia Molecular chaperones also remove damaged proteins through the action of CHIP, a ubiquitin ligase [6] Abbreviations ANS, 1-anilino-8-naphthalene-sulphonate; sHSP, small heat shock protein 1020 FEBS Journal 273 (2006) 1020–1034 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Sun et al sHSPs usually occur as oligomers composed of subunits ranging in molecular mass from 12 to 43 kDa, and they protect proteins from irreversible denaturation independent of ATP [3,7–13] The conserved a-crystallin domain of  90 amino acid residues, located towards the C terminus, is important for oligomer formation and chaperoning [14–16] The a-crystallin domain is preceded by a poorly conserved N-terminal region proposed to function in oligomer assembly, subunit exchange and substrate binding [17– 22], and is followed by a flexible, polar, C-terminal extension of variable sequence that influences solubility and oligomerization [17,21,23–25] sHSP secondary structure is dominated by b-pleated sheet, but the quaternary structure is variable [26,27] Hsp16.5 from the archaeon, Methanococcus jannaschii, and Hsp16.9 from wheat, Triticum aestivum, assemble monodisperse oligomers and they have been crystallized, revealing important sHSP structural attributes [14,16] sHSPs, most of which form polydisperse oligomers, interact with several substrates and a reservoir of intermediates accrues, a progression involving oligomer dissociation and subunit exchange [19,28–30], but which may also occur upon rearrangement of oligomer structure in the absence of dissociation [31] When stress is relieved, substrates are released and renatured, processes occurring spontaneously or with assistance from other molecular chaperones [32,33] The sHSPs influence cytoskeleton organization [34–36], apoptosis [37–40] and development [7,41], thereby playing important roles in cell activities Artemia females release offspring as swimming larvae (ovoviviparous development) or encysted gastrulae (oviparous development), termed cysts The cysts enter diapause, a resting stage where metabolic activity is extremely low, even under favourable conditions [41] Activation of encysted embryos by desiccation precedes reinitiation of development in the presence of appropriate hydration, temperature and aeration Artemia cysts are exceptionally resistant to harsh conditions, and when fully hydrated, either during diapause or in a postdiapause state of metabolic arrest, termed quiescence, they survive for several years without oxygen This is arguably the ultimate indifference to anoxia of any metazoan [42], and qualifies the organism, as other of its characteristics, as an extremophile Because activated Artemia embryos resume development immediately upon return to favourable circumstances, macromolecular components must be preserved in the presence of limiting ATP, an activity within sHSP functional capability Just such a protein, named p26, is synthesized in large quantities by oviparous, but not ovoviviparous, embryos [17,41,43–46] a-crystallin domain of p26 The p26 a-crystallin domain is similar in sequence to this region in other sHSPs, including wheat Hsp16.9; the protein confers thermotolerance on transformed Escherichia coli and it has chaperone activity in vitro The objectives of the work described here are to reveal structural and functional characteristics of polydisperse sHSPs by introducing single-site mutations in p26, and to better define the relationship between p26 and stress resistance in A franciscana The amino acids selected for study are highly conserved from species to species (Fig 1); at least one causes disease when mutated [47–52] and, as indicated by molecular modelling, they reside in a key structural interface, suggesting that their modification will affect oligomerization and chaperoning The role of b-strand in oligomerization was demonstrated in this work Additionally, as for other sHSPs, changing the conserved p26 a-crystallin domain arginine (R114) reduced chaperone activity, but in this case with only a minor effect on oligomerization This showed, in concert with analysis of the F112R mutation, that oligomerization and chaperoning are not linked in p26 The resistance of p26 chaperoning activity to single-site mutations suggests a stable protein and this, in concert with the large amount of p26 present during oviparous development, undoubtedly contributes to the remarkable stress resistance of encysted Artemia embryos Results Site-directed mutagenesis and purification of bacterially produced p26 cDNAs encoding the amino acid substitutions R110G, F112R, R114A and Y116D in the a-crystallin domain of p26 were cloned in the prokaryotic expression vector, pPROTet.E233, and used to transform E coli BL21PRO Sequencing demonstrated that each p26 cDNA contained only the introduced substitution p26 synthesized in bacteria possessed an N-terminal His-tag and an additional short N-terminal peptide (PRAAGIRHELVLK) encoded by the clone used for site-directed mutagenesis, but comparisons throughout the study to p26 from Artemia and transfected mammalian cells lacking these residues indicated that they had almost no effect on structure and function Cellfree extracts prepared from transformed bacteria induced with anhydrotetracycline (aTc) exhibited lightly stained bands, corresponding in size to p26 when electrophoresed in SDS polyacrylamide gels and stained with Coomassie blue, and these polypeptides reacted with anti-p26 immunoglobulin (Fig 2A,B) Expression levels in bacteria were very similar for all FEBS Journal 273 (2006) 1020–1034 ª 2006 The Authors Journal compilation ª 2006 FEBS 1021 a-crystallin domain of p26 Y Sun et al Fig Multiple sequence alignment of representative small heat shock proteins (sHSPs) The amino acid sequences of selected sHSPs were analyzed by CLUSTAL W (1.82) HHSP27, Homo sapiens Hsp27, P04792; MHSP25, Mus musculus Hsp25, P14602; HCRYAA, H sapiens aA-crystallin, P02489; HCRYAB, H sapiens aB-crystallin, P02511; Ap26, Artemia franciscana p26, AF031367; and WHSP16.9, wheat Hsp16.9, 1GME sHSP domains based on the sequence of p26 are indicated above the alignment, secondary structure elements based on the sequence of wheat Hsp16.9 are below the alignment, and the conserved a-crystallin domain amino acid residues selected for mutational analysis are shaded Residue number is indicated on the right –, no residue; *, identical residues; :, conserved substitution; , semiconserved substitution variants and there was no indication of protein degradation After purification on TALONtm affinity columns, a major polypeptide of the expected size recognized by anti-p26 immunoglobulin was obtained for each variant (Fig 2C,D) p26 in COS-1 cells Fig Purification of p26 synthesized in Escherichia coli BL21PRO Cell-free extracts from transformed E coli BL21PRO induced with anhydrotetracycline (aTc) were electrophoresed through SDS polyacrylamide gels and either stained with Coomassie blue (A) or blotted to nitrocellulose and reacted with antibody to p26 (B) Proteins purified by affinity chromatography were electrophoresed through SDS polyacrylamide gels and either stained with Coomassie blue (C), or blotted to nitrocellulose and reacted with antibody to p26 (D) Lane 1, R110G; lane 2, F112R; lane 3, R114A; lane 4, Y116D; lane 5, wild-type (WT) p26; lane 6, vector lacking p26 cDNA Lanes in panels A and B received 4.5–5.5 lg of protein and lanes in panels C and D received lg of protein Arrow, p26 M, molecular mass markers of 97, 66, 45, 31, 21 and 14 kDa 1022 COS-1 cells were transiently transfected with p26 cDNAs cloned in the eukaryotic expression vector, pcDNA4 ⁄ TO ⁄ myc-His.A, and p26 synthesis was verified by immunofluorescent staining and confocal laserscanning microscopy (Fig 3) Wild-type (WT) p26 was localized predominantly, if not exclusively, in the cytoplasm of transfected cells In contrast, all COS-1 cells transfected with cDNA containing the R114A mutation had p26 in nuclei as well as in the cytoplasm (Fig 3) p26 R110G, F112R and Y116D occurred in the cytoplasm and nuclei of transfected cells, although some nuclei lacked the protein (not shown) p26 was subsequently prepared from transfected COS-1 cells for determination of oligomer size FEBS Journal 273 (2006) 1020–1034 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Sun et al a-crystallin domain of p26 A Fig p26 synthesis in COS-1 cells COS-1 cells transiently transfected with the p26 cDNA-containing vector pcDNA4/TO/myc-His.A were incubated with antibody to p26 followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (green) Nuclei were stained with propidium iodide (red) The scale bar represents 100 lm and both figures are the same magnification B p26 oligomerization WT p26 produced the largest oligomers, while, for modified proteins, oligomer size was greatest for R114A and smallest for F112R (Fig 4; Table 1) The molecular mass of p26 variants synthesized in bacteria was unaffected by purification (Fig 4A,B), indicating that the methods employed had little effect on protein structure, an important observation in relation to analysis of chaperone function Except for WT p26, the maximum monomer number per oligomer was higher for p26 synthesized in COS-1 cells than in bacteria, but the variation, although observed consistently, was minor (Fig 4, Table 1), indicating little difference between the proteins from either source Of equal significance, the F112R substitution greatly reduced p26 oligomer size upon synthesis in COS-1 cells, demonstrating that results obtained upon synthesis in bacteria were not specific to the organism or the recombinant construct Amino acid substitutions in the p26 a-crystallin domain reduced chaperone activity Although all p26 variants conferred thermotolerance on bacteria, WT p26 was the most effective (Fig 5A) Thermotolerance levels induced by R110G, F112R and Y116D were similar to (P > 0.05) and significantly higher than those conferred by R114A (P < 0.05), which provided the least protection Bacteria lacking p26 failed to survive the 60 heat shock WT p26 at 1.6 lm, representing a chaperone to substrate molar ratio of 0.4 : if monomers are compared, almost completely prevented dithiothreitol-induced insulin aggregation at 25 °C, and even at 0.1 lm p26 aggregation was inhibited by more than 40% C Fig p26 oligomer formation Bacterially produced p26 either before (A) or after (B) purification, and p26 synthesized in transfected COS-1 cells (C), were centrifuged at 200 000 g for 12 h at °C in 10–50% (w ⁄ v) continuous sucrose gradients The gradients were fractionated and 15-lL samples from each fraction were electrophoresed in SDS polyacrylamide gels, blotted to nitrocellulose and reacted with antibody to p26 followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG The top of each gradient is to the right and fractions are numbered across the top The positions of the molecular mass markers alpha-lactalbumin, 14.2 kDa; carbonic anhydrase, 29 kDa; BSA, 66 kDa; alcohol dehydrogenase, 150 kDa; apoferritin, 443 kDa; and thyroglobulin, 669 kDa, are indicated by labeled arrows (Fig 5B) At all concentrations, WT p26 prevented insulin aggregation the most and R114A the least, followed by F112R, Y116D and R110G, with the latter two not significantly different from one another Chaperoning of insulin by p26 purified from Artemia [45] and E coli was very similar, whereas BSA and IgG at 1.6 lm had no effect on dithiothreitol-induced insulin aggregation (not shown) At 600 nm, a chaperone to target (monomer to dimer) molar ratio of : 1, WT p26 inhibited citrate synthase aggregation almost completely after h at 43 °C (Fig 5C) R110G, Y116D and F112R were progressively less effective in protecting citrate FEBS Journal 273 (2006) 1020–1034 ª 2006 The Authors Journal compilation ª 2006 FEBS 1023 a-crystallin domain of p26 Y Sun et al Table Characteristics of p26 oligomers The molecular mass of p26 oligomers produced in transformed Escherichia coli BL21PRO and transfected COS-1 cells was determined by sucrose density gradient centrifugation Monomer mass refers to the mass of p26 monomers and was calculated using a p26 molecular mass of 20.8 kDa, as determined by GENERUNNER (version 3.05, Hastings Software, Inc.) with corrections for protein modifications Oligomer mass range represents the smallest to largest oligomers observed, and maximum monomer number refers to the number of subunits in oligomers of maximum mass Oligomer mass range (kDa) Maximum monomer number Expression system p26 mutant Monomer mass (kDa) E coli R110G F112R R114A Y116D WT 25.4 25.5 25.4 25.5 25.5 29–443 29–150 29–500 29–443 29–669 17 20 17 26 COS-1 cells R110G F112R R114A Y116D WT 20.7 20.8 20.7 20.8 20.8 14.2–443 14.2–150 14.2–500 14.2–443 14.2–500 21 24 21 24 synthase, with the latter two not significantly different from one another However, the modified p26 variants exhibited appreciable chaperone activity, and at 1200 nm the variants were almost as good as WT p26 (Supplementary Fig 1) R114A provided the least protection, but was still about 60% as potent as WT p26 at 600 nm WT p26 also shielded citrate synthase enzyme activity against heat-induced inactivation better than mutated p26, and at 1200 nm the activity remaining was essentially the same as in unheated preparations (Fig 5D) R114A was the least effective of all p26 variants in protecting enzyme activity, although differences disappeared at lower concentrations Of the remaining p26 mutants, chaperone activity decreased from R110G to Y116D, which were similar, and then to F112R Chaperone activities of p26 from Artemia [45] and E coli with citrate synthase were similar, whereas BSA and IgG at 1200 nm neither prevented citrate synthase aggregation nor preserved enzyme activity (data not shown) To summarize, as determined by thermotolerance induction in E coli, dithiothreitol induced insulin aggregation at 25 °C, heat-induced citrate synthase aggregation at 43 °C, and maintenance of citrate synthase enzyme activity at 43 °C, WT p26 possessed the greatest chaperone activity and R114A the least It is noteworthy, however, that all p26 variants protected bacteria and substrate proteins in vitro 1024 Modification of p26 structure by amino acid substitutions Measurement of intrinsic fluorescence demonstrated that the maximum emission peak for each p26 mutant was less than for WT p26 (Fig 6A) Three variants (R110G, F112R and Y116D) had very similar emission spectra, whereas R114A possessed the lowest fluorescence and the emission was red-shifted The results indicate altered microenvironments for aromatic amino acid residues, such as tryptophan, of which two reside in the p26 N-terminal region at positions and 17, with the greatest change caused by the R114A modification All mutated versions of p26 exhibited less 1-anilino-8-naphthalene-sulphonate (ANS)-binding than WT p26, an indication of reduced surface hydrophobicity (Fig 6B), with R114A at the lowest level The enhancement of surface hydrophobicity by increasing the temperature from 25 °C to 43 °C was reduced for the p26 variants in comparison to WT p26 (Fig 6B) Additionally, as shown by far-UV CD, the spectra for p26 a-crystallin domain mutants possessed wider, more negative, shoulders ranging from 208 to 230 nm, and with one exception, a related positive shoulder peaking near 194 nm (Fig 7A,B) R114A exhibited the greatest variation from WT in CD spectra, this reflecting decreased b-structure content and an increase in a-helical constituents (Table 2) WT p26 from E coli and Artemia gave comparable fluorescence intensities in ANS-binding experiments at 25 °C and 43 °C (Fig 6B) The far-UV CD spectra of p26 from both sources were characteristic of b-sheet enrichment, with a negative shoulder near 214 nm and a positive shoulder near 194 nm (Fig 7A) The only indication of a difference was the slight red-shifted intrinsic fluorescence of bacterial WT p26; however, the fluorescence intensities for p26 from bacteria and Artemia were similar (Fig 6A) This structural resemblance, and the functional analysis mentioned previously, indicate data obtained by examining bacterially produced p26 are indicative of the protein synthesized in Artemia Localization of amino acid substitutions within p26 The p26 tetramer, modeled on the 3D crystal structure of wheat Hsp16.9, consists of two dimers, with monomers A and B in dimer and C and D in dimer (Fig 8) The a-crystallin domain of each monomer is composed of nine b-strands (labeled b2–b10), with the b6 strand situated in a large loop, L5 ⁄ 7, located between b-strands and b-strand 10 inhabits FEBS Journal 273 (2006) 1020–1034 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Sun et al a-crystallin domain of p26 A B C D Fig Chaperone activity of p26 (A) Transformed Escherichia coli BL21PRO cells were incubated at 54 °C for h with samples removed periodically, diluted, and plated in duplicate on Luria–Bertani (LB) agar followed by incubation at 37 °C for 16 h The log10 values of colonyforming units (CFU) per ml were plotted against heat shock in Bacteria containing the pPROTet.E233 vector lacking p26 cDNA did not survive the entire 60 Standard errors ranged from 3.3 to 7.1% (B) Bacterially produced p26 purified to apparent homogeneity was incubated with insulin for 30 in the presence of dithiothreitol, and solution turbidity was measured at 400 nm The p26 variants tested are indicated in the figure and they appear in the same order in each histogram group The standard error ranged from 4.2 to 5.8% (C) Purified, bacterially produced p26 at 600 nM was heated at 43 °C for h with 150 nM citrate synthase and the solution turbidity was measured at 360 nm The A360 values were multiplied by 1000 for construction of the curves The standard error ranged from 4.2 to 7.0% (D) Citrate synthase at 150 nM was heated at 43 °C for h in either the absence or presence of p26, and then enzyme activity was determined p26 concentrations are indicated and the p26 variants are in the same order in each histogram group The standard error ranged from 3.3 to 10.0% C-terminal extensions which extend to neighboring monomers, and the remaining b-strands, with the exception of strand 6, are arranged in two antiparallel beta sheets within the a-crystallin domain The interface between monomers of a dimer involves interaction between strands b2 and b6 of neighboring monomers The p26 modifications examined in this study are not located in either of these strands and they are not considered further As the basic p26 oligomer building block, dimers interact to form tetramers, the next level of structure Modeling indicates that tetramer formation depends upon contact of b-strand 10 from the C-terminal extensions of monomers A and D with b-strands and in the a-crystallin domain of monomers C and B, respectively (Fig 8) A more prominent dimer–dimer interface occurs with b-strand of monomer B interacting with loop L5 ⁄ of monomer C, and b-strand of monomer C reacting with L5 ⁄ of monomer B, regions of high similarity between p26 and Hsp16.9 (Fig 8) The p26 residues examined in this study are situated in b-strand 7, with mutations R110G and F112R directly in the dimer–dimer interface As a result of the spatial disposition of monomers and their b-strand elements in the a-crystallin domain, the amino acid substitution F112R introduces two changes at the dimer–dimer interface Although Y116 and R114 are in b-strand 7, neither is shown by the model to reside directly in the dimer–dimer interface Modification of these FEBS Journal 273 (2006) 1020–1034 ª 2006 The Authors Journal compilation ª 2006 FEBS 1025 a-crystallin domain of p26 Y Sun et al A B Fig Tertiary structure perturbation of p26 (A) The intrinsic fluorescence of purified p26 diluted in 10 mM NaH2PO4, pH 7.1, to 0.06 mgỈmL)1 was determined The excitation wavelength was 280 nm, with a 2-nm band pass, and fluorescence emission was detected from 310 to 400 nm The standard error ranged from 3.5 to 7.5% (B) Surface hydrophobicity of purified p26 at 0.06 mgỈmL)1 in 10 mM NaH2PO4, pH 7.1, was determined by oversaturation with 1-anilino-8-naphthalene-sulphonate (ANS) Fluorescence was measured at an excitation wavelength of 388 nm and band pass of nm, with emission wavelength at 473 nm and band pass of nm Measurements were made at either 25 °C (grey) or 43 °C (black) Fluorescence generated by buffer containing ANS, but no p26, was subtracted The standard error ranged from 6.7 to 10% Table Secondary structure elements of p26 The secondary element percentages were calculated using the CDNN v2.1 deconvolution program for each p26 variant generated by site-directed mutagenesis and for purified wild-type (WT) p26 from transformed Escherichia coli and Artemia embryos Structural elements WT WT R110G F112R R114A Y116D (E coli) (Artemia) (%) (%) (%) (%) (%) (%) a-helix b-antiparallel b-parallel b-turn Random coil 20.3 18.0 10.2 16.7 34.8 20.2 18.2 10.2 16.6 34.8 25.7 12.9 9.7 16.9 34.9 19.3 20.5 10.1 16.9 33.3 17.7 21.6 10.3 16.5 34.0 17.7 23.0 10.0 16.8 32.5 Discussion Fig Secondary structure of p26 Far-UV CD spectra were obtained for purified p26 dissolved in 10 mM NaH2PO4, pH 7.1, to 0.2 mgỈmL)1 The absorption data are expressed as molar ellipticity in degrees cm2Ỉdmol)1 (m deg), with each spectrum the average of three scans residues had little effect on oligomerization, although the R114A substitution reduced chaperoning to the greatest extent 1026 p26, an abundantly expressed, polydisperse sHSP thought to protect encysted Artemia embryos against physiological stress, was investigated by site-directed mutagenesis of a-crystallin domain residues and molecular modeling of protein structure The p26 a-crystallin domain contains nine b-strands arranged predominantly as paired b-sheets and possesses residues conserved in many other sHSPs, including those in the sequence 110REFRRRY116, where substitutions were generated Examination of mutations within the selected sequence indicated that b-strand is involved in dimer–dimer interactions, leading to higher-order oligomer structure In addition, it was concluded that p26 structural characteristics would promote Artemia survival during encystment, diapause and stress exposure FEBS Journal 273 (2006) 1020–1034 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Sun et al a-crystallin domain of p26 Fig Structural model of a p26 tetramer (A) Sequence alignment of amino acid residues 59–158 of Artemia p26 (AAB87967) and residues 45–151 of wheat, Triticum aestivum, Hsp16.9 (1GME) used to generate the 3D structural model of the p26 tetramer The proteins share 25.9% sequence identity and overall similarity of 69.4% in the regions compared The boxed residues labeled b2–b10 indicate the Hsp16.9 b-strand positions [14] and the corresponding residues in p26 Residues highlighted in yellow were modified in p26 by site-directed mutagenesis Residue numbers are given on the right (B) A structural model of the p26 tetramer generated by comparison to wheat Hsp16.9 is represented in a ribbon diagram Mutated residues Arg110, Phe112, Arg114 and Tyr116 are shown in gray in ball-and-stick and are labeled along the dimer–dimer interface by using the three-letter amino acid code in the color of the parent monomer Monomers A (green) and B (yellow) form dimer 1, while monomers C (red) and D (blue) form dimer L5 ⁄ 7, the loop between b-strands and which contains b-strand 6; N term, amino terminus of a p26 monomer; C term, carboxy terminus of a p26 monomer; the b-strands 2–10 are labeled in monomer A Oligomers for each exogenously produced p26 variant are composed of similar numbers of monomers when synthesized in mammalian and bacterial cells, and oligomerization is unaffected by protein purification, observations important for subsequent analysis of the protein in in vitro assays Single-site mutations to the p26 a-crystallin domain generally decreased oligomer size in comparison to WT p26, with mutation F112R reducing oligomerization most dramatically A tetramer model of p26 was constructed on the basis of the crystallin structure of wheat Hsp16.9 [14], a monodisperse sHSP used for modeling of human aA- and aB-crystallins [47], in order to position residues within the a-crystallin domain, better understand the consequences of amino acid substitutions, and identify protein regions involved in oligomer assembly The four modified a-crystallin domain residues are spatially close to one another in the p26 model, with R110 and F112 occupying central positions in the dimer–dimer interface The R110G mutation had relatively limited effect on oligomer size, indicating that p26, and by extrapolation, other polydisperse sHSPs tolerate charge reduction at the dimer–dimer interface The F112R modification, on the other hand, placed two positively charged residues in the dimer–dimer interface and the maximum oligomer mass dropped, as indicated by sucrose density gradient centrifugation, from 669 kDa, for WT p26, to150 kDa for the F112R variant Replacement of Y116 with negatively charged aspartic acid had limited effects on oligomerization, probably as a result of the residue’s location at the edge of the dimer–dimer interface The maximum oligomer size obtained with p26 R114A was  500 kDa, closer to the mass of the WT oligomer than the other variants This compares to oligomers of 2–4 MDa for mutation R116C of aA-crystallin and 0.7–2 MDa and larger for R120G aB-crystallin [48–53], both significant increases in mass when compared with oligomers of WT a-crystallins Modification of R114 in p26 obviously has less effect on oligomerization than equivalent substitutions in aA and aB-crystallin In agreement with the limited effect on oligomer mass and the proposed importance FEBS Journal 273 (2006) 1020–1034 ª 2006 The Authors Journal compilation ª 2006 FEBS 1027 a-crystallin domain of p26 Y Sun et al of tetramer formation in higher-order structure, the p26 model predicts R114 to be positioned slightly outside the dimer–dimer interface Interestingly, in Chinese hamster Hsp27, mutation R148G had a limited effect on chaperone activity and reduced oligomers to dimers [54], contrasting the results obtained with p26 R114A Whether this indicates fundamental differences between the two proteins awaits further study WT p26, purified from transformed bacteria, almost completely prevented heat-induced citrate synthase aggregation and loss of enzyme activity at a molar ratio of : (monomer to dimer), a result obtained previously [17] and which was similar to the activity of p26 from Artemia embryos (data not shown) Chemically induced insulin aggregation at 25 °C was inhibited at a monomer to monomer ratio of 0.4 : 1, the first measurement of p26 chaperone activity in vitro at a temperature near the optimum for Artemia growth Although it is difficult to compare chaperone activity across species owing to variation in experimental techniques, effective chaperone to substrate molar ratios determined by heating citrate synthase in the presence of other representative sHSPs are : for Bradyrhizobium japonicum sHSPs [55],  : for Caenorhabditis elegans Hsp16–2 [56], 15 : for Mycobacterium tuberculosis Hsp16.3, and : for human aB-crystallin [57] The bovine a-crystallin to substrate ratio for protection against dithiothreitol induced denaturation ranges from : for insulin and a-lactalbumin, : for BSA and 10 : for ovotransferrin, with the ratio rising as the molecular mass of the substrate increases [58] For human aB-crystallin, the ratio is : [48] The p26 chaperone activity therefore approximates that of other sHSPs and this, in concert with its abundance, provides a large capacity for storage of partially denatured proteins in oviparous Artemia embryos during diapause and quiescence Upon return of embryos to permissive conditions, proteins would be released from p26 and renatured, permitting rapid resumption of metabolism, cell growth and development, an advantage to the organism under most circumstances In contrast to a marginal impact on oligomerization, substitution R114A had the greatest detrimental effect on p26 chaperone activity in all assays R114 is probably buried within the a-crystallin domain, stabilized by a salt bridge with another charged residue(s) [59] The R114A substitution would destroy ionic linkages and expose negatively charged residues within monomer interiors, with ensuing structural changes and reduced chaperone activity In agreement with this idea, modified intrinsic fluorescence spectra and surface hydrophobicity – the latter an effector of sHSP chaperone activity [60] – indicate that p26 structural 1028 changes are greater for R114A than for other mutations Additionally, far-UV CD measurements showing decreased b-structure were most prominent for R114A, with similar observations reported for R120G in aB-crystallin [48–50] and R116C aA-crystallin at 37 °C but not 25 °C [51] Mutation R114A had the least effect on p26 oligomerization but the greatest consequence for function, demonstrating that chaperoning is independent of oligomerization All a-crystallin domain mutants, including R114A, retain significant amounts of chaperone activity In comparison, loss of chaperone activity reported upon introduction of substitution R116C into aA-crystallin ranges from 40% to almost 100% [49,51,52] The aBcrystallin mutation R120G promotes protein aggregation in in vitro turbidimetric assays, reduces in vitro chaperone activity [48–50], and decreases thermotolerance induction by 70% while promoting inclusion body formation [61], the latter not being observed for p26 R114A p26 chaperone activity appears to be more resistant to modification of this conserved a-crystallin domain arginine, suggesting that the residue is less critical than in mammalian a-crystallins where modification leads to disease [62–64] The ramifications of these observations for p26 are worthy of note For example, a-crystallins function in the mammalian lens for a lifetime, indicating, by comparison, that p26 is sufficiently stable to protect Artemia for long periods of time, as required in encysted embryos p26 oligomers synthesized in mammalian and bacterial cells are similar in size to one another and to Artemia p26, indicating that characteristics derived by studying bacterially produced p26 are reflective of the protein from Artemia Moreover, p26 localization in transfected cells is interesting because the protein migrates into Artemia nuclei during diapause and stress [65] Other sHSPs, such as Hsp20, aB-crystallin and Hsp27, access nuclei where they may be associated with speckles and nucleoli [66,67] The R120G mutation disrupts aB-crystallin speckle localization, with little of the modified protein entering nuclei [67], and there is a tendency for R120G aB-crystallin to form inclusion bodies in the cytosol [68], but this was not observed with R114A p26 Human R116C aA-crystallin occurs mainly in the cytoplasm of epithelial cells [53] How p26 enters nuclei is unknown, as is true for most, but not all, sHSPs [69] Oligomers of p26 R114A enter all COS-1 nuclei in which the protein is expressed and they are equivalent in mass to WT oligomers, which, in contrast to Hsp27 and a-crystallin [67], reside only in the cytoplasm of unstressed cells However, the much smaller F112R oligomers exhibit reduced translocation efficiency and they are not found FEBS Journal 273 (2006) 1020–1034 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Sun et al a-crystallin domain of p26 in the nuclei of all transfected COS-1 cells synthesizing this p26 variant; these results are in agreement with earlier work showing that p26 oligomers, reduced in size by C-terminal truncation, remain in the cytoplasm [17] p26 nuclear migration is apparently not accomplished by simple diffusion across the membrane upon oligomer size reduction, and why a-crystallin domain modifications promote translocation remains uncertain To summarize, analysis of individual amino acid substitutions, coupled with molecular modeling of protein structure, indicate that b-strand of the a-crystallin domain is an integral component of the p26 dimer–dimer interface in polydisperse sHSPs p26 chaperoning is not dependent upon oligomerization, and chaperone activity effectively tolerates structural perturbation, this potentially contributing to stress resistance in Artemia embryos The ability of p26 to prevent aggregation and loss of enzyme activity, in concert with its abundance, indicate a large protective capacity during oviparous development Proteins shielded by p26 would be readily available upon termination of diapause to initiate development, conferring a marked advantage on encysted Artemia embryos Experimental procedures Construction of p26 cDNAs p26 amino acid substitutions were generated by site-directed mutagenesis by using the QuikChangetm Site-directed Mutagenesis kit (Stratagene, La Jolla, CA, USA), using pRSET.C-p26–3-6-3 as template [46] and designated primers (Table 3) PCR mixtures were incubated for 30 s at 95 °C prior to 12 cycles of 30 s at 95 °C, at 55 °C and at 68 °C DNA products were digested with DpnI at 37 °C for h and used to transform E coli XL1- Table Primers for site-directed mutagenesis of p26 Single amino acid substitutions were generated within the p26 a-crystallin domain by site-directed mutagenesis using primers presented as sense and antisense, respectively, for each mutation p26 mutation R110G F112R R114A Y116D Primer 5¢-GGACACGTACAAGGAGAATTTCGACGACG-3¢ 5¢-CGTCGTCGAAATTCTCCTTGTACGTGTCC-3¢ 5¢-CACGTACAAAGAGAACGTCGACGACG-3¢ 5¢-CGTCGTCGACGTTCTCTTTGTACGTG-3¢ 5¢-GAGAATTTCGAGCACGATACAGACTCCC-3¢ 5¢-GGGAGTCTGTATCGTGCTCGAAATTCTC-3¢ 5¢-CGACGACGAGACAGACTCCCAGAACATGTC-3¢ 5¢-GACATGTTCTGGGAGTCTGTCTCGTCGTCG-3¢ blue supercompetent cells (Stratagene) p26 cDNA inserts were recovered from pRSET.C plasmids by digestion with BamHI and XhoI, electrophoresis in agarose and purification with the GFXtm PCR DNA and Gel Band purification kit (Amersham Biosciences, Piscataway, NJ, USA) before cloning in the eukaryotic expression vector, pcDNA4 ⁄ TO ⁄ myc-His.A (Invitrogen, San Diego, CA, USA) and transformation of E coli DH5a (Invitrogen, Carlsbad, CA, USA) The p26 cDNAs were also cloned into pPROTet.E233 (Clontech Laboratories, Inc., Palo Alto, CA, USA), a Histag-containing prokaryotic expression vector, using the BamHI and XbaI restriction sites Polypeptides encoded by pPROTet.E233 were longer than those encoded by pcDNA4 ⁄ TO ⁄ myc-His.A because the former employed a start codon upstream of the His-tag, while the latter initiated translation from the p26 start codon All p26 cDNA inserts were sequenced (DNA Sequencing Facility, Center for Applied Genomics, Hospital for Sick Children, Toronto, ON, Canada) Bacterial synthesis and purification of p26 p26 was synthesized in transformed E coli BL21PRO (Clontech Laboratories, Inc., Mississauga, ON, Canada) induced with 100 ngỈmL)1 anhydrotetracycline (aTc) (Clontech Laboratories) p26 was recovered from bacterial extracts using BD TALON resin (BD Biosciences Clontech, Mississauga, ON, Canada) and concentrated in CentriprepYM-10 centrifugal filter devices (Amicon Bioseparations, Billerica, MA, USA) [17] Protein samples were electrophoresed in 12.5% SDS polyacrylamide gels and either stained with Coomassie Brilliant Blue R-250 (Sigma) or blotted onto nitrocellulose (Bio-Rad, Hercules, CA, USA) for reaction with anti-p26 immunoglobulin [46] and Omniprobe (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), a monoclonal antibody recognizing the (His)6 tag Blots were then incubated with either horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, Mississauga, ON, Canada) and immunoconjugates were detected with Western Lightning Enhanced Chemiluminescence (ECL) Reagent Plus (PerkinElmer Life Sciences, Boston, MA, USA) p26 synthesis and localization in transiently transfected COS-1 cells Cloned p26 cDNA in SuperFecttm (Qiagen, Mississauga, ON, Canada) was employed to transiently transfect COS-1 cells [17] The cells were trypsinized 24 h after transfection for preparation of protein extract, centrifuged at 1500 g for min, washed with mL of phosphate-buffered saline (NaCl ⁄ Pi) (140 mm NaCl, 2.7 mm KCl, 8.0 mm Na2HPO4, 1.5 mm KH2PO4, pH 7.4), and incubated on ice for 20 in lysis buffer consisting of 50 mm Tris ⁄ HCl, pH 7.8, FEBS Journal 273 (2006) 1020–1034 ª 2006 The Authors Journal compilation ª 2006 FEBS 1029 a-crystallin domain of p26 Y Sun et al 150 mm NaCl, 0.5% Nonidet P-40, mm phenylmethanesulfonyl fluoride (Sigma, Oakville, ON, Canada), lgỈmL)1 pepstatin A (Sigma) and lgỈmL)1 leupeptin (Sigma) Lysates were centrifuged at 10 000 g for 10 min, supernatants were transferred to fresh tubes and protein concentrations were determined using the Bradford assay (Bio-Rad) p26 was detected on western blots, as described above, and localized in transfected COS-1 cells by staining with anti-p26 immunoglobulin and propidium iodide [17] p26 oligomerization p26, either purified from transfected bacteria or in extracts from transfected COS-1 cells and transformed bacteria, was centrifuged at 200 000 g for 12 h at °C in 10 mL of continuous 10–50% (w ⁄ v) sucrose gradients prepared in 0.l m Tris ⁄ glycine buffer, pH p26 was detected in gradient fractions by immunoprobing of western blots [17] Molar ratios were calculated using a p26 molecular mass of 20.8 kDa, as determined by generunner (version 3.05, Hastings Software, Inc., Hastings on Hudson, NY, USA) with corrections for protein modifications Molecular mass markers alphalactalbumin (14.2 kDa), carbonic anhydrase (29 kDa), BSA (66 kDa), alcohol dehydrogenase (150 kDa), apoferritin (443 kDa) and thyroglobulin (669 kDa) (Sigma) were centrifuged separately and localized in sucrose gradients by measuring the A280 of fractions p26 induced thermotolerance in E coli Transformed E coli, incubated overnight with shaking at 37 °C in mL of LB medium, containing spectinomycin, chloramphenicol and aTc, were diluted : 10 in fresh LB medium and incubated at 54 °C Samples were removed at timed intervals during heating, plated on LB agar and colonies were counted after incubation for 24 h at 37 °C All experiments were performed in triplicate Between data groups, two-sample t-tests were performed at a confidence level of 95% with the statistical software minitab 14.12.0 (Minitab Inc., State College, PA, USA) to evaluate the significance of difference, which was accepted at a P-value of < 0.05 The presence of p26 in bacteria that were stressed was confirmed by immunoprobing of western blots containing protein extracts obtained from aTc-induced cells prior to heating at 54 °C 10 mm oxaloacetic acid (Sigma), 10 lL of 10 mm 5,5¢-dithiobis (2-nitrobenzoic acid) (Sigma) and 30 lL of mm acetyl-CoA (Sigma) [70] Reactions initiated by adding 10 lL of 150 nm citrate synthase were monitored at 25 °C as an increase in absorption at 412 nm determined with a SPECTRAmax PLUS spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) Insulin (Sigma) at a final concentration of 4.0 lm in 10 mm phosphate buffer, pH 7.4, was mixed with p26, dithiothreitol (Sigma) was added to 20 mm and solution turbidity was measured at 400 nm in a SPECTRAmax PLUS spectrophotometer at 25 °C Assays were performed in triplicate p26 intrinsic fluorescence, ANS binding and secondary structure Purified p26 was diluted to 0.06 mgỈmL)1 in 10 mm NaH2PO4, pH 7.1, and fluorescence spectra were measured at 25 °C with a SPECTRAmax GEMINIXS fluorescence spectrophotometer (Molecular Devices) The emission wavelength was set initially at 340 nm with a 2-nm band pass, and fluorescence excitation was detected from 250 to 310 nm The excitation wavelength was then set to 280 nm with a 2-nm band pass, and fluorescence emission was detected from 310 to 400 nm To measure surface hydrophobicity, mixtures containing 80 lm ANS (Molecular Probes, Eugene, OR, USA) and 0.06 mgỈmL)1 p26 in 10 mm NaH2PO4, pH 7.1, were incubated for at either 25 °C or 43 °C The excitation wavelength was set to 388 nm with a band pass of nm, and emission wavelength was 473 nm with a band pass of nm Measurements were made with an AMINCO Bowman series z luminescence spectrometer (AMINCO, Rochester, NY, USA) equipped with a thermostated circulating water bath All spectra were recorded in duplicate using two independently prepared samples Far-UV CD spectra were recorded at 25 °C over 180–260 nm in a JASCO J-810 spectropolarimeter (Japan Spectroscopic, Tokyo, Japan) A 0.1-cm path length quartz cuvette containing 0.2 mgỈmL)1 p26 in 10 mm NaH2PO4, pH 7.1, was employed, and three scans were averaged for each spectrum Bandwidth was nm, with all scans corrected for buffer and smoothed to eliminate background noise Secondary structure parameters were calculated using the cdnn v2.1 deconvolution program (Martin-Luther-Universitat, Halle-Wittenberg, Germany) ă Modeling of p26 structure p26 chaperone activity in vitro Citrate synthase protection against heat-induced aggregation was determined [17], as was the ability of p26 to shield citrate synthase enzyme activity at 43 °C Reaction mixtures for measuring citrate synthase activity contained 940 lL of TE (50 mm Tris ⁄ HCl, pH 7.5, mm EDTA), 10 lL of 1030 The a-crystallin domains of p26, aA ⁄ aB-crystallin, Hsp27 and Hsp16.9 were aligned by clustal w followed by manual adjustment, according to secondary structure elements predicted with psipred [71] The Hsp16.9 tetramer, composed of subunits 1gme_1:A, 1gme_1:B, 1gme_1:G and 1gme_1:H, was obtained from the protein quaternary struc- FEBS Journal 273 (2006) 1020–1034 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Sun et al ture file server PQS at EMBL-EBI where the crystal structure coordinates of Hsp16.9 (accession number: 1GME) were used to reconstruct the Hsp16.9 oligomer by crystallographic symmetry transformations The p26 a-crystallin domain models were constructed using modeler [72] and the structure was obtained by optimizing the probability objective functions (pdfs) and simulated annealing minimization p26 a-crystallin domain tetramer models were based on the corresponding Hsp16.9 tetramer One hundred models were generated, and the structure displaying the lowest objective function value was used to represent p26 Model evaluation was made using verify3d without further energy minimization to preserve the conserved residue side chain conformation [73,74] Using the Hsp16.9 monomer as a template, the root mean square deviation (RMSD) [75] ˚ ˚ was 2.3 A, an acceptable value that decreased to 0.6 A when flexible protein regions were excluded Application of procheck [76] revealed that the stereochemical quality of the model was reliable, with 84% of the residues in the most favored regions of the tetramer model and none in disallowed regions Graphical representations were made using vmd [77] Acknowledgements We thank Dr Stephen Bearne and Dr Neil Ross for experimental support with biophysical studies and Mr Carey Isenor for expert assistance in image processing This work was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant, a Nova Scotia Health Research Foundation ⁄ Canadian Institutes of Health Research Regional Partnership Plan Grant, and a Heart and Stroke Foundation of Nova Scotia Grant to T.H.M and a NSHRF Student Fellowship to Y.S a-crystallin domain of p26 10 11 12 13 14 15 16 17 References Frydman J (2001) Folding of newly translated proteins in 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5¢-CGACGACGAGACAGACTCCCAGAACATGTC-3¢ 5¢-GACATGTTCTGGGAGTCTGTCTCGTCGTCG-3¢... investigated by site-directed mutagenesis of a- crystallin domain residues and molecular modeling of protein structure The p26 a- crystallin domain contains nine b-strands arranged predominantly as paired... stress proteins and a new family of proteins containing a- crystallin domains (Acd proteins) Cell Stress Chaperones 6, 225–2 37 Narberhaus F (2002) a- crystallin- type heat shock proteins: socializing