Báo cáo khoa học: Effect of mutations in the b5–b7 loop on the structure and properties of human small heat shock protein HSP22 (HspB8, H11) pptx

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Effect of mutations in the b5–b7 loop on the structureand properties of human small heat shock protein HSP22(HspB8, H11)Alexei S. Kasakov1,*, Olesya V. Bukach1,*, Alim S. Seit-Nebi1, Steven B. Marston2andNikolai B. Gusev11 Department of Biochemistry, School of Biology, Moscow State University, Russia2 National Heart and Lung Institute, Imperial College London, UKSmall heat shock proteins (sHsp) form a large super-family of ubiquitous proteins detected in all organ-isms, except for some bacteria [1–3]. The membersof this family range in size from 12–42 kDa andcontain a conservative so-called a-crystallin domainconsisting of 80–100 residues that is located in theC-terminal part of the polypeptide chain [1–3]. Thisconservative domain is flanked by the N-terminaldomain and short C-terminal extension with a differ-ent size and structure [4,5]. All sHsp tend to formflexible oligomers, ranging from a dimer to morethan 40 subunits, exchanging their subunits [6,7], andsome sHsp are able to form mixed oligomers consist-ing of subunits of different natures [8,9]. CrystalKeywordschaperone-like activity; intrinsicallydisordered regions; oligomeric structure;small heat shock proteinsCorrespondenceN. B. Gusev, Department of Biochemistry,School of Biology, Moscow StateUniversity, Moscow 119991, RussiaFax ⁄ Tel: +7 495 939 2747E-mail: nbgusev@mail.ru*These authors contributed equally to thiswork(Received 17 June 2007, revised 30 July2007, accepted 3 September 2007)doi:10.1111/j.1742-4658.2007.06086.xThe human genome encodes ten different small heat shock proteins, eachof which contains the so-called a-crystallin domain consisting of 80–100 residues and located in the C-terminal part of the molecule. Thea-crystallin domain consists of six or seven b-strands connected by differentsize loops and combined in two b-sheets. Mutations in the loop connectingthe b5 and b7 strands and conservative residues of b7inaA-, aB-crystallinand HSP27 correlate with the development of different congenital diseases.To understand the role of this part of molecule in the structure and func-tion of small heat shock proteins, we mutated two highly conservative resi-dues (K137 and K141) of human HSP22 and investigated the properties ofthe K137E and K137,141E mutants. These mutations lead to a decrease inintrinsic Trp fluorescence and the double mutation decreased fluorescenceresonance energy transfer from Trp to bis-ANS bound to HSP22. Muta-tions K137E and especially K137,141E lead to an increase in unorderedstructure in HSP22 and increased susceptibility to trypsinolysis. Bothmutations decreased the probability of dissociation of small oligomers ofHSP22, and mutation K137E increased the probability of HSP22 crosslink-ing. The wild-type HSP22 possessed higher chaperone-like activity thantheir mutants when insulin or rhodanase were used as the model substrates.Because conservative Lys residues located in the b5–b7 loop and in the b7strand appear to play an important role in the structure and properties ofHSP22, mutations in this part of the small heat shock protein moleculemight have a deleterious effect and often correlate with the development ofdifferent congenital diseases.Abbreviationsbis-ANS, 4,4¢-bis(1-anilinonaphtalene-8-sulfonate); DMS, dimethylsuberimidate; FRET, fluorescent resonance energy transfer; GuCl,guanidinium chloride; sHSP, small heat shock protein.5628 FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBSstructures are described in the literature for thehyperthermophile Methanococcus jannaschii Hsp16.5[10] and wheat (Triticum aestivum) Hsp16.9 [11], eachcontaining a single a-crystallin domain, and the par-asitic flatworm Taenia saginata Tsp36, containingtwo a-crystallin domains in the single polypeptidechain [12].Ten different sHsp are encoded in the human gen-ome and are differently expressed in human tissues[13,14]. None of these proteins has been crystallized;however, different experimental approaches (cryo-electron microscopy, electron spin resonance spectros-copy, protein pin array, etc.) [15–17] and proteinmodeling were used to reconstruct the structure ofmammalian aB-crystallin and Hsp27 (HspB1) [16–18]. According to these models, the a-crystallindomain of both proteins consists of seven b-strandspacked into two b-sheets [16–18]. The loop connect-ing b5 and b7 and the N-terminal part of b7appears to play an important role in the structure ofsHsp monomers [12,18] and intermonomer interac-tions [12,18,19], as well as in the binding of proteinsubstrates to sHsp [17]. The importance of this partof molecule of the sHsp is supported by the factthat mutations in the loop connecting the b5 and b7strands, or in the b7 strand of sHsp, often correlatewith the development of certain congenital diseases(congenital cataract, desmin related myopathy, distalhereditary motor neuropathy, amongst others)[20,21].A recently described protein with an apparentmolecular mass of 22 kDa, denoted as HSP22,HspB8 or H11 kinase, shares structural propertiestypical to all members of the family of sHsp [22].HSP22 possesses chaperone-like activity [23–25] andappears to be involved in the regulation of manyprocesses such as proliferation, myocardium hyper-trophy and apoptosis [26]. Missense mutations ofK141 (K141E, K141N) located at the beginningof b7 of HSP22 correlate with the development ofmotor neuropathy and Charcot–Marie–Tooth disease[27,28]. Another conservative residue of HSP22,namely K137, presumably located in the b5–b7 loop,is homologous to R136 of human HSP27 that ismutated in the case of Charcot–Marie–Tooth type 2disease [20,21]. Previously, we compared the structureand properties of the wild-type HSP22 and itsK141E mutant [29]. The present study analyses thestructure and properties of K137E and theK137,141E mutant of human HSP22, aiming to pro-vide new information on the structure of sHsp andto shed new light on their role in the developmentof human congenital diseases.ResultsPeculiarities of HSP22 structureUp to now, all attempts to crystallize mammaliansHsp have been unsuccessful. Therefore, all structuralinformation derives from a comparison of human sHspwith the crystal structures of M. jannaschii Hsp16.5[10] and T. aestivum Hsp16.9 [11]. The 3D structure ofthe monomer of T. aestivum Hsp16.9 is presented inFig. 1A (protein databank accession code 1GME) and,as shown in Fig. 1B, we aligned the structures ofM. jannaschii Hsp16.5 and T. aestivum Hsp16.9 withthe corresponding structures of three human sHsp [30].The elements of the secondary structure of M. janna-schii Hsp16.5 and T. aestivum Hsp16.9, as determinedby X-ray crystallography, are indicated by solid blue(a-helices) or solid red (b-strands) lines above andbelow the corresponding sequences (Fig. 1A). Boththese proteins contain a large number of well preservedb-strands that are predominantly (with the exceptionof the b10 strand) located in the a-crystallin domain[10–12].The models built for two mammalian sHsp (aB-crys-tallin [16] and HSP27 [18]) predict that both these pro-teins contain short a-helices in the N-terminal part ofmolecule (dashed blue lines denoted a1–a3 above theaB-crystallin and below the HSP27 sequences inFig. 1B). According to these models, both aB-crystallinand HSP27 contain seven b-strands (b2–b9) (dashedred lines) located in positions homologous to the cor-responding strands of two crystallized nonmetazoansHsp. Two predictions slightly differ with respect tothe location and length of specific b-strands. Forexample, in the model of aB-crystallin, the b7 strand isonly four residues long [16] whereas, in the model ofHSP27, the same strand is ten residues long [18]. How-ever, the overall structures of aB-crystallin and HSP27predicted by these two models are very similar, andthe positions of the b-strands correlate well with thecorresponding positions of the b-strands in M. janna-schii Hsp16.5 and T. aestivum Hsp16.9 (Fig. 1B).Predictions of the secondary structure of HSP22 per-formed with the jpred program (http://www.combio.dundee.ac.uk) indicate that this protein contains verysmall quantities of a-helices and is enriched in unor-dered structure and b-strands. The residues of HSP22that are predicted to form b-strands are indicated bywide dashed red lines in Fig. 1B and are located inpositions corresponding to the b3, b4, b5, b7 and b9strands. jpred failed to predict the formation of a b2strand in the HSP22 structure. According to thisprediction, residues 153–155 of HSP22 tend to form anA. S. Kasakov et al. Point mutations of the b5– b7 loop of human HSP22FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS 5629ABFig. 1. Comparison of the structure of human HSP22 and other sHsp. (A) Ribbon diagram of T. aestivum Hsp16.9 monomer (protein data-bank accession code 1GME). The N- and C-terminal domains are indicated by N and C correspondingly. All b-strands are numbered andthe b5 and b7 strands are shown in red and blue, respectively. G104 (equivalent to K137 of human HSP22) and R108 (equivalent to K141of human HSP22) are shown in purple and grey, respectively. (B) Alignment of human HSP22 with human aB-crystallin and HSP27 andM. jannaschii Hsp16.5 and T. aestivum Hsp16.9 made withCLUSTALW [30] using the default settings. The residues shown in black areidentical in at least four sequences; residues in dark grey are conservative in at least four or identical in at least three sequences; resi-dues in light grey are homologous at three or identical in at least two sequences. Solid blue and red lines above M. jannaschii Hsp16.5and below T. aestivum Hsp16.9 sequences indicate a-helices and b-strands detected in the crystal structure of the corresponding proteins[10,11]. Dashed blue and red lines above human aB-crystallin and below human HSP27 sequences indicate a-helices and b-strands pre-dicted in the models of the corresponding proteins [16,18]. Residues of HSP22 predicted to form b-strands according toJPRED are indi-cated by wide dashed red lines and K137 and K141 are shown in red. Numbers in parenthesis correspond to NCBI-Entrez-Proteindatabase accession numbers.Point mutations of the b5–b7 loop of human HSP22 A. S. Kasakov et al.5630 FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBSa-helix, whereas residues 156 and 157 tend to form avery short b-strand that might correspond to the b8strand of the other sHsp.The primary structure of the a-crystallin domain ofhuman sHsp is very conservative and the loop connect-ing the b5 and b7 strands is shorter than the corre-sponding loop connecting the b5 and b7 strands ofnonmetazoan sHsp (Fig. 1B). Moreover, the structureof human sHsp lacks the b6 strand that is involved indimer formation of nonmetazoan sHsp (Fig. 1A).Although the b5–b7 loop is very short, it is not com-pletely deleted in any human sHsp. This part of themolecule has a very conservative primary structure andappears to play a diverse and important role. Forexample, mutation of a highly conservative positivelycharged residue (R116 of aA-crystallin, R120 of aB-crystallin or K141 of HSP22 located in homologousposition; Fig. 1B) correlates with the development ofcongenital cataract and ⁄ or desmin related myopathy[20,21], whereas mutations of R127, S135 and R136 ofhuman HSP27 are associated with distal hereditarymotor neuropathy and Charcot–Marie–Tooth disease[20,21]. Therefore, it is advisable to analyze the effectof a mutation in this part of the molecule on the struc-ture and properties of human sHsp.Oligomeric structure of HSP22 and its mutantsAll samples of recombinant HSP22 and its mutantspurified by the method described previously [23] werehomogeneous according to SDS gel electrophoresis(Fig. 2). HSP22 and its mutants are highly susceptibleto proteolysis [23,24,29] and occasionally containedsmall quantities of proteolytic fragments. Under theconditions used, the wild-type HSP22 and its K137Eand K141E mutants migrated on the SDS gel electro-phoresis [31] as a band with an apparent molecularmass of 25.4 kDa, whereas the apparent molecularmass of the double mutant K137,141E was 30.4 kDa.The calculated molecular mass of human wild-typeHSP22 is close to 21.6 kDa [22]. The unusually highapparent molecular mass determined by SDS gel elec-trophoresis can be due to anomalous binding of SDSto acidic HSP22 and this effect is especially pro-nounced in the case of the particularly acidic doublemutant K137,141E of HSP22. On native gel electro-phoresis performed both at neutral [32] and alkalinepH [33], the wild-type HSP22 and its mutants migratedas a single band with an apparent molecular mass ofapproximately 60 kDa (data not shown), thus indicat-ing that, under these conditions, HSP22 and itsmutants form small oligomers.Size-exclusion chromatography was used for furtherinvestigation of the quaternary structure of HSP22 andits mutants. When 200 lg of the wild-type HSP22 wasloaded on the column, a single peak was detected witha Stokes radius equal to 26.2 A˚, corresponding to anapparent molecular mass of 36.1 kDa (Fig. 3A). Thesedata agree well with the previously published data[23,24,29]. On size-exclusion chromatography, bothK141E and K137,141E were eluted as symmetricalpeaks and the width at the respective half-height oftheir peaks was similar to that of the wild-type HSP22.The Stokes radii and apparent molecular masses of theK141E and K137,141E mutants were similar: 26.7 A˚and 37.9 kDa (Fig. 3A) [29]. At the same time, theK137E mutant of HSP22 was eluted as a broad peakwith a trailing end, with a Stokes radius and apparentmolecular mass of 28.2 A˚and 43.9 kDa, respectively(Fig. 3A). Taking into account that the molecular massof HSP22 monomer is 21.6 kDa [22], it might beassumed that, under conditions of size-exclusion chro-matography, HSP22 and its mutants are either highlyasymmetric (or intrinsically unfolded) or presented inthe form of a mixture of monomers and dimers. Thedata presented indicate that mutations in the b5–b7loop (and especially K137E) affect either folding orextension of oligomerization of HSP22.To test this suggestion, we performed size-exclusionchromatography on the Superdex 200 HR10 ⁄ 30 col-umn in the presence of 6 m guanidinium chloride(GuCl) and, under these conditions, calibrated the col-umn with a set of protein standards (BSA, ovalbumin,chymotrypsin A and RNAse) [34] (Fig. 3B). Underdenaturating conditions, all samples of HSP22 wereFig. 2. SDS electrophoresis of the wild-type HSP22 (1) and itsK137E (2), K141E (3) and K137,141E (4) mutants. The positions ofthe molecular mass standards (in kDa) are indicated by arrows.A. S. Kasakov et al. Point mutations of the b5– b7 loop of human HSP22FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS 5631eluted in the form of symmetrical peaks with an appar-ent molecular mass of 22.8 kDa, which is close to thecalculated value of the HSP22 monomer (21.6 kDa).The data presented agree with the suggestion that,under native conditions, HSP22 and its mutants formdimers that dissociate to monomers in the presence of6 m GuCl.If this suggestion is correct, we might assume that adecrease in protein concentration will result in the dis-sociation of small HSP22 oligomers and the formationof protein species with smaller apparent molecularmass. Indeed, if the quantity of the wild-type HSP22loaded on the column was decreased from 200 lgto10 lg, the elution volume of the protein peak wasincreased from approximately 11.3 mL to 11.8 mL(Fig. 3C). This increase in elution volume correspondsto a decrease in the apparent molecular mass fromapproximately 36.9 kDa to 29.3 kDa. A similardecrease in the apparent molecular mass was observedfor the K137,141E mutant of HSP22; however, at allconcentrations, the apparent molecular mass of thismutant was slightly larger than the molecular mass ofthe wild-type protein (Fig. 3C). At high concentration,the K137E mutant formed oligomers with an apparentmolecular mass of approximately 44 kDa whereas, atvery low concentration, the molecular mass of oligo-mers formed by this mutant was close to 32 kDa(Fig. 1C). The data presented mean that mutations ofK137 and K141 might affect either folding or dissocia-tion of HSP22 oligomers.There are many examples indicating that certainpoint mutations do not dramatically affect the quater-nary structure but, at the same time, induce destabili-zation of the overall structure of the sHsp [35,36].Therefore, we analyzed the effect of point mutations inthe linker connecting the b5 and b7 strands of HSP22on its thermal stability. The wild-type protein or itsmutants were heated for 30 min at 70 °C and, afterFig. 3. Size-exclusion chromatography of the wild-type HPS22 andits point mutants. (A) Size-exclusion chromatography of the wild-type HSP22 (1, 2) and its K137E (3, 4) and K137,141E (5, 6)mutants on Superdex 75 column under native conditions. The sam-ples were either kept on ice (solid curves 1, 3, 5) or heated for30 min at 70 °C (dashed curves 2, 4, 6). Equal volumes (150 lL) ofeach protein (210 lg) were subjected to chromatography on a Su-perdex 75 HR10 ⁄ 30 column. For clarity, elution profiles of unheatedand heated proteins are shifted from each other by 10 mAu andelution profiles between different proteins are shifted from eachother by 30 mAu. Arrows above the panel indicate the elution vol-ume of protein standards and their apparent molecular masses. (B)Size-exclusion chromatography of the wild-type HSP22 (1) and itsK137E (2), K141E (3) and K137,141E (4) mutants on the Super-dex 200 HR10 ⁄ 30 column in the presence of 6M GuCl. Equalvolumes (150 lL) of each protein (150 lg) were subjected tochromatography. For clarity, elution profiles are shifted from eachother by 20 mAu. Arrows above the panel indicate the elution vol-ume of protein standards and their apparent molecular masses. (C)Dependence of elution volume on the quantity of protein loaded ona Superdex 75 HR10 ⁄ 30 column. Equal volumes (150 lL) contain-ing 10–200 lg of the wild-type protein (1) and its K137E (2) orK137,141E (3) mutants were subjected to chromatography undernative conditions. The data are representative of three independentexperiments.Point mutations of the b5–b7 loop of human HSP22 A. S. Kasakov et al.5632 FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBScooling for 20 min and centrifugation, were subjectedto size-exclusion chromatography (Fig. 3A). Prolongedheating at 70 °C did not affect the elution profile ofany of the proteins analyzed. The amplitude, positionand the width of the protein peaks were not dependenton the transient heating. These data suggest that thewild-type HSP22 and its mutants belong to the groupof the so-called intrinsically disordered proteins withlong stretches of unordered structure [37] and thisis one of the reasons for their unusual high thermalstability.To further investigate the oligomeric structure ofHSP22, we employed chemical crosslinking. HSP22and its mutants at three different concentrations (0.1,0.5 and 2.0 mgÆmL)1) were incubated in the presenceof 3.5 mm dimethylsuberimidate (DMS) for 1 h at37 °C and the protein composition of the sample thusobtained was analyzed by means of SDS gel electro-phoresis. In good agreement with the previously pub-lished data [23,29], we found that incubation of thewild-type HSP22 with the bifunctional reagent resultedin the formation of an additional protein band with anapparent molecular mass of 50 kDa, which presumablycorresponds to the HSP22 dimer (Fig. 4A). Similarresults were observed in the case of the K137E mutantof HSP22 (Fig. 4B); however, in this case, the intensityof the band corresponding to the HSP22 dimer wasmore intense than in the case of the wild-type protein.Thus, although mutation K137E eliminates one poten-tial site of chemical modification, the probability ofcrosslinking of the K137E mutant by DMS is higherthan the probability of crosslinking of the wild-typeprotein. This fact agrees well with the size-exclusionchromatography data indicating that the K137Emutant forms larger oligomers than the wild-type pro-tein (Fig. 3C). If the double mutant K137,141E wassubjected to crosslinking, we detected only a very faintband corresponding to dimer and this band wasdetected only at a rather high protein concentration(Fig. 4C). The decreased probability of crosslinking ofthe K137,141E mutant might be due to replacementsof Lys residues being potential sites of crosslinking or,more likely, to the overall changes in the structure ofHSP22 that are induced by replacing two closely sepa-rated positively charged Lys residues by negativelycharged Glu (see below).Effect of K137E and K137,141E mutations on thestructure of HSP22The data presented might indicate that the analyzedmutations affect the secondary and tertiary structureof HSP22. To check this suggestion, we analyzed somespectral properties of the wild-type protein and its twomutants.The maximum of intrinsic Trp fluorescence of thewild-type HSP22 was located at 342 nm and the posi-tion of this maximum was not changed by mutationsK137E or K137,141E (Fig. 5). Similar results wereobtained previously with the K141E mutant of HSP22[29]. The fluorescence spectrum of HSP22 was decom-posed into discrete components characteristic of Trplocated in different environments [38]. For thisABCFig. 4. Crosslinking of the wild-type HSP22 (A) and its K137E (B)and K137,141E (C) mutants by DMS. HSP22 was incubated eitherin the absence of DMS (0), or in the presence of 3.5 mM of DMS(1–3). The protein concentration was equal to 0.10 (1), 0.50 (2) or2.0 (3) mgÆmL)1and, after incubation, equal quantities (2.5 lg) ofprotein were loaded onto the gel. The positions of the molecularmass standards (in kDa) are indicated by arrows on the right.A. S. Kasakov et al. Point mutations of the b5– b7 loop of human HSP22FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS 5633purpose, the fluorescence spectra were fitted as a sumof three polynomial distributions of the fourth of fifthorder, corresponding to three classes of Trp residuesdiffering in their environment, accessibility to solventand position of the fluorescent spectrum. Using thisapproach, we estimated the portion of each class offluorophores in the protein spectrum and found thatHSP22 contains Trp residues belonging to the so-calledclasses I, II and III. Class I corresponds to indolelocated inside the protein globule, forming a 2 : 1 exci-plex with neighboring polar groups and having maxi-mum fluorescence at 330–332 nm. Class II correspondsto Trp at the protein surface in contact with boundwater molecules (maximum fluorescence at 340–342 nm). Finally, class III corresponds to indolelocated at the protein surface in contact with freewater molecules (maximum fluorescence at 350–355 nm). Approximately 44% of Trp residues ofHSP22 belong to class I, approximately 18% belong toclass II and approximately 38% belong to class III.Point mutations K137E or K137,141E do not signifi-cantly affect the distribution of Trp residues betweenthese classes (data not shown). This may be due tothe fact that three out of four Trp residues are locatedin the N-terminal end (Trp48, Trp51, Trp60) and thefourth Trp residue (Trp96) are located at the verybeginning of the a-crystallin domain, far apart fromthe mutated Lys residues. Although the point muta-tions do not affect the position of maximumfluorescence, they slightly decrease the amplitude offluorescence and this decrease was more pronouncedfor the K141E [29] and K137,141E mutants than forthe K137E mutant (Fig. 5). The small decrease inthe amplitude of fluorescence detected for the pointmutants of HSP22 might reflect small changes instructure, leading to an altered Trp environment ortheir accessibility to quencher or water molecules.Hydrophobic interactions appear to play an impor-tant role in oligomer formation and in the interactionof sHsp with their protein substrates [1,10–12]. Hydro-phobic surfaces of HSP22 and its mutants were probedby using bis-ANS. In the isolated state, this hydropho-bic probe has a very low quantum yield that is dramat-ically increased after its binding to hydrophobic siteson the protein molecules [24,29]. Titration of HSP22with bis-ANS was accompanied by an increase in fluo-rescence at 495 nm, indicating binding of the fluores-cence probe to the protein [24,29]. In agreement withthe previously published data [29], we were unable toachieve saturation and, in the range of 0–10 lm bis-ANS, the fluorescence at 495 nm was approximatelyproportional to the concentration of the fluorescentprobe added. These data indicate that HSP22 containsmany low affinity bis-ANS binding sites that cannotbe completely saturated in the range of bis-ANS con-centrations used. This is to be expected if HSP22belongs to the group of intrinsically disordered pro-teins lacking well-organized hydrophobic sites. Toobtain more information on the structure, we analyzedfluorescence resonance energy transfer (FRET) fromTrp residues of HSP22 and its mutants to the boundbis-ANS. As indicated in Fig. 6, titration of the wild-type HSP22 and its K137,141E mutant with bis-ANSwas accompanied by a decrease in intrinsic Trp fluo-rescence at 342 nm and a concomitant increase in thefluorescence of bis-ANS at 495 nm. Because, at anybis-ANS concentration, the ratio of fluorescence at 342to fluorescence at 495 nm (F342⁄ F395) was lower for thewild-type protein than for its K137,141E mutant, weconclude that the probability of FRET is higher forthe wild-type protein than for its mutant. This mayindicate that the mutation K137,141E affects themutual orientation, overall flexibility and ⁄ or distancesbetween Trp and bis-ANS bound to HSP22.To obtain more detailed information on the struc-ture of HSP22 mutants, we employed CD spectros-copy. The far-UV CD spectra of the wild-type proteinhas a negative maximum at 208 nm and its molar ellip-ticity at this wavelength is rather low (Fig. 7). Thisspectrum is characteristic for proteins with a lowa-helix content and a high content of unordered andb-structures. Mutation K137E had no dramatic effecton the far-UV CD spectra and a blue shift of only 2–3 nm was observed in the position of the negativemaximum (Fig. 7). Previously, we have found thatmutation K141E induces a rather large increase in theamplitude of the negative maximum on the far-UVCD spectrum of HSP22 [29]. Even larger changes wereFig. 5. Intrinsic Trp fluorescence of the wild-type HSP22 (1) and itsK137E (2) and K137,141E (3) mutants. Fluorescence was excited at295 nm. The protein concentration was 0.1 mgÆmL)1.Point mutations of the b5–b7 loop of human HSP22 A. S. Kasakov et al.5634 FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBSobserved in the case of the double K137,141E mutant.Indeed, the double mutation results in a blue shift of5–6 nm with respect to the position of negative maxi-mum and a significant increase in the amplitude of thismaximum. This change of the far-UV CD spectra canreflect pronounced changes in the secondary structure.Using the approach developed by Sreerama andWoody [39], we attempted to estimate the changesinduced by the point mutations in the secondary struc-ture of HSP22.According to this estimation, the a-helix content isequally low (approximately 5–6%) in the structure ofboth the wild-type HSP22 and its two mutants. Asexpected, the secondary structure of HSP22 and itsmutants was characterized by a high content ofb-strands (approximately 31–37%) and turns andunordered structures (approximately 58–63%). Muta-tion K137E induced only very moderate changes in thesecondary structure. At the same time, mutationK141E [29] and especially double mutation K137,141Ewere accompanied by a simultaneous decrease in thecontent of b-structure (from 37% to 31%) and anincrease in the content of turns and unordered structure(from 58% to 63%). These data might indicate thatmutations in the b5–b7 loop and in the N-terminal partof the b7 strands destabilize the structure of HSP22.Limited trypsinolysis of the wild-type HSP22 andits K137E and K137,141E mutantsThe method of limited trypsinolysis was used to checkthe suggestion that the analyzed mutations affect thestability of HSP22. The available literature [23,24,29]indicate that HSP22 is highly susceptible to proteoly-sis. Indeed, even at a weight ratio for HSP22 ⁄ trypsinequal to 12 000 : 1, the sHsp was rapidly hydrolyzed(Fig. 8A). Trypsinolysis of the wild-type HSP22 wasaccompanied by disappearance of the band corre-sponding to intact protein that migrated with anapparent molecular mass of 25.4 kDa and accumula-tion of peptides with apparent molecular masses equalto 16.5, 18.0, 19.0, 22.0 and 23 kDa, respectively(Fig. 8A). The same set of peptides was observed ifK137E and K137,141E mutants were subjected totrypsinolysis. To compare the apparent rates of tryp-sinolysis of the wild-type HSP22 and its mutants, weplotted ln(At⁄ Ao) (where Aoand Atare the intensitiesof the band of intact protein at the beginning of tryp-sinolysis and at the fixed time of trypsinolysis) againstthe time of incubation (Fig. 8D). The apparent rateconstants of trypsinolysis under these conditions wereequal to 0.0496 ± 0.0027, 0.068 ± 0.026, 0.0863 ±0.0039Æmin)1(n ¼ 7) for the wild-type HSP22, and itsK137E and K137,141E mutants, respectively. The dataFig. 6. Fluorescence resonance energy transfer from Trp residues of the (A) wild-type HSP22 and (B) its K137,141E mutant to the boundbis-ANS. All experiments were performed at a protein concentration of 0.03 mgÆmL)1(1.5 lM of HSP22 monomer) and bis-ANS (in lM) indi-cated above each spectrum.Fig. 7. Far-UV CD spectra of the wild-type HPS22 (1) and its K137E(2) and K137,141E (3) mutants. The spectra were recorded at theconcentration 0.65 mgÆmL)1of each species with a cell path of0.05 cm. The spectra reported are the average of eight determina-tions.A. S. Kasakov et al. Point mutations of the b5– b7 loop of human HSP22FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS 5635presented mean that K137E and especially K137,141Emutants were more susceptible to proteolysis than thewild-type HSP22. Mutations K137E and K137,141Eshould eliminate one or two potential sites of trypsin-olysis and, in this way, were expected to decrease therate of proteolysis. Instead of decreasing susceptibility,these mutations increased the susceptibility of HSP22to trypsinolysis and this finding agrees well withthe data of far-UV CD indicating that the ana-lyzed mutations induce destabilization of the HSP22structure.Chaperone-like activity of wild-type HSP22 andits mutantsThe data presented indicate that the point mutationsof residues 137 and 141 affect the structure and sta-bility of HSP22. Therefore, it can be expected thatthese mutations might change the chaperone-likeactivity of HSP22. To investigate this idea, we usedtwo different model protein substrates. Reduction ofthe disulfide bonds of insulin results in dissociation ofits peptide chains and aggregation of chain B. Addi-tion of the wild-type HSP22 retarded the onset ofaggregation and decreased the amplitude of light scat-tering induced by insulin aggregation (Fig. 9A,curves 3 and 3¢). K137E (Fig. 9A, curves 1 and 1¢)and K137,141E (Fig. 9A, curves 2 and 2¢) alsoretarded the onset of insulin aggregation anddecreased the amplitude of light scattering; however,their effects were less pronounced than the corre-sponding effects of the wild-type protein. For exam-ple, the aggregation curve in the presence of0.2 mgÆmL)1of K137E was comparable to the aggre-gation curve observed in the presence of 0.1 mgÆmL)1of the wild-type HSP22 (compare curves 1¢ and 3 inFig. 9). The double mutant (K137,141E) possessedhigher chaperone-like activity than the K137Emutant. However, both at low and high concentra-tions, the double mutant possessed slightly lowerchaperone-like activity than the wild-type protein(curves 2 and 3 in Fig. 9).Heating of rhodanase at 43 °C induces its denatur-ation, which is followed by aggregation. The wild-type HSP22 and its mutants decreased the rate ofrhodanase aggregation and the amplitude of lightscattering (Fig. 9B). In good agreement with the dataobtained for insulin, we found that the K137Emutant was much less effective than the wild-typeprotein in preventing rhodanase aggregation (com-pare curves 1 and 3 in Fig. 9B). The chaperone-likeactivity of K137,141E mutant was lower than, butcomparable to, the chaperone activity of the wild-type protein. Thus, on two different protein sub-strates, the chaperone-like activity of the wild-typeHSP22 was higher than the corresponding activity ofthe two mutants analyzed and, among these mutants,the chaperone activity of the K137E mutant wasespecially low.ABCDFig. 8. Limited trypsinolysis of the wild-type HSP22 and its K137Eand K137,141E mutants. Kinetics of trypsinolysis of the wild-typeHSP22 (A) and its K137E (B) and K137,141E (C) mutants. The timeof incubation (in min) is indicated below each track and the arrowsshow the positions of molecular mass markers. (D) Determinationof apparent rate constants of trypsinolysis of the wild-type HSP22(1, squares), K137E mutant (2, circles) and K137,141E mutant (3,triangles). The data are representative of three independent experi-ments.Point mutations of the b5–b7 loop of human HSP22 A. S. Kasakov et al.5636 FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBSDiscussionAll sHsp are characterized by the presence of a highlyconservative a-crystallin domain consisting of six orseven b-strands combined in two b-sheets [1,10–12,40].The detailed location and orientation of theseb-strands is known only for Hsp16.5 of M. jannaschii[10], Hsp16.9 of wheat [11] and Tsp36 of T. saginata[12] that were all obtained in crystallized form. Thetertiary structure of other sHsp (including all humanproteins) is unknown; however, several models of theirstructure have been proposed in the literature [16–18,40]. These models are based on a comparison of theprimary structure of different sHsp, predictions oftheir secondary structure and on superposition of themammalian protein sequence on the 3D structure ofalready crystallized sHsp [16–18,40,41]. The a-crystallindomain of human sHsp lacks the b6 strand detected inthe structure of Hsp16.5 of M. jannaschii and wheatHsp16.9 and the loop connecting b5 and b7 is muchshorter than the corresponding loop of bacterial orplant sHsp (Fig. 1) [10–12,40,41]. This loop appears toplay an important role in the structure and propertiesof the sHsp [10–12,17,19,40,41] and mutations insidethis loop or in the b7 strand correlate with the devel-opment of different congenital diseases [20,21,27,28].Because, at present, only two mammalian sHsp(a-crystallin and HSP27) have been investigated indetail, we were interested in analyzing the role of theb5–b7 loop in the structure of recently describedhuman HSP22.The data obtained with respect to far-UV CD(Fig. 7), extra high susceptibility to proteolysis (Fig. 8)and resistance to thermal denaturation (Fig. 3) indicatethat HSP22 has a predominantly unordered structure.Therefore, we might assume that HSP22 belongs tothe group of intrinsically disordered proteins. Accord-ing to the predictions, K137 is located either in theC-terminal part of the b5–b7 loop or in the N-terminalpart of the b7 strand, whereas K141 is located insidethe b7 strand (Fig. 1A). Predictions of disorderedregions using two different programs (http://www.strubi.ox.ac.uk/RONN and http://iupred.enzim.hu) indi-cate that residues 137–141 of HSP22 are located onthe border of the unordered and ordered regions ofHSP22 in the so-called downward spike [37] (Fig. 10).Very often, these parts of the molecules are involved ininter- or intramolecular interactions and play animportant role in recognition and cell signaling [37].Fig. 9. Chaperone activity of the wild-type HSP22 and its K137E andK137,141E mutants using insulin (A) and rhodanase (B) as a modelprotein substrates. (A) Reduction induced aggregation of insulin(0.2 mgÆmL)1) in the absence of HSP22 (curve 0) or in the presenceof 0.1 mgÆmL)1(empty symbols) or 0.2 mgÆmL)1(filled symbols) ofHSP22 (curves 3 and 3¢), or its K137E (curve 1 and 1¢) andK137,141E mutant (curves 2 and 2¢). (B) Heat-induced aggregation ofrhodanase (0.14 mgÆmL)1) in the absence of HSP22 (curve 0) or inthe presence of 0.07 mgÆmL)1(empty symbols) or 0.14 mgÆmL)1(filled symbols) of HSP22 or its mutants. Curve numbers andsymbols are same as given in (A).Fig. 10. RONN plot of disorder probability of the wild-type HSP22(NCBI-Entrez-Protein database accession number Q9UKS3). Thehorizontal line indicates the threshold for disorder prediction. Posi-tions of K137 and K141 are marked by black dots.A. S. Kasakov et al. Point mutations of the b5– b7 loop of human HSP22FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS 5637[...]... predictions, K137 and K141 are located on the border of the unordered loop and the b7 strand of HSP22 Mutations K137E and especially K137,141E lead to an increase in the proportion of unordered structure in HSP22 (Fig 7) and increased susceptibility to trypsinolysis (Fig 8) These effects can be due to the overall changes in the flexibility of HSP22 (e.g the abovementioned movement of the N-terminal end)... crosslinking of the wild-type protein (Fig 4) The data available in the literature indicate that the b5–b7 loop can be involved in the interaction of the sHsp with different unfolded target proteins [17,40] Therefore, it is desirable to analyze the effect of K137E and K137,141E mutations on the chaperonelike activity of HSP22 The data shown in Fig 9 indicate that the K137E mutant possesses lower chaperone... conclude that mutations in the b5– b7 loop located on the border of an intrinsically disordered region and the b7 strand affect the structure of HSP22 and its chaperone-like activity This explains why mutations in this part of different sHsp (aA-, aBcrystallin, HSP27 and HSP22) induce deleterious effects and are associated with different congenital diseases Experimental procedures Cloning, expression... changes in the flexibility of the b5–b7 loop itself The data available in the literature indicate that the b5–b7 loop is involved in a-crystallin intersubunit contacts [19] Data obtained via size-exclusion chromatography (Fig 3) and chemical crosslinking (Fig 4) indicate that HSP22 is presented in the form of an equilibrium mixture of monomers and dimers Mutation K137E decreased the probability of the dissociation... are the average of 8–10 accumulations The method of Sreerama and Woody [39] was used to estimate the secondary structure All calculations were performed by using the continll program with the reference set of 12 proteins containing a high proportion of b-strand (including Hsp16.5 of M jannaschii) and five denatured proteins Limited proteolysis HSP22 and its mutants (0.6 mgÆmL)1) dissolved in buffer containing... composition was determined by SDS gel electrophoresis performed on gradient (5–20%) polyacrylamide gel The apparent rate constant of trypsinolysis was determined by plotting ln(At ⁄ Ao) (where Ao and At denote the intensity of protein bands of intact unhydrolized protein at the beginning and at the fixed time of incubation) against the time of hydrolysis 5640 Chaperone-like activity The chaperone-like... spectrophotometrically using A280 ¼ 1.09 for 0.1% solution Stock solution of insulin was added to 100 mm phosphate buffer containing 100 mm NaCl so that the final pH of the mixture became equal to 7.1 and the insulin concentration was equal to 0.40 mgÆmL)1 Some 120 lL of this insulin solution was mixed with 120 lL of buffer B containing different quantities of HSP22 so that the final concentration of HSP22 varied... 0–0.20 mgÆmL)1 After incubation for 10 min at 37 °C, the reaction was started by the addition of 20 lL of 260 mm solution of dithiothreitol In the second case, 150 lL of rhodanase (Sigma) (0.28 mg mL)1) in 100 mm phosphate pH 7.0 containing 100 mm NaCl were mixed with 150 lL of buffer B containing different quantities of HSP22 or its mutants The final concentration of HSP22 varied in the range 0–0.14 mgÆmL)1... flexibility in the small heat shock protein Hsp26 Structure 14, 1197–1204 Bova MP, Huang Q, Ding L & Horwitz J (2002) Subunit exchange, conformational stability, and chaperonelike function of the small heat shock protein 16.5 from Methanococcus jannaschii J Biol Chem 277, 38468– 38475 Sobott F, Benesch JL, Vierling E & Robinson CV (2002) Subunit exchange of multimeric protein complexes Real-time monitoring of. .. mutations of the b5–b7 loop of human HSP22 17 Ghosh JG, Estrada MR & Clark JI (2005) Interactive domains for chaperone activity in the small heat shock protein, human alphaB crystallin Biochemistry 44, 14854–14869 18 Theriault JR, Lambert H, Chavez-Zobel AT, Charest G, Lavigne P & Landry J (2004) Essential role of the NH2-terminal WD ⁄ EPF motif in the phosphorylationactivated protective function of mammalian . Effect of mutations in the b5–b7 loop on the structure and properties of human small heat shock protein HSP22 (HspB8, H11) Alexei S. Kasakov1,*,. in the b5–b7 loop and in the b7strand appear to play an important role in the structure and properties of HSP22, mutations in this part of the small heat
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