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

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,*, Olesya V Bukach1,*, Alim S Seit-Nebi1, Steven B Marston2 and Nikolai B Gusev1 Department of Biochemistry, School of Biology, Moscow State University, Russia National Heart and Lung Institute, Imperial College London, UK Keywords chaperone-like activity; intrinsically disordered regions; oligomeric structure; small heat shock proteins Correspondence N B Gusev, Department of Biochemistry, School of Biology, Moscow State University, Moscow 119991, Russia Fax ⁄ Tel: +7 495 939 2747 E-mail: nbgusev@mail.ru *These authors contributed equally to this work (Received 17 June 2007, revised 30 July 2007, accepted September 2007) doi:10.1111/j.1742-4658.2007.06086.x The human genome encodes ten different small heat shock proteins, each of which contains the so-called a-crystallin domain consisting of 80– 100 residues and located in the C-terminal part of the molecule The a-crystallin domain consists of six or seven b-strands connected by different size loops and combined in two b-sheets Mutations in the loop connecting the b5 and b7 strands and conservative residues of b7 in aA-, aB-crystallin and HSP27 correlate with the development of different congenital diseases To understand the role of this part of molecule in the structure and function of small heat shock proteins, we mutated two highly conservative residues (K137 and K141) of human HSP22 and investigated the properties of the K137E and K137,141E mutants These mutations lead to a decrease in intrinsic Trp fluorescence and the double mutation decreased fluorescence resonance energy transfer from Trp to bis-ANS bound to HSP22 Mutations K137E and especially K137,141E lead to an increase in unordered structure in HSP22 and increased susceptibility to trypsinolysis Both mutations decreased the probability of dissociation of small oligomers of HSP22, and mutation K137E increased the probability of HSP22 crosslinking The wild-type HSP22 possessed higher chaperone-like activity than their mutants when insulin or rhodanase were used as the model substrates Because conservative Lys residues located in the b5–b7 loop and in the b7 strand appear to play an important role in the structure and properties of HSP22, mutations in this part of the small heat shock protein molecule might have a deleterious effect and often correlate with the development of different congenital diseases Small heat shock proteins (sHsp) form a large superfamily of ubiquitous proteins detected in all organisms, except for some bacteria [1–3] The members of this family range in size from 12–42 kDa and contain a conservative so-called a-crystallin domain consisting of 80–100 residues that is located in the C-terminal part of the polypeptide chain [1–3] This conservative domain is flanked by the N-terminal domain and short C-terminal extension with a different size and structure [4,5] All sHsp tend to form flexible oligomers, ranging from a dimer to more than 40 subunits, exchanging their subunits [6,7], and some sHsp are able to form mixed oligomers consisting of subunits of different natures [8,9] Crystal Abbreviations bis-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 FEBS A S Kasakov et al structures are described in the literature for the hyperthermophile Methanococcus jannaschii Hsp16.5 [10] and wheat (Triticum aestivum) Hsp16.9 [11], each containing a single a-crystallin domain, and the parasitic flatworm Taenia saginata Tsp36, containing two a-crystallin domains in the single polypeptide chain [12] Ten different sHsp are encoded in the human genome and are differently expressed in human tissues [13,14] None of these proteins has been crystallized; however, different experimental approaches (cryoelectron microscopy, electron spin resonance spectroscopy, protein pin array, etc.) [15–17] and protein modeling were used to reconstruct the structure of mammalian aB-crystallin and Hsp27 (HspB1) [16– 18] According to these models, the a-crystallin domain of both proteins consists of seven b-strands packed into two b-sheets [16–18] The loop connecting b5 and b7 and the N-terminal part of b7 appears to play an important role in the structure of sHsp monomers [12,18] and intermonomer interactions [12,18,19], as well as in the binding of protein substrates to sHsp [17] The importance of this part of molecule of the sHsp is supported by the fact that mutations in the loop connecting the b5 and b7 strands, or in the b7 strand of sHsp, often correlate with the development of certain congenital diseases (congenital cataract, desmin related myopathy, distal hereditary motor neuropathy, amongst others) [20,21] A recently described protein with an apparent molecular mass of 22 kDa, denoted as HSP22, HspB8 or H11 kinase, shares structural properties typical to all members of the family of sHsp [22] HSP22 possesses chaperone-like activity [23–25] and appears to be involved in the regulation of many processes such as proliferation, myocardium hypertrophy and apoptosis [26] Missense mutations of K141 (K141E, K141N) located at the beginning of b7 of HSP22 correlate with the development of motor 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 is mutated in the case of Charcot–Marie–Tooth type disease [20,21] Previously, we compared the structure and properties of the wild-type HSP22 and its K141E mutant [29] The present study analyses the structure and properties of K137E and the K137,141E mutant of human HSP22, aiming to provide new information on the structure of sHsp and to shed new light on their role in the development of human congenital diseases Point mutations of the b5–b7 loop of human HSP22 Results Peculiarities of HSP22 structure Up to now, all attempts to crystallize mammalian sHsp have been unsuccessful Therefore, all structural information derives from a comparison of human sHsp with the crystal structures of M jannaschii Hsp16.5 [10] and T aestivum Hsp16.9 [11] The 3D structure of the monomer of T aestivum Hsp16.9 is presented in Fig 1A (protein databank accession code 1GME) and, as shown in Fig 1B, we aligned the structures of M jannaschii Hsp16.5 and T aestivum Hsp16.9 with the corresponding structures of three human sHsp [30] The elements of the secondary structure of M jannaschii Hsp16.5 and T aestivum Hsp16.9, as determined by X-ray crystallography, are indicated by solid blue (a-helices) or solid red (b-strands) lines above and below the corresponding sequences (Fig 1A) Both these proteins contain a large number of well preserved b-strands that are predominantly (with the exception of the b10 strand) located in the a-crystallin domain [10–12] The models built for two mammalian sHsp (aB-crystallin [16] and HSP27 [18]) predict that both these proteins contain short a-helices in the N-terminal part of molecule (dashed blue lines denoted a1–a3 above the aB-crystallin and below the HSP27 sequences in Fig 1B) According to these models, both aB-crystallin and HSP27 contain seven b-strands (b2–b9) (dashed red lines) located in positions homologous to the corresponding strands of two crystallized nonmetazoan sHsp Two predictions slightly differ with respect to the location and length of specific b-strands For example, in the model of aB-crystallin, the b7 strand is only four residues long [16] whereas, in the model of HSP27, the same strand is ten residues long [18] However, the overall structures of aB-crystallin and HSP27 predicted by these two models are very similar, and the positions of the b-strands correlate well with the corresponding positions of the b-strands in M jannaschii Hsp16.5 and T aestivum Hsp16.9 (Fig 1B) Predictions of the secondary structure of HSP22 performed with the jpred program (http://www.combio dundee.ac.uk) indicate that this protein contains very small quantities of a-helices and is enriched in unordered structure and b-strands The residues of HSP22 that are predicted to form b-strands are indicated by wide dashed red lines in Fig 1B and are located in positions corresponding to the b3, b4, b5, b7 and b9 strands jpred failed to predict the formation of a b2 strand in the HSP22 structure According to this prediction, residues 153–155 of HSP22 tend to form an FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS 5629 Point mutations of the b5–b7 loop of human HSP22 A S Kasakov et al A B Fig Comparison of the structure of human HSP22 and other sHsp (A) Ribbon diagram of T aestivum Hsp16.9 monomer (protein databank accession code 1GME) The N- and C-terminal domains are indicated by N and C correspondingly All b-strands are numbered and the b5 and b7 strands are shown in red and blue, respectively G104 (equivalent to K137 of human HSP22) and R108 (equivalent to K141 of human HSP22) are shown in purple and grey, respectively (B) Alignment of human HSP22 with human aB-crystallin and HSP27 and M jannaschii Hsp16.5 and T aestivum Hsp16.9 made with CLUSTALW [30] using the default settings The residues shown in black are identical in at least four sequences; residues in dark grey are conservative in at least four or identical in at least three sequences; residues in light grey are homologous at three or identical in at least two sequences Solid blue and red lines above M jannaschii Hsp16.5 and 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 predicted in the models of the corresponding proteins [16,18] Residues of HSP22 predicted to form b-strands according to JPRED are indicated by wide dashed red lines and K137 and K141 are shown in red Numbers in parenthesis correspond to NCBI-Entrez-Protein database accession numbers 5630 FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS A S Kasakov et al a-helix, whereas residues 156 and 157 tend to form a very short b-strand that might correspond to the b8 strand of the other sHsp The primary structure of the a-crystallin domain of human sHsp is very conservative and the loop connecting the b5 and b7 strands is shorter than the corresponding loop connecting the b5 and b7 strands of nonmetazoan sHsp (Fig 1B) Moreover, the structure of human sHsp lacks the b6 strand that is involved in dimer formation of nonmetazoan sHsp (Fig 1A) Although the b5–b7 loop is very short, it is not completely deleted in any human sHsp This part of the molecule has a very conservative primary structure and appears to play a diverse and important role For example, mutation of a highly conservative positively charged residue (R116 of aA-crystallin, R120 of aBcrystallin or K141 of HSP22 located in homologous position; Fig 1B) correlates with the development of congenital cataract and ⁄ or desmin related myopathy [20,21], whereas mutations of R127, S135 and R136 of human HSP27 are associated with distal hereditary motor neuropathy and Charcot–Marie–Tooth disease [20,21] Therefore, it is advisable to analyze the effect of a mutation in this part of the molecule on the structure and properties of human sHsp Oligomeric structure of HSP22 and its mutants All samples of recombinant HSP22 and its mutants purified by the method described previously [23] were homogeneous according to SDS gel electrophoresis (Fig 2) HSP22 and its mutants are highly susceptible Fig SDS electrophoresis of the wild-type HSP22 (1) and its K137E (2), K141E (3) and K137,141E (4) mutants The positions of the molecular mass standards (in kDa) are indicated by arrows Point mutations of the b5–b7 loop of human HSP22 to proteolysis [23,24,29] and occasionally contained small quantities of proteolytic fragments Under the conditions used, the wild-type HSP22 and its K137E and K141E mutants migrated on the SDS gel electrophoresis [31] as a band with an apparent molecular mass of 25.4 kDa, whereas the apparent molecular mass of the double mutant K137,141E was 30.4 kDa The calculated molecular mass of human wild-type HSP22 is close to 21.6 kDa [22] The unusually high apparent molecular mass determined by SDS gel electrophoresis can be due to anomalous binding of SDS to acidic HSP22 and this effect is especially pronounced in the case of the particularly acidic double mutant K137,141E of HSP22 On native gel electrophoresis performed both at neutral [32] and alkaline pH [33], the wild-type HSP22 and its mutants migrated as a single band with an apparent molecular mass of approximately 60 kDa (data not shown), thus indicating that, under these conditions, HSP22 and its mutants form small oligomers Size-exclusion chromatography was used for further investigation of the quaternary structure of HSP22 and its mutants When 200 lg of the wild-type HSP22 was loaded on the column, a single peak was detected with ˚ a Stokes radius equal to 26.2 A, corresponding to an apparent molecular mass of 36.1 kDa (Fig 3A) These data agree well with the previously published data [23,24,29] On size-exclusion chromatography, both K141E and K137,141E were eluted as symmetrical peaks and the width at the respective half-height of their peaks was similar to that of the wild-type HSP22 The Stokes radii and apparent molecular masses of the ˚ K141E and K137,141E mutants were similar: 26.7 A and 37.9 kDa (Fig 3A) [29] At the same time, the K137E mutant of HSP22 was eluted as a broad peak with a trailing end, with a Stokes radius and apparent ˚ molecular mass of 28.2 A and 43.9 kDa, respectively (Fig 3A) Taking into account that the molecular mass of HSP22 monomer is 21.6 kDa [22], it might be assumed that, under conditions of size-exclusion chromatography, HSP22 and its mutants are either highly asymmetric (or intrinsically unfolded) or presented in the form of a mixture of monomers and dimers The data presented indicate that mutations in the b5–b7 loop (and especially K137E) affect either folding or extension of oligomerization of HSP22 To test this suggestion, we performed size-exclusion chromatography on the Superdex 200 HR10 ⁄ 30 column in the presence of m guanidinium chloride (GuCl) and, under these conditions, calibrated the column with a set of protein standards (BSA, ovalbumin, chymotrypsin A and RNAse) [34] (Fig 3B) Under denaturating conditions, all samples of HSP22 were FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS 5631 Point mutations of the b5–b7 loop of human HSP22 A S Kasakov et al Fig Size-exclusion chromatography of the wild-type HPS22 and its point mutants (A) Size-exclusion chromatography of the wildtype HSP22 (1, 2) and its K137E (3, 4) and K137,141E (5, 6) mutants on Superdex 75 column under native conditions The samples were either kept on ice (solid curves 1, 3, 5) or heated for 30 at 70 °C (dashed curves 2, 4, 6) Equal volumes (150 lL) of each protein (210 lg) were subjected to chromatography on a Superdex 75 HR10 ⁄ 30 column For clarity, elution profiles of unheated and heated proteins are shifted from each other by 10 mAu and elution profiles between different proteins are shifted from each other by 30 mAu Arrows above the panel indicate the elution volume of protein standards and their apparent molecular masses (B) Size-exclusion chromatography of the wild-type HSP22 (1) and its K137E (2), K141E (3) and K137,141E (4) mutants on the Superdex 200 HR10 ⁄ 30 column in the presence of M GuCl Equal volumes (150 lL) of each protein (150 lg) were subjected to chromatography For clarity, elution profiles are shifted from each other by 20 mAu Arrows above the panel indicate the elution volume of protein standards and their apparent molecular masses (C) Dependence of elution volume on the quantity of protein loaded on a Superdex 75 HR10 ⁄ 30 column Equal volumes (150 lL) containing 10–200 lg of the wild-type protein (1) and its K137E (2) or K137,141E (3) mutants were subjected to chromatography under native conditions The data are representative of three independent experiments eluted in the form of symmetrical peaks with an apparent molecular mass of 22.8 kDa, which is close to the calculated value of the HSP22 monomer (21.6 kDa) The data presented agree with the suggestion that, under native conditions, HSP22 and its mutants form dimers that dissociate to monomers in the presence of m GuCl If this suggestion is correct, we might assume that a decrease in protein concentration will result in the dissociation of small HSP22 oligomers and the formation 5632 of protein species with smaller apparent molecular mass Indeed, if the quantity of the wild-type HSP22 loaded on the column was decreased from 200 lg to 10 lg, the elution volume of the protein peak was increased from approximately 11.3 mL to 11.8 mL (Fig 3C) This increase in elution volume corresponds to a decrease in the apparent molecular mass from approximately 36.9 kDa to 29.3 kDa A similar decrease in the apparent molecular mass was observed for the K137,141E mutant of HSP22; however, at all concentrations, the apparent molecular mass of this mutant was slightly larger than the molecular mass of the wild-type protein (Fig 3C) At high concentration, the K137E mutant formed oligomers with an apparent molecular mass of approximately 44 kDa whereas, at very low concentration, the molecular mass of oligomers formed by this mutant was close to 32 kDa (Fig 1C) The data presented mean that mutations of K137 and K141 might affect either folding or dissociation of HSP22 oligomers There are many examples indicating that certain point mutations not dramatically affect the quaternary structure but, at the same time, induce destabilization of the overall structure of the sHsp [35,36] Therefore, we analyzed the effect of point mutations in the linker connecting the b5 and b7 strands of HSP22 on its thermal stability The wild-type protein or its mutants were heated for 30 at 70 °C and, after FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS A S Kasakov et al cooling for 20 and centrifugation, were subjected to size-exclusion chromatography (Fig 3A) Prolonged heating at 70 °C did not affect the elution profile of any of the proteins analyzed The amplitude, position and the width of the protein peaks were not dependent on the transient heating These data suggest that the wild-type HSP22 and its mutants belong to the group of the so-called intrinsically disordered proteins with long stretches of unordered structure [37] and this is one of the reasons for their unusual high thermal stability To further investigate the oligomeric structure of HSP22, we employed chemical crosslinking HSP22 and its mutants at three different concentrations (0.1, 0.5 and 2.0 mgỈmL)1) were incubated in the presence of 3.5 mm dimethylsuberimidate (DMS) for h at 37 °C and the protein composition of the sample thus obtained was analyzed by means of SDS gel electrophoresis In good agreement with the previously published data [23,29], we found that incubation of the wild-type HSP22 with the bifunctional reagent resulted in the formation of an additional protein band with an apparent molecular mass of 50 kDa, which presumably corresponds to the HSP22 dimer (Fig 4A) Similar results were observed in the case of the K137E mutant of HSP22 (Fig 4B); however, in this case, the intensity of the band corresponding to the HSP22 dimer was more intense than in the case of the wild-type protein Thus, although mutation K137E eliminates one potential site of chemical modification, the probability of crosslinking of the K137E mutant by DMS is higher than the probability of crosslinking of the wild-type protein This fact agrees well with the size-exclusion chromatography data indicating that the K137E mutant forms larger oligomers than the wild-type protein (Fig 3C) If the double mutant K137,141E was subjected to crosslinking, we detected only a very faint band corresponding to dimer and this band was detected only at a rather high protein concentration (Fig 4C) The decreased probability of crosslinking of the K137,141E mutant might be due to replacements of Lys residues being potential sites of crosslinking or, more likely, to the overall changes in the structure of HSP22 that are induced by replacing two closely separated positively charged Lys residues by negatively charged Glu (see below) Effect of K137E and K137,141E mutations on the structure of HSP22 The data presented might indicate that the analyzed mutations affect the secondary and tertiary structure of HSP22 To check this suggestion, we analyzed some Point mutations of the b5–b7 loop of human HSP22 A B C Fig Crosslinking of the wild-type HSP22 (A) and its K137E (B) and K137,141E (C) mutants by DMS HSP22 was incubated either in 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) or 2.0 (3) mgỈmL)1 and, after incubation, equal quantities (2.5 lg) of protein were loaded onto the gel The positions of the molecular mass standards (in kDa) are indicated by arrows on the right spectral properties of the wild-type protein and its two mutants The maximum of intrinsic Trp fluorescence of the wild-type HSP22 was located at 342 nm and the position of this maximum was not changed by mutations K137E or K137,141E (Fig 5) Similar results were obtained previously with the K141E mutant of HSP22 [29] The fluorescence spectrum of HSP22 was decomposed into discrete components characteristic of Trp located in different environments [38] For this FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS 5633 Point mutations of the b5–b7 loop of human HSP22 A S Kasakov et al Fig Intrinsic Trp fluorescence of the wild-type HSP22 (1) and its K137E (2) and K137,141E (3) mutants Fluorescence was excited at 295 nm The protein concentration was 0.1 mgỈmL)1 purpose, the fluorescence spectra were fitted as a sum of three polynomial distributions of the fourth of fifth order, corresponding to three classes of Trp residues differing in their environment, accessibility to solvent and position of the fluorescent spectrum Using this approach, we estimated the portion of each class of fluorophores in the protein spectrum and found that HSP22 contains Trp residues belonging to the so-called classes I, II and III Class I corresponds to indole located inside the protein globule, forming a : exciplex with neighboring polar groups and having maximum fluorescence at 330–332 nm Class II corresponds to Trp at the protein surface in contact with bound water molecules (maximum fluorescence at 340– 342 nm) Finally, class III corresponds to indole located at the protein surface in contact with free water molecules (maximum fluorescence at 350– 355 nm) Approximately 44% of Trp residues of HSP22 belong to class I, approximately 18% belong to class II and approximately 38% belong to class III Point mutations K137E or K137,141E not significantly affect the distribution of Trp residues between these classes (data not shown) This may be due to the fact that three out of four Trp residues are located in the N-terminal end (Trp48, Trp51, Trp60) and the fourth Trp residue (Trp96) are located at the very beginning of the a-crystallin domain, far apart from the mutated Lys residues Although the point mutations not affect the position of maximum fluorescence, they slightly decrease the amplitude of fluorescence and this decrease was more pronounced for the K141E [29] and K137,141E mutants than for the K137E mutant (Fig 5) The small decrease in the amplitude of fluorescence detected for the point mutants of HSP22 might reflect small changes in 5634 structure, leading to an altered Trp environment or their accessibility to quencher or water molecules Hydrophobic interactions appear to play an important role in oligomer formation and in the interaction of sHsp with their protein substrates [1,10–12] Hydrophobic surfaces of HSP22 and its mutants were probed by using bis-ANS In the isolated state, this hydrophobic probe has a very low quantum yield that is dramatically increased after its binding to hydrophobic sites on the protein molecules [24,29] Titration of HSP22 with bis-ANS was accompanied by an increase in fluorescence at 495 nm, indicating binding of the fluorescence probe to the protein [24,29] In agreement with the previously published data [29], we were unable to achieve saturation and, in the range of 0–10 lm bisANS, the fluorescence at 495 nm was approximately proportional to the concentration of the fluorescent probe added These data indicate that HSP22 contains many low affinity bis-ANS binding sites that cannot be completely saturated in the range of bis-ANS concentrations used This is to be expected if HSP22 belongs to the group of intrinsically disordered proteins lacking well-organized hydrophobic sites To obtain more information on the structure, we analyzed fluorescence resonance energy transfer (FRET) from Trp residues of HSP22 and its mutants to the bound bis-ANS As indicated in Fig 6, titration of the wildtype HSP22 and its K137,141E mutant with bis-ANS was accompanied by a decrease in intrinsic Trp fluorescence at 342 nm and a concomitant increase in the fluorescence of bis-ANS at 495 nm Because, at any bis-ANS concentration, the ratio of fluorescence at 342 to fluorescence at 495 nm (F342 ⁄ F395) was lower for the wild-type protein than for its K137,141E mutant, we conclude that the probability of FRET is higher for the wild-type protein than for its mutant This may indicate that the mutation K137,141E affects the mutual orientation, overall flexibility and ⁄ or distances between Trp and bis-ANS bound to HSP22 To obtain more detailed information on the structure of HSP22 mutants, we employed CD spectroscopy The far-UV CD spectra of the wild-type protein has a negative maximum at 208 nm and its molar ellipticity at this wavelength is rather low (Fig 7) This spectrum is characteristic for proteins with a low a-helix content and a high content of unordered and b-structures Mutation K137E had no dramatic effect on the far-UV CD spectra and a blue shift of only 2– nm was observed in the position of the negative maximum (Fig 7) Previously, we have found that mutation K141E induces a rather large increase in the amplitude of the negative maximum on the far-UV CD spectrum of HSP22 [29] Even larger changes were FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS A S Kasakov et al Point mutations of the b5–b7 loop of human HSP22 Fig Fluorescence resonance energy transfer from Trp residues of the (A) wild-type HSP22 and (B) its K137,141E mutant to the bound bis-ANS All experiments were performed at a protein concentration of 0.03 mgỈmL)1 (1.5 lM of HSP22 monomer) and bis-ANS (in lM) indicated above each spectrum secondary structure At the same time, mutation K141E [29] and especially double mutation K137,141E were accompanied by a simultaneous decrease in the content of b-structure (from 37% to 31%) and an increase in the content of turns and unordered structure (from 58% to 63%) These data might indicate that mutations in the b5–b7 loop and in the N-terminal part of the b7 strands destabilize the structure of HSP22 Limited trypsinolysis of the wild-type HSP22 and its K137E and K137,141E mutants Fig Far-UV CD spectra of the wild-type HPS22 (1) and its K137E (2) and K137,141E (3) mutants The spectra were recorded at the concentration 0.65 mgỈmL)1 of each species with a cell path of 0.05 cm The spectra reported are the average of eight determinations observed in the case of the double K137,141E mutant Indeed, the double mutation results in a blue shift of 5–6 nm with respect to the position of negative maximum and a significant increase in the amplitude of this maximum This change of the far-UV CD spectra can reflect pronounced changes in the secondary structure Using the approach developed by Sreerama and Woody [39], we attempted to estimate the changes induced by the point mutations in the secondary structure of HSP22 According to this estimation, the a-helix content is equally low (approximately 5–6%) in the structure of both the wild-type HSP22 and its two mutants As expected, the secondary structure of HSP22 and its mutants was characterized by a high content of b-strands (approximately 31–37%) and turns and unordered structures (approximately 58–63%) Mutation K137E induced only very moderate changes in the The method of limited trypsinolysis was used to check the suggestion that the analyzed mutations affect the stability of HSP22 The available literature [23,24,29] indicate that HSP22 is highly susceptible to proteolysis Indeed, even at a weight ratio for HSP22 ⁄ trypsin equal to 12 000 : 1, the sHsp was rapidly hydrolyzed (Fig 8A) Trypsinolysis of the wild-type HSP22 was accompanied by disappearance of the band corresponding to intact protein that migrated with an apparent molecular mass of 25.4 kDa and accumulation of peptides with apparent molecular masses equal to 16.5, 18.0, 19.0, 22.0 and 23 kDa, respectively (Fig 8A) The same set of peptides was observed if K137E and K137,141E mutants were subjected to trypsinolysis To compare the apparent rates of trypsinolysis of the wild-type HSP22 and its mutants, we plotted ln(At ⁄ Ao) (where Ao and At are the intensities of the band of intact protein at the beginning of trypsinolysis and at the fixed time of trypsinolysis) against the time of incubation (Fig 8D) The apparent rate constants of trypsinolysis under these conditions were equal to 0.0496 ± 0.0027, 0.068 0.026, 0.0863 0.0039ặmin)1 (n ẳ 7) for the wild-type HSP22, and its K137E and K137,141E mutants, respectively The data FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS 5635 Point mutations of the b5–b7 loop of human HSP22 A S Kasakov et al these mutations increased the susceptibility of HSP22 to trypsinolysis and this finding agrees well with the data of far-UV CD indicating that the analyzed mutations induce destabilization of the HSP22 structure A Chaperone-like activity of wild-type HSP22 and its mutants B C D Fig Limited trypsinolysis of the wild-type HSP22 and its K137E and K137,141E mutants Kinetics of trypsinolysis of the wild-type HSP22 (A) and its K137E (B) and K137,141E (C) mutants The time of incubation (in min) is indicated below each track and the arrows show the positions of molecular mass markers (D) Determination of 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 experiments presented mean that K137E and especially K137,141E mutants were more susceptible to proteolysis than the wild-type HSP22 Mutations K137E and K137,141E should eliminate one or two potential sites of trypsinolysis and, in this way, were expected to decrease the rate of proteolysis Instead of decreasing susceptibility, 5636 The data presented indicate that the point mutations of residues 137 and 141 affect the structure and stability of HSP22 Therefore, it can be expected that these mutations might change the chaperone-like activity of HSP22 To investigate this idea, we used two different model protein substrates Reduction of the disulfide bonds of insulin results in dissociation of its peptide chains and aggregation of chain B Addition of the wild-type HSP22 retarded the onset of aggregation and decreased the amplitude of light scattering induced by insulin aggregation (Fig 9A, curves and 3¢) K137E (Fig 9A, curves and 1¢) and K137,141E (Fig 9A, curves and 2¢) also retarded the onset of insulin aggregation and decreased the amplitude of light scattering; however, their effects were less pronounced than the corresponding effects of the wild-type protein For example, the aggregation curve in the presence of 0.2 mgỈmL)1 of K137E was comparable to the aggregation curve observed in the presence of 0.1 mgỈmL)1 of the wild-type HSP22 (compare curves 1¢ and in Fig 9) The double mutant (K137,141E) possessed higher chaperone-like activity than the K137E mutant However, both at low and high concentrations, the double mutant possessed slightly lower chaperone-like activity than the wild-type protein (curves and in Fig 9) Heating of rhodanase at 43 °C induces its denaturation, which is followed by aggregation The wildtype HSP22 and its mutants decreased the rate of rhodanase aggregation and the amplitude of light scattering (Fig 9B) In good agreement with the data obtained for insulin, we found that the K137E mutant was much less effective than the wild-type protein in preventing rhodanase aggregation (compare curves and in Fig 9B) The chaperone-like activity of K137,141E mutant was lower than, but comparable to, the chaperone activity of the wildtype protein Thus, on two different protein substrates, the chaperone-like activity of the wild-type HSP22 was higher than the corresponding activity of the two mutants analyzed and, among these mutants, the chaperone activity of the K137E mutant was especially low FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS A S Kasakov et al Fig Chaperone activity of the wild-type HSP22 and its K137E and K137,141E mutants using insulin (A) and rhodanase (B) as a model protein substrates (A) Reduction induced aggregation of insulin (0.2 mgỈmL)1) in the absence of HSP22 (curve 0) or in the presence of 0.1 mgỈmL)1 (empty symbols) or 0.2 mgặmL)1 (lled symbols) of HSP22 (curves and 3Â), or its K137E (curve and 1¢) and K137,141E mutant (curves and 2¢) (B) Heat-induced aggregation of rhodanase (0.14 mgỈmL)1) in the absence of HSP22 (curve 0) or in the presence of 0.07 mgỈmL)1 (empty symbols) or 0.14 mgỈmL)1 (filled symbols) of HSP22 or its mutants Curve numbers and symbols are same as given in (A) Point mutations of the b5–b7 loop of human HSP22 their secondary structure and on superposition of the mammalian protein sequence on the 3D structure of already crystallized sHsp [16–18,40,41] The a-crystallin domain of human sHsp lacks the b6 strand detected in the structure of Hsp16.5 of M jannaschii and wheat Hsp16.9 and the loop connecting b5 and b7 is much shorter than the corresponding loop of bacterial or plant sHsp (Fig 1) [10–12,40,41] This loop appears to play an important role in the structure and properties of the sHsp [10–12,17,19,40,41] and mutations inside this loop or in the b7 strand correlate with the development of different congenital diseases [20,21,27,28] Because, at present, only two mammalian sHsp (a-crystallin and HSP27) have been investigated in detail, we were interested in analyzing the role of the b5–b7 loop in the structure of recently described human 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) indicate that HSP22 has a predominantly unordered structure Therefore, we might assume that HSP22 belongs to the group of intrinsically disordered proteins According to the predictions, K137 is located either in the C-terminal part of the b5–b7 loop or in the N-terminal part of the b7 strand, whereas K141 is located inside the b7 strand (Fig 1A) Predictions of disordered regions using two different programs (http://www strubi.ox.ac.uk/RONN and http://iupred.enzim.hu) indicate that residues 137–141 of HSP22 are located on the border of the unordered and ordered regions of HSP22 in the so-called downward spike [37] (Fig 10) Very often, these parts of the molecules are involved in inter- or intramolecular interactions and play an important role in recognition and cell signaling [37] Discussion All sHsp are characterized by the presence of a highly conservative a-crystallin domain consisting of six or seven b-strands combined in two b-sheets [1,10–12,40] The detailed location and orientation of these b-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 The tertiary structure of other sHsp (including all human proteins) is unknown; however, several models of their structure have been proposed in the literature [16– 18,40] These models are based on a comparison of the primary structure of different sHsp, predictions of Fig 10 RONN plot of disorder probability of the wild-type HSP22 (NCBI-Entrez-Protein database accession number Q9UKS3) The horizontal line indicates the threshold for disorder prediction Positions of K137 and K141 are marked by black dots FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS 5637 Point mutations of the b5–b7 loop of human HSP22 A S Kasakov et al Our results indicate that mutations K137E and K137,141E affect the structure of HSP22 We found that the four Trp residues of HSP22 belong to three classes that differ in their accessibility to water (Fig 5) Our data agree well with the recently published data of Chowdary et al [42] who also detected three classes of Trp residues in rat HSP22 with a distribution of Trp residues between these three classes which was very similar to that determined in our investigation Three out of four Trp residues of HSP22 are located in the N-terminal part of the molecule This part of the molecule is assumed to be very flexible and appears to interact with the a-crystallin domain [18] through hydrogen bonds formed between certain residues of the N-terminal tail and Arg (or Lys) homologous to Lys141 of HSP22 [12] We might suppose that mutation of K141 or closely separated K137 makes the interaction of the N-terminal tail with the a-crystallin domain less probable This might result in the movement of the N-terminal tail away from the hydrophobic cluster that is formed by hydrophobic residues belonging to the b2, b3 and b7 strands [12,18] If this suggestion is correct, then three Trp residues located in the N-terminal tail will change their environment and their distance from one of the hydrophobic clusters of HSP22 This might explain the decrease in the intensity of Trp fluorescence (Fig 5) and the decrease in fluorescence resonance energy transfer (Fig 6) observed for K137E and K137,141E mutants of HSP22 As previously mentioned, according to 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) or to 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 of dimers (Fig 3) and therefore the K137E mutant probably is more easily crosslinked with DMS than the wild-type protein (Fig 4) Double mutation K137,141E also decreased the probability of dissociation of HSP22 dimers (Fig 2) However, because the double mutant K137,141E has a very flexible structure (Figs and 8), the probability of its crosslinking is lower than the 5638 probability of 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 indicate that the K137E mutant possesses lower chaperone activity than the wild-type protein The chaperone-like activity of the K137,141E mutant was lower than, but comparable to, the corresponding activity of the wildtype protein (Fig 9) The decreased chaperone-like activity of K137E mutant can be due to its tendency to form more stable oligomers than the wild-type protein (Figs and 4) and ⁄ or to changes in its hydrophobic properties The question arises as to why the double mutant K137,141E has a higher chaperone activity than the single K137E mutant We propose that this is explained by an increased flexibility of the K137,141E mutant (Figs and 8) and by its lower ability to form high molecular mass oligomers compared with the single K137E mutant (Fig 4) It is postulated [37,43] that both these factors can increase the chaperone-like activity of sHsp In summary, we 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 and purification of HSP22 and its mutants BL21-DE3 cells were transformed with the pET23b construct carrying the full sequence of the wild-type HSP22 or its mutants and cultured in LB media containing 0.1 mgỈmL)1 of ampicillin overnight at 25 °C Two hundred millilitres of the overnight culture were inoculated with L of LB media containing 0.1 mgỈmL)1 of ampicillin and cultured at 37 °C until an attenuance of 0.6 at D600 nm was reached Expression of HSP22 was induced by the addition of isopropyl thio-b-d-galactoside up to a final concentration 0.5 mm and the culture was grown for a further h The cells were collected by centrifugation, washed with the lysis buffer (50 mm Tris ⁄ HCl pH 8.0, 0.1 m NaCl, mm EDTA, 15 mm 2-mercaptoethanol, 0.5 mm phenylmethanesulfonyl fluoride), suspended in 30–40 mL of this buffer and frozen FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS A S Kasakov et al After thawing, lysozyme was added up to a final concentration 0.05 mgỈmL)1 and the suspension was incubated for 30 at °C Subsequently, magnesium chloride and DNAse I were added to the final concentration at mm and lgỈmL)1, respectively, and the suspension was incubated for another 15 at °C The suspension thus obtained was sonicated and subjected to centrifugation (22 000 g, 20 min) The supernatant was collected and the pellet was suspended in 20 mL of lysis buffer and subjected to sonication and centrifugation This operation was repeated twice more and the supernatants obtained were combined and subjected to ammonium sulfate fractionation (0–30% saturation) The pellet obtained was dissolved and dialyzed overnight against buffer A (20 mm Tris-acetate pH 8.0, containing 10 mm NaCl, 0.1 mm EDTA, 15 mm 2-mercaptoethanol and 0.1 mm phenylmethanesulfonyl fluoride) The dialysate was subjected to ultracentrifugation (105 000 g, 60 min) and the supernatant was used as the starting material for HSP22 purification Ammonium sulfate was added to the supernatant up to a final concentration of 0.3 m and the sample obtained was loaded onto a mL phenyl-sepharose High Trap column (Amersham Pharmacia, Helsinki, Finland) equilibrated with buffer A, containing 0.3 m ammonium sulfate After washing, the column was developed with 15 column volumes of a descending gradient of ammonium sulfate (0.3–0.005 m) The fractions containing HSP22 were collected and concentrated by ultrafiltration The samples obtained were subjected to size-exclusion chromatography on a Sephacryl S100 (16 ⁄ 60) column (Amersham Pharmacia) equilibrated with buffer A containing 150 mm NaCl The fractions containing highly purified HSP22 were concentrated by ultrafiltration, dialyzed against buffer B (20 mm Tris-acetate pH 7.6, containing 10 mm NaCl, 0.1 mm EDTA, 15 mm 2-mercaptoethanol and 0.1 mm phenylmethanesulfonyl fluoride) and kept frozen Size-exclusion chromatography Variable quantities (from 10 lg to 200 lg) of the wild-type HSP22 or its mutants in 150 lL of buffer C1 (20 mm Trisacetate pH 7.6, containing 150 mm NaCl, 0.1 mm EDTA, 15 mm 2-mercaptoethanol and 0.1 mm phenylmethanesulfonyl fluoride) were loaded via a 500 lL loop onto Superdex 75 HR10 ⁄ 30 column connected to Acta FPLC (Amersham Pharmacia) and eluted with the same buffer The following protein markers (with Stokes radii and molecular masses given in parentheses) were used for cali˚ bration of the column: BSA (33 A, 68 kDa), ovalbumin ˚ , 43 kDa), chymotrypsinogen A (22.54 A, 25 kDa) ˚ (26.8 A ˚ and ribonuclease (17.7 A, 13.7 kDa) To analyze thermal stability, the wild-type HSP22 and its mutants were heated in buffer C2 (20 mm Tris-acetate pH 8.0, containing 150 mm NaCl, 0.1 mm EDTA, 15 mm 2-mercaptoethanol and 0.1 mm phenylmethanesulfonyl fluoride) at 70 °C for Point mutations of the b5–b7 loop of human HSP22 30 min, cooled on ice for 20 min, centrifuged (12 000 g, 10 min) and subjected to size-exclusion chromatography at room temperature on a Superdex 75 HR10 ⁄ 30 column equilibrated with buffer C2 Size-exclusion chromatography in the presence of GuCl was performed on a Superdex 200 10 ⁄ 30 column according to Mann and Fish [34] All samples were incubated overnight in 50 mm Tris-acetate pH 7.6, containing m GuCl and 200 mm 2-mercaptoethanol and subjected to chromatography on a Superdex 200 10 ⁄ 30 column in the same buffer containing 15 mm 2-mercaptoethanol The column was calibrated with BSA (68 kDa), ovalbumin (43 kDa), rabbit glyceraldehyde-3phosphate dehydrogenase (36 kDa) and ribonuclease (13.7 kDa) that were subjected to chromatography in the presence of GuCl under the above-mentioned conditions Electrophoretic methods SDS gel electrophoresis on homogeneous (12.5% or 15%) or gradient (5–20%) polyacrylamide gels was performed by the method of Laemmli [31] Native gel electrophoresis run on gradient (5–20%) polyacrylamide gel using buffer systems with initial pH values equal to 7.0 and 8.9 was performed according to Schagger et al [32] or Davis [33], respectively Ferritin (880 kDa and 440 kDa), thyroglobulin (670 kDa), catalase (230 kDa), BSA (136 kDa and 68 kDa), ovalbumin (43 kDa) and troponin C (18 kDa) were used as standard markers Chemical crosslinking Crosslinking was performed at three different protein concentrations (0.10, 0.50 and 2.00 mgỈmL)1) and a fixed concentration of DMS equal to 3.5 mm Twenty microlitres of protein solution in 20 mm Tris-acetate pH 7.6, containing 10 mm NaCl, 0.1 mm EDTA, 0.1 mm phenylmethanesulfonyl fluoride and 30 mm 2-mercaptoethanol, were mixed with an equal volume of mm DMS in 400 mm triethanolamine ⁄ HCl (pH 8.0) and incubated at 37 °C for 60 The reaction was stopped by the addition of SDS sample buffer and the protein composition of samples thus obtained was analyzed by means of SDS gel electrophoresis on gradient (5–20%) polyacrylamide gel [31] Fluorescence spectroscopy Intrinsic protein fluorescence was measured on an Hitachi F3000 spectrofluorometer (Hitachi Corp Tokyo, Japan) and fluorescence was excited at 295 nm (slit width ¼ nm) and recorded in the range 300–400 nm (slit width ¼ 1.5 nm) All measurements were performed in buffer F (50 mm phosphate buffer, pH 7.5, containing 150 mm NaCl and mm dithiothreitol) at a protein concentration of 0.08–0.12 mgỈmL)1 at 25 °C The fluorescence spectra were FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS 5639 Point mutations of the b5–b7 loop of human HSP22 A S Kasakov et al decomposed into components corresponding to discrete states of Trp that differ in their environment and accessibility for the solvent [38] Bis-ANS was used for probing hydrophobic sites of HSP22 and its mutants Proteins (0.03–0.04 mgỈmL)1) in buffer F were titrated with the stock solution of bis-ANS (200 lm in buffer F) so that the final concentration of the fluorescence probe in the sample varied in the range 0–8 lm All experiments were performed at room temperature (25 °C) Fluorescence of bis-ANS bound to HSP22 and its mutants was excited at 385 nm (slit width ¼ nm) and recorded in the range 400–600 nm (slit width ¼ nm) To estimate FRET, intrinsic Trp fluorescence of HSP22 was excited at 295 nm (slit width ¼ nm) and fluorescence was recorded in the range 300–600 nm (slit width ¼ nm) The decrease in fluorescence at 342 nm (intrinsic Trp fluorescence) and the concomitant increase in fluorescence at 495 nm (fluorescence of bis-ANS bound to HSP22) indicated fluorescence resonance transfer from excited Trp to bis-ANS CD spectroscopy Far-UV CD measurements were performed on Mark V autodichrograph in the cells with an optical path of 0.05 cm in the range 200–250 nm HSP22 and its mutants were dissolved in buffer F and dialyzed against the same buffer overnight After dialysis, the samples were subjected to centrifugation (12 000 g, 20 min) and the supernatant was used for the optical measurements All proteins were used at identical concentrations of 0.6–0.8 mgỈmL)1 The data presented 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 mgỈmL)1) dissolved in buffer containing 20 mm Tris-acetate pH 7.4, 10 mm NaCl and 30 mm 2-mercaptoethanol were mixed with N-tosyl-l-phenylalanine chloromethyl ketone-treated trypsin (Sigma, St Louis, MO, USA) at a weight ratio for Hsp20 ⁄ trypsin equal to 12 000 : and incubated at 37 °C for different times The reaction was stopped by the addition of phenylmethanesulfonyl fluoride up to a final concentration 0.2 mm and the protein 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 activity of HSP22 and its mutants was determined by their ability to retard or prevent aggregation of model protein substrates In the first case, insulin (Sigma) was dissolved in 2.5% acetic acid and, after overnight incubation, was subjected to centrifugation (12 000 g, 20 min) The pellet was discarded and the insulin concentration in supernatant (usually 5–7 mgỈmL)1) was determined 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 mgỈmL)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 from 0–0.20 mgỈmL)1 After incubation for 10 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 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IUBMB Life 55, 661–668 FEBS Journal 274 (2007) 5628–5642 ª 2007 The Authors Journal compilation ª 2007 FEBS ... Ao and At are the intensities of the band of intact protein at the beginning of trypsinolysis and at the fixed time of trypsinolysis) against the time of incubation (Fig 8D) The apparent rate constants... to provide new information on the structure of sHsp and to shed new light on their role in the development of human congenital diseases Point mutations of the b5–b7 loop of human HSP22 Results... analyzed the effect of point mutations in the linker connecting the b5 and b7 strands of HSP22 on its thermal stability The wild-type protein or its mutants were heated for 30 at 70 °C and, after