Báo cáo khoa học: Molecular metamorphosis in polcalcin allergens by EF-hand rearrangements and domain swapping docx

Molecular metamorphosis in polcalcin allergens by EF-hand rearrangements and domain swapping Iris Magler 1 , Dorota Nu ¨ ss 1 , Michael Hauser 2 , Fatima Ferreira 2 and Hans Brandstetter 1 1 Division of Structural Biology, Department of Molecular Biology, University of Salzburg, Austria 2 Division of Allergy, Department of Molecular Biology, University of Salzburg, Austria Introduction Allergy is a health problem that is growing at an almost epidemic rate, with approximately 20% of the population being affected by type I allergy worldwide [1–6]. Allergies appear in many versions, including pol- len and food allergies, and mite dust and environmen- tally caused allergies. Pollen allergens represent the largest subgroup, and can be classified into 29 protein families; most of them belong to the expansin, profilin or calcium-binding protein families [7]. Massive efforts have been directed at elucidating the characteristics and causative mechanisms underlying the action of allergens. Among the biophysical proper- ties shared by allergens with the ability to breach phys- ical defense mechanisms in a susceptible host are: (a) small size, typically ranging from 5 to 30 kDa; (b) high effective concentration, implying high solubility and stability; and (c) foreignness to the affected host [8]. Additionally, allergens elicit an IgE response and a Keywords covalently locked conformation; EF-hand protein; protein engineering; structure; temperature-dependent oligomerization Correspondence H. Brandstetter, Billrothstr. 11, 5020 Salzburg, Austria Fax: +43 662 8044 7209 Tel: +43 662 8044 7270 E-mail: hans.brandstetter@sbg.ac.at (Received 5 February 2010, revised 17 March 2010, accepted 7 April 2010) doi:10.1111/j.1742-4658.2010.07671.x Polcalcins such as Bet v 4 and Phl p 7 are pollen allergens that are con- structed from EF-hand motifs, which are very common and well character- ized helix–loop–helix motifs with calcium-binding functions, as elementary building blocks. Being members of an exceptionally well-characterized protein superfamily, these allergens highlight the fundamental challenge in explaining what features distinguish allergens from nonallergenic proteins. We found that Bet v 4 and Phl p 7 undergo oligomerization transitions with characteristics that are markedly different from those typically found in proteins: transitions from monomers to dimers and to distinct higher oligomers can be induced by increasing temperature; similarly, low concen- trations of destabilizing agents, e.g. SDS, induce oligomerization transitions of Bet v 4. The changes in the quaternary structure, termed molecular metamorphosis, are induced and controlled by a combination of EF-hand rearrangements and domain swapping rather than by the classical law of mass action. Using an EF-hand-pairing model, we provide a two-step model that consistently explains and substantiates the observed metamor- phosis. Moreover, the unusual oligomerization behavior suggests a straight- forward explanation of how allergens can accomplish the crosslinking of IgE on mast cells, a hallmark of allergens. Structured digital abstract l MINT-7718612: Bet v 4 (uniprotkb:Q39419) and Bet v 4 (uniprotkb:Q39419) bind (MI:0407) by molecular sieving ( MI:0071) l MINT-7718648: Phl p 7 (uniprotkb:O82040) and Phl p 7 (uniprotkb:O82040) bind (MI:0407) by molecular sieving ( MI:0071) Abbreviations GFP, green fluorescent protein; TEV, tobacco etch virus. 2598 FEBS Journal 277 (2010) 2598–2610 ª 2010 The Authors Journal compilation ª 2010 FEBS clinical response, which may represent an immediate and ⁄ or late-phase response [9]. Many allergens show proteolytic activity, for which, in selected cases, a cau- sal connection has been demonstrated [10–12]; addi- tionally, surface-exposed hydrophobic patches have been suggested to provide allergen-typical danger sig- nals that are recognizable to the innate immune system [13,14]; similarly, glycosylation patterns present on allergen surfaces are believed to be involved in recogni- tion and endocytotic internalization by innate immune cells [15]. For a recent review, see [16]. The increased biological knowledge is accompanied by an enormous increase in the available structural database on aller- gens, accomplished by crystallography and NMR projects [17–32]. Despite this progress, our mechanistic understanding of the molecular principles of allergen- icity remains unsatisfactory. This is highlighted by the fact that we are unable to predict the allergenic behav- ior of a protein on the basis of biophysical properties, such as its crystal structure [33,34]. We lack reliable structural motifs that could serve as hallmarks of aller- genicity – such as a catalytic triad and an oxyanion hole that could identify proteolytic activity. The investigations in the current study were aimed at the identification of a biophysical hallmark that could distinguish allergens from other proteins and could ultimately reveal a causative mechanism acting in a subfamily of allergens. To this end, we investi- gated the hypothesis that the ability to undergo con- formational changes represents a distinguishing feature of allergens. The concept of molecular metamorphosis is receiving increasing attention [35,36]. We have iden- tified and characterized this unexpected molecular metamorphosis in the pollen allergens Bet v 4 from the white birch and Phl p 7 from timothy grass. These allergens are built from EF-hand motifs, which are exceptionally well-studied building blocks [37,38]. We have identified physicochemical parameters that con- trol the oligomerization transitions, and provide a model relating the oligomerization to the ability of the allergens to crosslink already synthesized IgE antibod- ies on mast cells. Results Bet v 4 can be expressed in a soluble, SDS-stable dimeric form Bet v 4 and the related Phl p 7 were expressed in Escherichia coli BL21(DE3) cells. Typically, SDS⁄ PAGE analysis of intact cells indicated the expression of monomeric proteins with approximate sizes of 12.5 kDa and 11.7 kDa, as shown for Bet v 4 in Fig. 1A and Phl p 7 in Fig. 1C, respectively. Purifica- tion to almost homogeneity was achieved in a single step by employing immobilized metal affinity chroma- tography (Fig. 1B,C). Under standard storage conditions, both Bet v 4 and Phl p 7 were also monomeric under native condi- tions, as judged by gel filtration chromatography (Fig 3A). Surprisingly, we observed spontaneous dimerization of Bet v 4 with a size of 25 kDa by SDS ⁄ PAGE (Fig. 2A). Although we repeated the expression of dimeric Bet v 4 more than 10 times, the underlying mechanism of dimerization is partly statistical in nat- ure, because we observed dimerization in $ 1–2% of the expression trials only. However, when dimerization 72 55 43 72 55 43 72 55 43 12345 678 1 2 3 4 5 6 1 2 3 4 5 6 7 8910 34 26 34 26 34 26 17 17 10 17 10 10 ABC Fig. 1. Bet v 4 and Phl p 7 protein samples appear exclusively as monomers on SDS ⁄ PAGE when expressed and purified under standard conditions. (A) Expression of Bet v 4 under standard conditions. Lane 1: protein standard (Fermentas). Lane 2: sample before induction. Lanes 3–8: Samples 4 h after induction. All samples were drawn from different expression flasks. (B) Bet v 4 purification by affinity chroma- tography. Lane 1: protein standard. Lane 2: Bet v 4 cell lysate. Lane 3: flow-through. Lane 4: wash step. Lanes 5 and 6: eluted protein with- out impurities. (C) Expression and purification of Phl p 7. Lane 1: protein standard. Lane 2: sample before induction. Lanes 3–6: samples from different expression flasks 4 h after induction. Lane 7: unbound protein impurities (flow-through) after Ni 2+ –nitrilotriacetic acid treat- ment. Lane 8: wash fraction. Lanes 9 and 10: purified Phl p 7 protein. I. Magler et al. Molecular metamorphosis in allergens FEBS Journal 277 (2010) 2598–2610 ª 2010 The Authors Journal compilation ª 2010 FEBS 2599 was observed at all, it was apparently 100% complete. The statistical nature of the dimerization is puzzling, and cannot be explained by obvious factors, e.g. the presence or absence of metal factors such as Ca 2+ or EDTA, as described in more detail below. To exclude the possibility of artefacts and to confirm the identity of the protein, we cleaved the N-terminal His 6 -tag by utilizing the tobacco etch virus (TEV) protease cleavage site. Figure 2B shows that, upon removal of the N-terminal His 6 -tag plus linker ($ 3 kDa), the migration of the protein on SDS ⁄ PAGE corresponds to a molecular mass reduced by approxi- mately 6 kDa, as expected. As a consequence, we can conclude that the dimer contact is not mediated by, but is independent from, the N-terminus. The identity of the Bet v 4 protein was unambiguously confirmed by ESI-MS. The dimerization is reversible, because Bet v 4 monomers were observed by SDS ⁄ PAGE after several weeks of storage at 4 °C. This finding, in particular, shows that dimerization can take place at $ 37 °C. Spontaneous in vitro dimerization of Bet v 4 When Bet v 4 was expressed as a monomer, it remained in the monomeric state when stored at 4 °C or 20 °C(Fig. 3A). By serendipity, we identified spon- taneous dimerization of a Bet v 4 sample that was left on the bench in the summer for weeks, as analyzed by gel filtration chromatography. These findings prompted us to systematically investigate possible mechanisms that govern the unexpected and intriguing oligomerization behavior of Bet v 4. Given the storage at elevated temperatures over a very long time period, we hypothesized that temperature and incubation time may affect the oligomerization behavior. Distinct oligomerization transitions in Bet v 4 and Phl p 7 can be induced by temperature changes We systematically studied the temperature dependence of the oligomerization state of Bet v 4 under native conditions by using gel filtration chromatography. Oligo- merization was observed at $ 30 ° C, but only over time intervals of several months. These long incubation times effectively excluded the option to conduct sys- tematic experiments at 30 °C. However, when heated to 75 °C, a mixture of monomeric and dimeric proteins appeared quite rapidly (Fig. 3B). When the tempera- ture was further increased to 95 °C, the dimeric form of the protein was observed exclusively (Fig. 3C). Con- sequently, temperature is one key parameter that induces Bet v 4 oligomerization transitions in vitro. Naturally, the question arises of whether the structure of Bet v 4 remains intact at high temperatures; we con- firmed the structural integrity by performing overnight CD measurements at 75 °C, as detailed below. Oligomerization depends on incubation time The oligomerization transitions did not occur instanta- neously, but required some incubation time. To quan- tify the required time scale, we analyzed protein oligomerization after distinct incubation times. We found approximately 75% of the protein to be monomeric after 24 h at 75 °C and the rest of the protein to be dimeric, whereas the situation was 12345678 1234567 43 55 72 72 55 43 34 26 34 17 26 17 11 11 AB Fig. 2. Spontaneous and complete dimerization of Bet v 4 can be observed during protein expression. (A) Expression of the SDS-stable dimer of Bet v 4 in E. coli BL21(DE3) cells. Lane 1: mass standard. Lane 2: cells before induction. Lanes 3–8: samples 4 h after induction. Note that the samples in the different lanes were drawn from different expression flasks and showed complete dimerization in each case. (B) Cleavage of the N-terminal His 6 -tag. Following the mass standard (lane 1), the His 6 -tagged Bet v 4 is shown before addition of the TEV protease (lane 2). The protein migrates at an apparent size of 25 kDa ($ 2 · 12.5 kDa). Lanes 3–7: TEV-digested Bet v 4 at different time points; TEV protease is visible at $ 30 kDa. Lane 3: TEV digest at time zero. Lane 4: digest after 1 h. Lane 5: digest after 6 h. Lane 6: digest after 12 h. Lane 7: digest after 24 h. TEV protease cleavage releases the N-terminal His 6 -tag and thus shifts the size of the protein to about 19 kDa ($ 2 · 9.5 kDa). Molecular metamorphosis in allergens I. Magler et al. 2600 FEBS Journal 277 (2010) 2598–2610 ª 2010 The Authors Journal compilation ª 2010 FEBS reversed after 48 h (Fig. 3D–F). After 72 h, all of the monomeric protein was converted to higher oligomers. The process of oligomerization does not stop with dimer formation. Instead, higher oligomeric forms were observed by size exclusion chromatography after 72 h of incubation (Fig. 3F). As mentioned earlier, we found dimerization of Bet v 4 that was stored at room temperature for approximately 4–10 weeks, whereas Bet v 4 stored at 4 °C was always found to be monomeric. Significantly, the observed oligomerization does not correspond to an unspecific aggregation phenomenon, but reflects a reversible transition between distinct oligomerization states; in particular, monomer formation can be induced by lowering the temperature to 4 °C within few days. Therefore, we conclude that Bet v 4 oligo- merization depends on both incubation temperature and time in a multiplicative manner. SDS induces instantaneous monomer-to-dimer transitions in Bet v 4 at room temperature In contrast to monomer-to-dimer transitions in vitro, dimerization in E. coli cells can take place rapidly, within a few hours (Fig. 2A). We hypothesized that dimerization can be efficiently catalyzed by compounds that are presumably present in E. coli in trace amounts. Therefore, we systematically screened a vari- ety of chemicals, including Ca 2+ and other metals, for their effect on oligomerization, both in expression con- ditions and with purified protein. Ca 2+ is known to affect the 3D structure of the Bet v 4 monomer [32]. Interestingly, addition of neither 10 mm Ca 2+ nor EDTA had a direct effect on the dimerization behavior, as judged by SDS⁄ PAGE and gel filtration chromatography, which gave results iden- tical to those shown in Fig. 3. These findings were further corroborated by CD measurements, as described below. Surprisingly, we found that SDS led to partial dimer formation in Bet v 4 at 20 and 4 °C. The addition of 0.05% SDS led to equal amounts of the monomeric and dimeric states, as reflected by two prominent peaks at approximately 13 and 11.4 mL (Fig. 4A). To a lesser extent ($ 10%), a highly oligo- meric species was observed at an elution volume of $ 8 mL, represented by a broad peak. At an SDS con- centration of 0.5%, nearly all of the protein aggregates and only approximately 10% of the protein remained in the monomeric or dimeric state, as shown by the dashed line in Fig. 4A. The bimodal oligomerization behavior of Bet v 4 contrasts with what is seen for most soluble proteins, as confirmed by a control experiment with green fluo- rescent protein (GFP). Whereas native GFP migrates 10.0 20.0 30.0 40.0 50.0 60.0 mAU 0.0 5.0 10.0 15.0 20.0 ml 12.15 13.56 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 mAU 0.0 5.0 10.0 15.0 20.0 25.0 30.0 ml 11.95 13.56 10.0 20.0 30.0 40.0 mAU 0.0 5.0 10.0 15.0 20.0 ml 9.19 11.63 24 h 48 h 72 h 0.0 20.0 40.0 60.0 80.0 mAU 0.0 5.0 10.0 15.0 20.0 25.0 ml 13.75 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 mAU 0.0 5.0 10.0 15.0 ml 11.97 13.58 5.0 10.0 15.0 20.0 25.0 30.0 35.0 mAU 0.0 5.0 10.0 15.0 20.0 ml 11.78 4 °C A 75 °C 95 °C Monomer Dimer Monomer Dimer Dimer Monomer Dimer Monomer Tetramer Dimer BC DEF Fig. 3. Temperature and time affect the dimerization of Bet v 4. (A–C) The temperature dependence of Bet v 4 oligomerization was analyzed by gel filtration chromatography. Bet v 4 samples were incubated for 48 h at (A) 20 °C, (B) 75 °C, and (C) 95 °C. The experiments showed a monomer-to-dimer transition as a function of incubation temperature. Further details are given in Experimental procedures. (D–F). The time dependence of Bet v 4 oligomerization as analyzed by gel filtration chromatography. Bet v 4 was incubated at 75 °C and analyzed every 24 h for 3 days. I. Magler et al. Molecular metamorphosis in allergens FEBS Journal 277 (2010) 2598–2610 ª 2010 The Authors Journal compilation ª 2010 FEBS 2601 exclusively as a monomer on gel filtration (Fig. 4B, continuous line), addition of 0.05% SDS induced aggregation of $ 10% of the protein (Fig. 4B, dashed line). In summary, 0.05% SDS generates an equilib- rium between native and aggregated GFP, resembling the situation for Bet v 4, with the exception that Bet v 4 can be monomeric or dimeric in its native state. Bet v 4 can be conformationally locked in the monomeric state The oligomerization properties of Bet v 4 revealed unexpected and unique features, such as its dependence on temperature and chemicals. Moreover, the dimer- ization is apparently independent of protein concentra- tion in the range from 0.1 to 25 mgÆmL )1 . These unique properties suggested that, in Bet v 4, oligomeri- zation could involve not only intermolecular recogni- tion events, governed by the law of mass action, but also intramolecular conformational rearrangements. To investigate this hypothesis, we constructed a Bet v 4 variant containing a K25C ⁄ F60C double muta- tion. On the basis of the NMR structure of monomeric Bet v 4 [32], we devised these point mutations to form an intramolecular disulfide bond that stabilizes the conformation by covalently linking both EF-hand motifs in Bet v 4 (Fig. 5). This covalent linkage is absent in the presence of dithiothreitol. If an intramolecular rearrangement does indeed accompany the oligomerization of Bet v 4, the oligomerization behavior of oxidized (disulfide-linked) Bet v 4-K25C ⁄ F60C should deviate markedly from that of the wild type. By contrast, reduced Bet v 4-K25C ⁄ F60C should show oligomerization behavior identical to that of the wild type. We carried out experiments to test both the tem- perature and time dependence of the oligomerization by incubating the disulfide-linked Bet v 4 double mutant at 20 °C for 7 days and at 75 °C for 24 h. Under both conditions, the monomer was stable over the observation period, as monitored by gel filtration (Fig. 6A). As a control experiment, we carried out similar experiments under reducing conditions using 5 mm dithiothreitol. The reduced Bet v 4 double mutant was incubated at 75 °C for 24 h, and subsequently ana- lyzed by gel filtration. The reduced protein revealed the native-like induction of higher oligomer formation (Fig. 6B, continuous line), clearly contrasting with the behavior of the oxidized double mutant (Fig. 6B, broken line). 0.0 20 40 60 80 mAU 0.0 2.0 4.0 6.0 8.0 10.0 12.0 ml 8.01 11.41 12.77 7.50 11.40 12.74 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 mAU 5.0 6.0 7.0 8.0 9.0 10.0 11.0 ml 8.71 10.67 10.70 AB Fig. 4. SDS induces dimerization in Bet v 4. (A) Solid line: at 0.05% SDS and 20 °C, most of the Bet v 4 elutes at retention volumes corre- sponding to monomers and dimers, with only a small ($ 10%) aggregated fraction eluting near the void volume. Dashed line: at 0.5% SDS, most (90%) of the Bet v 4 aggregates (eluting at the void volume: 7.5 mL), and only 10% elutes at volumes corresponding to monomers and dimers. (B) Solid line: Control experiment using GFP at 0.05% SDS and 20 °C reveals a predominantly native monomeric form, corre- sponding to a retention volume of 10.67 mL, and a small ($ 10%) aggregated fraction eluting near the void volume (retention volume of 8.71 mL). Dashed line: at 0% SDS, GFP migrates exclusively as a monomer. C25 C60 NH 2 COOH Fig. 5. Engineering of a disulfide bridge intended to lock the mono- mer conformation of Bet v 4. The introduced K25C ⁄ F60C double mutation promotes disulfide bond formation between the two anti- parallel b-strands, and thus crosslinks the first Ca 2+ -binding EF-hand (shown in red) with the second EF-hand (blue). Molecular metamorphosis in allergens I. Magler et al. 2602 FEBS Journal 277 (2010) 2598–2610 ª 2010 The Authors Journal compilation ª 2010 FEBS Consequently, the formation of Bet v 4 dimers and higher molecular mass oligomers does indeed involve an intramolecular conformational rearrangement. Temperature-induced oligomerization may be universally conserved in polcalcins Next, we tested whether the intriguing temperature- dependent and time-dependent oligomerization is spe- cific to Bet v 4 or could also be found in structurally related proteins. We selected Phl p 7 as a further repre- sentative of Ca 2+ -binding EF-hand proteins, and car- ried out oligomerization analyses analogous to those described for Bet v 4. We found that Phl p 7 is indeed able to undergo temperature-dependent oligomeriza- tion: At 4 °C, Phl p 7 formed monomers exclusively, whereas significant amounts of dimeric Phl p 7 accumu- lated after overnight incubation at 75 °C. Overnight incubation at 95 °C completely converted the mono- meric form of Phl p 7 to higher oligomer forms (Fig. 7). The temperature dependence of the oligomeri- zation state of Phl p 7 thus parallels the behavior observed with Bet v 4. The broadness of the 95° C peak may be partly related to heat-induced denaturation. The secondary structure content is independent of the oligomerization state and is conserved at 75 °C We employed CD spectroscopy to investigate whether the secondary structure of Bet v 4 at room temperature was dependent on its oligomeric state. Furthermore, as we used heating of Bet v 4 as a tool to speed up the conformational transition, we wished to clarify whether the structure becomes disrupted at 75 °C. Finally, we used an engineered disulfide-containing variant to test the nature of the conformer transforma- tion, which raises the question of how well this mutant resembles the wild type. CD is ideally suited for pro- viding answers to these questions. To investigate the first question, we used Bet v 4 stored at 4 °C, corresponding to the monomeric spe- cies, and Bet v 4 that had been heated to 75 °C over- night, corresponding to the dimeric species. The 0.0 20.0 40.0 60.0 80.0 mAU 6.0 8.0 10.0 12.0 14.0 16.0 18.0 ml 10.66 12.80 0.0 20.0 40.0 60.0 80.0 mAU 6.0 8.0 10.0 12.0 14.0 16.0 18.0 ml 10.66 12.80 AB Fig. 6. Disulfide bond inhibits the monomer-dimer transformation. (A) The gel filtration chromatogram of the disulfide-containing (oxidizing) Bet v 4 mutant; the protein was incubated at 4 °C (continuous line) and at 75 °C (dashed line) for 24 h. The chromatogram did not change over an incubation period of up to 7 days, indicating an exclusively monomeric state. (B) The gel filtration chromatogram of the reduced Bet v 4 mutant (no disulfide bond); the protein was incubated at 4 °C (continous line) and at 75 °C (dashed line) for 24 h. The heat-treated protein eluted at a retention volume corresponding to the dimer, resembling the wild-type protein in this respect. 20 40 60 80 100 120 mAU 0.0 5.0 10.0 15.0 20.0 25.0 ml 12.62 0 100 200 300 400 500 600 mAU 0.0 5.0 10.0 15.0 20.0 ml 10.12 12.45 0 100 200 300 400 mAU 0.0 5.0 10.0 15.0 ml 10.08 11.50 Monomer Dimer Monomer Dimer AB C Fig. 7. Oligomerization of Phl p 7. Gel filtration chromatograms indicate the conversion from monomer to higher oligomerization states at 4 °C (A), 75 °C (B) and 95 °C (C) over an incubation period of 24 h, qualitatively resembling the behavior of Bet v 4. I. Magler et al. Molecular metamorphosis in allergens FEBS Journal 277 (2010) 2598–2610 ª 2010 The Authors Journal compilation ª 2010 FEBS 2603 oligomerization states were further confirmed by gel filtration chromatography. Both protein samples yielded CD spectra that revealed well-folded a-helical proteins (Figs 8A and S1). Therefore, the secondary structure in the monomeric and dimeric species is qualitatively identical. Next, we tested the a-helical content of Bet v 4 at 75 °C at three time points: after 15 min, after 16 h, and after 20 h. The sample was kept at 75 °C. In all three samples, the a-helical content was preserved, and qualitatively coincided with that indicated by the spec- tra measured at 20 °C (Fig. 8B). The double minimum structure characteristic of a-helices was less pro- nounced in the heated samples, however. Similarly, a quantitatively reduced mean residual weight ellipticity indicated increased flexibility in the secondary struc- ture of the heated samples (Fig. S1). As a third experiment, we tested the disulfide-engi- neered Bet v 4 mutant (K25C ⁄ F60C). The disulfide mutant stored at 4 C resembled the native monomer and gave CD spectra qualitatively identical to those of the unmodified protein, independently of whether the disulfide bond was formed or reduced (Fig. 8C, CC oxi ⁄ red 4 °C). Interestingly, whereas the overall secondary structure content was also conserved after heating at 75 °C, there appeared to be significant disorder in these protein variants (Fig. 8C). The reduction in a-helix content is most prominent in ‘CC oxi 75 °C’, in which the monomeric state is enforced by the intact disulfide bond. These findings are paralleled by the quantitative representation of the ellipticity in Fig. S1. Finally, we confirmed that Ca 2+ is tightly bound by Bet v 4 and cannot be extracted by the addition of 10 mm EDTA, as demonstrated by the qualitatively unchanged CD spectrum in the presence of EDTA (Fig. S2). Discussion Bet v 4 forms monomers, dimers, and higher oligomers We identified several distinct oligomerization states for Bet v 4. Although these findings appear to be in con- flict with those from previous experiments, these dis- agreements may be reconciled by considering the settings of the particular experiments [32]. This is of particular relevance for experiments with measurement times of days, such as NMR and ultracentrifugation, which were run at a constant temperature of 20 °Cor 4 °C, respectively. In fact, also in our hands, the pro- tein’s oligomerization behavior over time was tempera- ture-dependent. Even when stored for several months at 4 °C, Bet v 4 remained in a monomeric conforma- tion. If expressed in monomeric form, Bet v 4 remained monomeric over days to weeks at 20 °C. However, after months, Bet v 4 adopted a dimeric con- formation at room temperature. Temperature is a universal inducer of oligomerization transitions The consistent observations made with Phl p 7 and Bet v 4 suggest to us that temperature acts as an important order parameter controlling oligomer forma- tion in polcalcins. The fact that an increase in temper- Fig. 8. CD measurements document the structural integrity of diverse Bet v 4 species. Data are presented as baseline-corrected mean resi- due molar ellipticity [Q] MRW at a given wavelength. (A) The spectra of monomeric Bet v 4 protein stored 4 °C (continuous line) and after heat-induced dimerization (dashed line) qualitatively coincide, indicating a near-identical secondary structure content. (B) Time series of CD spectra of Bet v 4 kept and analyzed at 75 °C for 15 min (continuous line), 16 h (dashed line with dots), and 20 h (dashed line). The second- ary structure is mostly conserved, and does not noticeably vary over time. (C) CD spectra of Bet v 4-K25C ⁄ F60C (CC) with the disulfide bond formed (oxi) or reduced (red), each stored at either 4 °Cor75°C (overnight). When stored at 4 °C, the (monomeric) CC mutant adopted a native-like ellipticity spectrum, independently of the status of the disulfide bond (oxidized or reduced), indicating a native like 3D structure. After heat treatment, the qualitative form of the spectrum remained conserved, albeit with a significantly reduced amplitude. Molecular metamorphosis in allergens I. Magler et al. 2604 FEBS Journal 277 (2010) 2598–2610 ª 2010 The Authors Journal compilation ª 2010 FEBS ature induces transitions to high molecular mass oligo- mers is surprising: with increasing temperature (T), the entropy (S) of the protein becomes more important for its Gibbs free energy (G) than the enthalpic contribu- tion (E ): G = E ) TS. Thus, dissociation of oligomers should be favored at high temperature, because the degrees of freedom are maximized for monomers. However, the observed behavior contrasts with these fundamental physicochemical principles, and points to the existence of temperature-induced intramolecular rearrangements in Bet v 4. In other words, although CD spectra indicate that the secondary structures of monomers and dimers are alike (Fig. 8), their detailed conformations differ in a subtle way (Fig. 10). Only the excited conformation is able to form dimers; the ground state conformation is monomeric. Importantly, although, for practical reasons, we performed the experiments shown in Figs 3, 6 and 7 at unphysiologi- cally high temperatures, these transitions do also occur at ambient temperatures. Additionally, the tempera- ture-induced transitions may well be catalyzed by other components present in the pollen, as discussed below. As an additional cautionary remark, we must point out that structural integrity could be demonstrated for temperatures up to 75 °C only (Fig. 8); the sample at 95 °C may be partially unfolded. Like temperature, chemical substances induce metamorphosis by stabilizing or destabilizing local free energy minima SDS is known to destabilize the quaternary and or ternary structure of proteins [39,40]. This effect is also observed in Bet v 4 and our control protein, GFP, leading to a broad peak near the void volume of the gel filtration column (Fig. 4A,B). Significantly, how- ever, we found that SDS induced specific dimerization of Bet v 4, as reflected by a sharp elution peak (Fig. 4A). It is very likely that a number of other physiological chemicals will affect the oligomerization of Bet v 4. In fact, sodium chloride at 0.5 m favors the monomeric state of Bet v 4 over the dimeric state. These findings support the notion that oligomer transformations are relevant in the physiological environment of the pollen. This observation can be explained by assuming a multimodal free energy surface of Bet v 4 with several distinct substates; in a simplified version, this surface can be represented by two isothermal free energy graphs (Fig. 9). The ratio of the free energy minima representing the monomeric and dimeric states changes with temperature. This property reflects the surprising fact that dimers and higher oligomers are preferred over monomers at high temperature. The differences in free energy can be quantitatively estimated by exploiting the fact that the statistics of oligomer formation are governed by Boltzmann’s law p ¼ 1 Z e ÀDG=RT where the probability p corresponds to the likelihood of a dimer and is estimated to be $ 1%, reflecting the frequency of observation of spontaneous dimerization (see Results). Z represents the partition function, which we roughly estimate, from the number of acces- sible states, to be Z = 3 (monomer, dimer, and tetra- mer). R is the gas constant [8.3 JÆ(mol K) )1 ], T is the absolute temperature (300 K), and DG is the change in free energy associated with the monomer-to-dimer transition. Reformulation of Boltzmann’s law yields DG ¼ÀRT ln ðZ Á 0:01Þ%RT ln ð3 Á 0:01Þ%9kJ We admit that our estimations of both P and Z are quite inaccurate; however, as both parameters (P, Z) affect DG via logarithmic dependence, errors will trans- late to the resulting DG value only in a dampened manner. In addition to this phenomenological consideration, we tried to develop a structural model that could pro- vide a mechanism that explains this counterintuitive behavior. Such a model is developed below. High temperature Low temperature 12 Monomer Dimer Oligomerization State ΔG Fig. 9. Schematic free energy diagram of Bet v 4 governing the occupancy of its conformational substates. The free energy depends most prominently on the temperature: at low temperature (i.e. 4 °C), the monomer is preferred, whereas higher oligomers are preferred at higher temperatures. Additionally, the free energy depends on parameters such as ionic strength or SDS, which together result in a more complex hypersurface, as illustrated. I. Magler et al. Molecular metamorphosis in allergens FEBS Journal 277 (2010) 2598–2610 ª 2010 The Authors Journal compilation ª 2010 FEBS 2605 Dimerization is a two-step process involving an excited conformation of Bet v 4 The anomalous dependence of the oligomerization on temperature and ⁄ or chemical substances had already indicated that changes in the tertiary structure of the Bet v 4 subunits precede the monomer-to-dimer transi- tion. Proof for this hypothesis was obtained by engi- neering a disulfide-containing variant that locks the known 3D structure of the monomeric substate. This protein is unable to undergo dimerization or higher oligomer formation under oxidizing conditions in which the disulfide bridge is conserved. This model also explains the different modes of action of tempera- ture and SDS. The latter slightly destabilizes the monomeric state and effectively lowers the separating energy barrier, leading to a population of both mono- mers and dimers (Fig. 4A). The effect of temperature is more sophisticated: although it also helps to over- come the separating energy barrier, an additional mechanism is required to explain why dimers are pre- ferred over monomers at high temperature. Our model involves a two-step process. First, elevated temperatures will induce a conformational transition within the Bet v 4 subunit from the ground state to an excited state, whereby the ground state represents a closed conformation and theexcited state an open con- formation with no EF-hand pairing. Both the ground state and excited state are monomeric (Fig. 5). Two hydrogen bonds in the short antiparallel b-sheet have to be broken in the excited state, representing an energetic barrier that matches that derived previously from Boltz- mann’s distribution ($ 9 kJ). In a second step, this excited form is now able to gain enthalpy by intermolec- ular pairing of the EF-hands, and thus forms dimers (Fig. 10A,B). Such an extended conformation has been observed in the crystal structure of Phl p 7 [41]. It should be noted that a straightforward extension of the EF-hand-pairing model, shown in Fig. 10C, explains the existence of higher order oligomers. Here, we assume that an initial dimer is formed by monomer pairing with one EF-hand rather than two. This gener- ates a ‘sticky overhang’ dimer that provides two addi- tional EF-hand docking sites. These docking sites can attract additional Bet v 4 subunits, and thus provide a mechanism to generate trimers, tetramers, and higher oligomers. This model further explains why Bet v 4 proteins migrating as dimers during gel filtration may differ on SDS ⁄ PAGE. We propose that single EF-hand-paired dimers are less stable and dissociate to form monomers on SDS ⁄ PAGE, whereas double EF-hand-paired dimers stay intact on SDS ⁄ PAGE. The proposed model (Fig. 10) has a qualitatively unchanged secondary structure content in the mono- meric and dimeric states, consistent with the recorded CD spectra (Fig. 8). Fig. 10. (A, B) EF-hand pairing as a mechanism for dimerization. Bet v 4 consists of two EF-hand motifs, EF1 (blue) and EF2 (red), which are connected by a flexible connecting segment (green). A C-terminal helix (a5; gray) presumably contributes to stabilization of the EF-hand pair- ing. In the experimentally determined monomer structure, intramolecular EF-hand pairing occurs via strands b1 and b2, forming a central antiparallel b-sheet. This structure represents the ground state conformation. On the basis of the crystal structure of dimeric Phl p 7 [41], we propose that dimerization is mediated by intermolecular EF-hand pairing via strands b1 and b2¢ and strands b2 and b1¢. For this dimeriza- tion to occur, we propose the existence of an excited state intermediate (open form) that will be increasingly common at high temperature. (C) Alternatively, a singly EF-hand-paired dimer may form, as shown here, via strands b1 and b1¢; possible alternative dimers would involve strands b2 and b2¢, b1 and b2¢,orb2 and b1¢. Singly EF-hand-paired dimers will be less stable than doubly paired dimers. Molecular metamorphosis in allergens I. Magler et al. 2606 FEBS Journal 277 (2010) 2598–2610 ª 2010 The Authors Journal compilation ª 2010 FEBS CD measurements further showed that the second- ary structure of Bet v 4 is mostly conserved during the overnight heat treatments (Fig. 8). This lends support to the notion that the heating protocol only accelerates the transformation from monomer to dimer, and does not change the reaction path of the transformation. In particular, the heat-induced dimerization does not occur via an unfolding–folding process. Finally, the CD measurements of the disulfide- containing variant indicate that the Bet v 4 dimer structure will deviate in subtle details from the pro- posed Phl p 7 dimer structure (Fig. 10). A remaining puzzle is why Bet v 4 is mostly expressed as a monomer in E. coli, but sometimes as a dimer; moreover, if Bet v 4 is expressed as dimer, it is exclusively dimeric. It seems quite plausible that the difference in the observed oligomerization relates to the presence of a dimerization catalyst. We propose that a chaperone, such as a heat shock protein, could account for the observed dimerization behavior. The expression rate of heat shock proteins varies drastically upon subtle and difficult-to-control changes. Molecular metamorphosis provides a framework to explain the ability of polcalcins to crosslink IgE antibodies on mast cells Naturally, the question arises of whether the intrigu- ing biophysical behavior of Bet v 4 and Phl p 7 relates to their allergenic properties. The molecular metamorphosis model provides a straightforward explanation for one allergenic key feature, namely, the ability to crosslink IgEs on mast cells. Addition- ally, it is very plausible that the oligomerization status of an allergen will affect its endocytosis and endoso- mal processing. Significantly, dimerization or multi- merization has been reported for numerous allergens [42–51]. Clearly, IgE binding is necessary, but not suf- ficient, to induce a Th2 immune response, which is characteristic of allergy. In fact, there is conclusive evidence for selected allergens that the allergenicity, including antibody-binding capacity, differs for mono- mers and dimers: Scho ¨ ll et al. have shown, for the birch pollen allergen Bet v 1, that dimers (34 kDa), and not monomers (17 kDa), represent the allergenic Bet v 1 species [51]. The same basic mechanism has been reported by Reese et al. for the carrot allergen Dau c 1 [45]. According to the molecular metamor- phosis hypothesis presented here, we suggest that con- formationally locked allergens (e.g. disulfide- stabilized) will cause drastically reduced allergic reac- tions. Clearly, the validation of this hypothesis awaits further experiments. Experimental procedures Materials Plasmids coding for Bet v 4 and Phl p 7 (Uniprot Database accession numbers are Q39419 for Bet v 4 and O82040 for Phl p 7) were isolated from pollen, as described previously [52,53]. Restriction enzymes and T4 ligase were obtained from Fermentas (St Leon-Rot, Germany). Pfu Ultra II Fusion HS DNA polymerase was obtained from Stratagene (La Jolla, CA, USA). Custom-made primers were obtained and sequence analyses were performed at Eurofins MWG Operon (Germany). E. coli strain XL1 Blue (Stratagene) was used for subcloning. Strain BL21(DE3) (Novagen, Madison, WI, USA) was used as host strain for protein expression. For expression, LB-Lennox (Roth, Karlsruhe, Germany) was used. All reagents were of the highest stan- dard available from Sigma-Aldrich (Mu ¨ nchen, Germany) or AppliChem (Darmstadt, Germany). Cloning The plasmids were cloned in the pHIS parallel II vector with an NcoI site at the 5¢-end and an EcoRI site at the 3¢-end [54]. To engineer the disulfide mutant of Bet v 4, a double mutation K25C ⁄ F60C was constructed by site- directed mutagenesis using the QickChange method [55]. The following primers were used: 5¢ -gccaatggcgatggt TGCat- ctcAgcagcagag-3¢ [K25C forward primer, bases exchanged are underlined, silent control restriction (PstI) site is in bold]; 5¢-ctctgctgcTgagat GCAaccatcgccattggc-3¢ (K25C reverse primer, bases exchanged are underlined, control restriction site is in bold); 5¢-accgatggcgacggA TGCatt- tcgttccaagag-3¢ [F60C forward primer, bases exchanged are underlined, control restriction site (NsiI) is in bold]; and 5¢-ctcttggaacgaaat GCATccgtcgccatcggt-3¢ (F60C reverse primer, bases exchanged are underlined, control restriction site is in bold). The PCR product was digested with the meth- ylation-sensitive enzyme DpnI for 1 h at 37 °C. Products were purified (Qiagen, Hilden, Germany) and transformed into XL1-blue cells by electroporation; cells were plated on LB agar containing ampicillin. Plasmid Mini Preparation (Qiagen) was performed and the obtained plasmids were digested with the appropriate control restriction enzymes (PstI and NsiI) for 2 h at 37 °C to screen for plasmids with the correct mutation. The correctness of the restriction- positive plasmids was finally confirmed by sequencing. Protein expression Plasmids were transformed into E. coli strain BL21(DE3) via electroporation, and grown overnight in 100 mL of LB medium containing 100 lgÆmL )1 ampicillin. Large-scale expression cultures (12 · 600 mL) were inoculated with I. Magler et al. Molecular metamorphosis in allergens FEBS Journal 277 (2010) 2598–2610 ª 2010 The Authors Journal compilation ª 2010 FEBS 2607 [...].. .Molecular metamorphosis in allergens I Magler et al 2 mL of preculture The cells were grown at 37 °C to a D600 nm of 0.8, or in the case of Phl p 7 to a D600 nm of 0.4, when protein expression was induced by adding 1 mm isopropyl thio-b-d-galactoside Cells were harvested 4 h after induction by centrifugation (5000 g for 10 min.), resuspended in buffer A (50 mm NaH2PO4, 10... g 1: molecular analysis of cross-reactivity J Mol Biol 351, 1101–1109 Molecular metamorphosis in allergens 24 Gustchina A, Li M, Wunschmann S, Chapman MD, Pomes A & Wlodawer A (2005) Crystal structure of cockroach allergen Bla g 2, an unusual zinc binding aspartic protease with a novel mode of self-inhibition J Mol Biol 348, 433–444 25 Ichikawa S, Takai T, Inoue T, Yuuki T, Okumura Y, Ogura K, Inagaki... 2598–2610 ª 2010 The Authors Journal compilation ª 2010 FEBS 2609 Molecular metamorphosis in allergens I Magler et al 35 Tokuriki N & Tawfik DS (2009) Protein dynamism and evolvability Science 324, 203–207 36 Tuinstra RL, Peterson FC, Kutlesa S, Elgin ES, Kron MA & Volkman BF (2008) Interconversion between two unrelated protein folds in the lymphotactin native state Proc Natl Acad Sci USA 105, 5057–5062 37 Herzberg... 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MA, USA) The resulting curves were baseline-corrected and presented as mean residue molar elipticity [Q]MRW at a given wavelength Protein concentrations (typically at 20 lgÆmL)1) were detected using UV light at 280 nm In order to have well-defined oligomerization states of the individual protein samples, we employed gel filtration as a preparative step, and selected fractions according to their retention... 633–642 Chruszcz M, Chapman MD, Vailes LD, Stura EA, Saint-Remy JM, Minor W & Pomes A (2009) Crystal structures of mite allergens Der f 1 and Der p 1 reveal differences in surface-exposed residues that may in uence antibody binding J Mol Biol 386, 520–530 Jin T, Guo F, Chen YW, Howard A & Zhang YZ (2009) Crystal structure of Ara h 3, a major allergen in peanut Mol Immunol 46, 1796–1804 Schweimer K, Petersen... Wangorsch A, Randow S & Vieths S (2007) Allergenicity and antigenicity of wildtype and mutant, monomeric, and dimeric carrot major allergen Dau c 1: destruction of conformation, not oligomerization, is the roadmap to save allergen vaccines J Allergy Clin Immunol 119, 944–951 46 Gieras A, Focke-Tejkl M, Ball T, Verdino P, Hartl A, Thalhamer J & Valenta R (2007) Molecular determinants of allergen-induced effector . induced and controlled by a combination of EF-hand rearrangements and domain swapping rather than by the classical law of mass action. Using an EF-hand- pairing. Molecular metamorphosis in polcalcin allergens by EF-hand rearrangements and domain swapping Iris Magler 1 , Dorota Nu ¨ ss 1 ,