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)

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

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

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

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

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

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

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

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

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)

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

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

(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-

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

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

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

600 nm
of 0.8, or in the case of Phl p 7 to a D
600 nm
of 0.4,
when protein expression was induced by adding 1 mm iso-
propyl thio-b-d-galactoside. Cells were harvested 4 h after
induction by centrifugation (5000 g for 10 min.), resus-
pended in buffer A (50 mm NaH
2
PO
4
,10mm Tris, 150 mm
NaCl, 10 mm imidazole, pH 8.0), and sonicated (Sonicator,
Bandelin Sonopuls).
Purification
Immobilized metal affinity chromatography
Recombinant protein lysate carrying an N-terminal His
6
-
tag was purified using Ni
2+
–nitrilotriacetic acid resin
(Qiagen) [56]. Bound protein was washed twice (50 mm
NaH
2
PO
4
, pH 8.0, 300 mm NaCl, 20 mm imidazole). The
target protein was eluted with a highly concentrated imid-
azole buffer (50 mm NaH

was pretreated with dithiothreitol, it was separated by using
a desalting column (illustra NAP-columns, GE Healthcare)
prior to the gel filtration run. Typical buffer conditions for
Bet v 4 samples were either 25 mm Tris (pH 7.5) and
50 mm NaCl, or 25 mm Hepes (pH 7.5) and 50 m m NaCl,
or 20 mm Mops (pH 7.5) and 10 mm CaSO
4
; for Phl p 7,
the running buffer contained 40 mm NH
4
Cl and 20 mm Tris
(pH 4.7). The observed retention times (corresponding to
distinct oligomeric states) were identical with sample
concentrations ranging from 1 to $ 80 mgÆmL
)1
.
Circular Dichroism
CD spectra of Bet v 4 were recorded in 4 mm Mops
(pH 7.5) and 2 mm CaSO
4
with a Jasco J-815 spectropola-
rimeter (Jasco, Tokyo, Japan), equipped with a Neslab
RTE-111M temperature control system (Thermo Fischer
Scientific, Waltham, MA, USA). The resulting curves were
baseline-corrected and presented as mean residue molar
elipticity [Q]
MRW
at a given wavelength. Protein concentra-
tions (typically at 20 lgÆmL
)1

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