From the Lehrstuhl für Biopolymere,
Universität Bayreuth, D-95440 Bayreuth, Germany and the
¶ Abteilung Allergologie, Paul-Ehrlich-Institut,
Paul-Ehrlich-Stra
e 51-59, D-63225 Langen, Germany
Received for publication, February 22, 2001, and in revised form, April 2, 2001
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ABSTRACT |
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Birch pollinosis is often accompanied by
hypersensitivity to fruit as a consequence of the cross-reaction of
pollen allergen-specific IgE antibodies with homologous food
proteins. To provide a basis for examining the cross-reactivity on a
structural level, we used heteronuclear multidimensional NMR
spectroscopy to determine the high-resolution three-dimensional
structure of the major cherry allergen, Pru av 1, in solution. Based
on a detailed comparison of the virtually identical structures of
Pru av 1 and Bet v 1, the major birch pollen allergen, we propose
an explanation for a significant aspect of the observed
cross-reactivity pattern among the family of allergens under
consideration. The large hydrophobic cavity expected to be important
for the still unknown physiological function of Bet v 1 is conserved
in Pru av 1. Structural homology to a domain of human MLN64
associated with cholesterol transport suggests phytosteroids as
putative ligands for Pru av 1. NMR spectroscopy provides experimental
evidence that Pru av 1 interacts with phytosteroids, and molecular
modeling shows that the hydrophobic cavity is large enough to
accommodate two such molecules.
Birch pollinosis is one of the prevailing allergic diseases in
regions with birch trees, such as Northern and Central Europe and
Northern America. Up to 70% of birch pollen allergic patients who
suffer from clinical syndromes like hay fever and asthma also show
hypersensitivity to fresh fruit or vegetables (1). The allergic
reactions after ingestion of foodstuff are predominantly oropharyngeal,
for example itching and swelling of the lips, tongue, and throat, but
in rare cases even severe anaphylactic reactions are possible. The
symptoms of these type I allergies are caused by an immune response
that is triggered when two receptor-bound IgE antibodies on the surface
of a mast cell or basophil are cross-linked by simultaneous binding of
an otherwise harmless antigen, the so-called allergen (2).
Pollen-associated food allergies are a consequence of the
cross-reaction of pollen allergen-specific IgE antibodies with highly
homologous proteins contained in foodstuff. The 17.4-kDa major birch
(Betula verrucosa) pollen allergen, Bet v 1, is
responsible for IgE binding in more than 95% of birch pollen allergic
patients (3). A series of allergens with high sequence identity to
Bet v 1 have been reported in the literature, pollen allergens from
other trees belonging to the Fagales order as well as food
allergens like, for example, Api g 1 from celery (Apium
graveolens (4)), Mal d 1 from apple (Malus domestica (5)), Pru av 1 (formerly Pru a 1) from cherry (Prunus
avium (6)), Pyr c 1 from pear (Pyrus communis (7)),
and Cor a 1.0401 from hazelnut (Corylus avellana (8))
(Fig. 1). In contrast to the
three-dimensional structure of Bet v 1, which has been studied
extensively in recent years (9-12), as yet no high-resolution structure of any of the corresponding food allergens is available. Because this is a prerequisite for a detailed understanding of the
observed immune cross-reactivity on a structural level, we determined
the three-dimensional structure of the major cherry allergen Pru av 1
in solution. Like Bet v 1, Pru av 1 is produced as a 160-residue
precursor protein that is processed by cleavage of the
NH2-terminal methionine (13), yielding a protein with a
calculated molecular mass of 17.5-kDa and a calculated isoelectric point of 5.9. The physiological function of these allergens is still
unknown. They show high sequence similarity to pathogenesis-related and
stress-induced proteins (14, 15) but seem to be expressed constitutively, even though the expression of several genes related to
Bet v 1 has been reported to be induced upon contact with
microorganisms (16). A potential ribonuclease activity of Bet v 1 was
also discussed (17). Three highly conserved regions on the surface of
the Bet v 1 molecule were proposed as candidates for IgE antibody binding epitopes (10); one of them, the glycine-rich P-loop around
Glu45, was recently confirmed by the crystal structure of
Bet v 1 in complex with an Fab fragment of a monoclonal murine IgG
antibody with high capacity to inhibit binding of serum IgE from
allergic patients to Bet v 1 (18). Additional information on
potential epitopes is provided by biochemical data like the study of
low IgE-binding isoforms or mutants for both Bet v 1 (12, 19) and
Pru av 1 (20). A thorough knowledge of the IgE binding epitopes is
the key to the development of hypoallergenic allergen variants that can
be used as vaccines for a patient-tailored specific immunotherapy with
reduced anaphylactic side effects (21).
NMR Sample Preparation--
We employed two different strategies
to purify recombinant Pru av 1 from Escherichia coli
lysates. The samples used for the structure determination were prepared
as described previously (22, 23). For the samples used to measure
{1H}15N nuclear Overhauser effect
(NOE)1 values and to
investigate the interaction with homocastasterone, a completely native
purification protocol of non-fusion Pru av 1 based on
chromatofocusing (24) was carried out. Final purification was achieved
by anion exchange chromatography. The NMR spectra of the uniformly
15N-labeled sample used for these measurements
were virtually identical to those of the
samples used for the structure determination except for minor changes
for the NH2-terminal residues up to Phe3 and
the loop from Thr122 to Lys129, which is
located next to the NH2 terminus in the three-dimensional structure. Part of the sample retained the NH2-terminal
methionine in the course of the native purification protocol as
verified by NH2-terminal sequencing and observed in the NMR
spectra. Homocastasterone was purchased from CIDtech Research Inc.,
Cambridge, Ontario, Canada,
Me2SO-d6 from euriso-top,
Gif-sur-Yvette, France. The NMR samples contained 0.8-1.2
mM uniformly 15N- or
13C/15N-labeled Pru av 1 and 10 mM potassium phosphate (pH 7.0) in
H2O/D2O (9:1) or
H2O/Me2SO-d6 (9:1).
NMR Spectroscopy--
All NMR spectra were recorded on a Bruker
DRX 600 NMR spectrometer with pulsed field gradient capabilities at a
temperature of either 25 or 35 °C. In addition to the experiments
described previously (23) the following experiments were conducted to collect NOESY data: three-dimensional 15N NOESYHSQC (120-ms
mixing time (25)), 13C NOESYHSQC (120-ms mixing time (26)),
15N HMQCNOESYHSQC (150-ms mixing time (27, 28)),
13C,15N HMQCNOESYHSQC (120-ms mixing time
(29)), and 15N-filtered two-dimensional
[1H,1H] NOESY (120-ms mixing time
(23)). In the amide-detected experiments a binomial 3-9-19 WATERGATE
sequence (30) with water flip-back and in the
13C-edited NOESY experiments gradient coherence selection
(31) was employed for water suppression. Quadrature detection in the indirect dimensions was achieved by the States-TPPI
(time-proportional phase incrementation) method (32). Slowly exchanging
amide protons were identified from a [1H,15N]
HSQC recorded after the sample had been dialyzed against 10 mM potassium phosphate (pH 7.0) in D2O for
4 h. {1H}15N NOE values were measured
using the pulse sequence of Dayie and Wagner (33) with a relaxation
delay of 4 s. For proton saturation a train of 120° high-power
pulses was applied for the final 3 s of the relaxation delay. The
NMR data were processed using software written in-house and analyzed
with the program packages NMRView (34) and NDEE (SpinUp Inc., Dortmund,
Germany). {1H}15N NOEs were corrected for
signal decrease because of minor sample precipitation during the
experiment and averaged over two independent data sets.
Structure Calculation--
Based on the almost complete
assignment of the 1H, 13C, and 15N
resonances of Pru av 1 published previously (23), a total of 2299 distance restraints could be derived from the two- and
three-dimensional NOESY spectra in an iterative procedure. NOE
cross-peaks were classified manually as strong, medium, or weak
according to their intensities and converted into distance restraints
of less than 2.7, 3.5, or 5.0 Å, respectively. 23 of the 97 3JHNH Complete Cross-validation--
For complete cross-validation
(45) the NOE distance restraints were randomly partitioned into 10 test
sets of roughly equal size (between 211 and 245). 10 sets of 60 structures were calculated, each of the sets having one of the distance
restraint test sets left out. For those 16 structures of each set
showing the lowest energy values, the r.m.s.d. from the distance
restraints not used for their calculation were determined after
assigning the restraints from the nine working sets a relative weight
of 10 compared with those from the test set to prioritize them during
floating assignment of prochiral groups.
Patients' Sera--
Sera from patients allergic to birch pollen
and with an oral allergy syndrome after ingestion of fresh fruits
(cherry, apple, pear, hazelnut) and vegetables (celery) were selected
for this study. Most of the sera showed positive CAP or EAST
(enzyme allergo-sorbent test) classes (greater than class 2) to the
major allergens of birch pollen (Bet v 1), celery (Api g 1), cherry
(Pru av 1), apple (Mal d 1), and pear (Pyr c 1). Sera were taken
from the serum collection of the Paul-Ehrlich-Institut or kindly
supplied by Dr. H. Aulepp (Hospital Borkum Riff, Borkum, Germany).
Recombinant Allergens for Immunoblot Experiments--
The
recombinant major allergens from birch pollen, Bet v 1 isoform a,
apple, Mal d 1, and celery tuber, Api g 1, were obtained from
BIOMAY, Linz, Austria. The recombinant major allergen from sweet
cherry, Pru av 1, was purified as described elsewhere (6). The major
allergens from pear, Pyr c 1 (GenBankTM accession
number AF057030), and from hazelnut, Cor a 1.0401 (GenBankTM accession number AF136945), were purified as
non-fusion proteins and kindly supplied by Dr. F. Karamloo and
D. Lüttkopf (Paul-Ehrlich-Institut, Langen, Germany).
SDS-Gel Electrophoresis, Immunoblotting, and Immunoblot
Inhibition--
The purified recombinant allergens were analyzed by
SDS-polyacrylamide gel electrophoresis under nonreducing conditions
according to Laemmli (46). IgE immunoblotting was performed by a
modification of a previously described procedure (47). Briefly, for
immunoblot analysis the allergens (0.5 µg/cm slot) were
electrophoretically transferred onto nitrocellulose membranes (0.45 µm, Schleicher & Schuell) by tank blotting. For immunoblot
inhibition 20 µl of a pooled patient serum was preincubated with 15 µg of Bet v 1a, 10 µg of Pru av 1, 10 µg of Api g 1, and
buffer as control for 5 h. Thereafter, samples were diluted to 0.6 ml (1 ml for the samples with Bet v 1a as an inhibitor) and added to
the blot strips (3 mm width). After overnight incubation, bound IgE was
detected with a rabbit anti-human IgE antiserum (1:4000, 1 h;
DAKO, Glostrup, Denmark) followed by a biotin-labeled goat anti-rabbit
immunoglobulin antibody (1:6000, 1 h; DAKO) as a secondary
antibody and a streptavidin-horseradish peroxidase (Amersham Pharmacia
Biotech, Little Chalfont, Buckinghamshire, UK) incubation (1:10000, 30 min). Visualization was performed with the ECLTM Western
blotting detection reagents (Amersham Pharmacia Biotech).
Modeling of Allergen-Steroid Complexes--
Cavities were
examined with SURFNET 1.5 (48) using a grid separation of 1.0 Å and a
minimum and maximum gap sphere radius of 1.4 and 3.5 Å, respectively.
Topology and parameter files for cholesterol were generated by the
HIC-Up server (49) based on the cholesteryl linoleate moiety of a
crystal structure (50) from which the linoleate atoms were removed and
subsequently modified to obtain topology and parameter files for
castasterone. The simulated annealing protocol described above was also
used to model Bet v 1 complexes with castasterone. To this end the
atom positions of Bet v 1 as given by its crystal structure (10) were
kept fixed, whereas a set of 33 distance restraints between the
C-8 atom of castasterone and those C Structure Determination--
Analysis of the NMR spectra of
Pru av 1 yielded a total of 2438 experimental restraints for the
structure calculation. In particular, the good dispersion of the amide
proton resonances (23) allowed the identification of 1908 15N NOESYHSQC cross-peaks, thus providing the largest
contribution. The 13C NOESYHSQC suffered from an
unsatisfactory signal-to-noise ratio because of the relatively low
sample concentration, which was worsened by the presence of some
degradation products in the sample. As a consequence, only 54 distance
restraints were derived from this spectrum. A 15N-filtered
NOESY could, however, provide 337 additional distance restraints which
were particularly valuable because many of them are based on long-range
NOEs involving aromatic side-chains expected to form hydrophobic cores
(Fig. 2). 71 dihedral angle restraints derived from 3JHNH Description of the Structure--
Pru av 1 shows a well defined
structure in solution (Fig. 3) with
average atomic r.m.s.d. from the average structure of 0.60 Å for the
backbone and 0.93 Å for all heavy atoms. A schematic representation of
the solution structure of Pru av 1 (Fig.
4) reveals that a folded seven-stranded
antiparallel Comparison with Bet v 1--
The folding topology of Pru av 1
has already been observed for the major birch pollen allergen
Bet v 1, and a backbone overlay of the lowest energy structure of
Pru av 1 with the crystal structure of Bet v 1 (Fig.
5; steroid molecules are modeled into
these structures as discussed below) confirms that indeed both the
secondary structure elements and the tertiary fold of these two
allergens are virtually identical. More precisely, a comparison of the
average solution structure of Pru av 1 with the crystal structure of
Bet v 1 (10) and the average solution structure of Bet v 1 (Ref.
11; only residues 1-154 are taken into account, because the COOH
terminus is less well defined experimentally) yields backbone atomic
r.m.s.d. of 1.85 and 2.23 Å, respectively, which is of the same order
as the difference of 2.06 Å found upon comparing these two Bet v 1
structures with each other. Together with the considerable sequence identity between Pru av 1 and Bet v 1, the conserved backbone conformation leads to a very similar molecular surface as far as shape
and charge distribution are concerned, rendering the existence of
cross-reactive IgE-binding epitopes most likely. In particular, the
glycine-rich P-loop around Glu45 is structurally conserved
in Pru av 1. For Bet v 1, this region was recently identified as
the binding epitope of a monoclonal murine IgG antibody (18) whose high
capacity to inhibit binding of serum IgE from allergic patients to
Bet v 1 indicates that the P-loop is also one of the IgE binding
epitopes. The introduction of four point mutations including the
substitution of Glu45 by serine indeed resulted in a
Bet v 1 mutant with severalfold reduced IgE binding capacity (12). In
the crystal structure of the complex of Bet v 1 with the IgG Fab
fragment, the negatively charged side-chain of Glu45 is
located in a binding pocket of the antibody with a positive electrostatic potential, where it forms two hydrogen bonds. In addition
to Glu45, which is found to be solvent-exposed in all 22 accepted structures of Pru av 1 (Fig. 3), 14 of the remaining 15 residues forming the interaction surface between Bet v 1 and the IgG
Fab fragment are either conserved (Glu42,
Gly46, Gly48, Gly49,
Pro50, Gly51, Thr52,
Asp72, Ile86, and Lys97) or
substituted conservatively (Ile44 by Leu, Asn47
by Asp, Arg70 by Lys, and His76 by Lys) in
Pru av 1 (Fig. 1), which strongly suggests that this region is a
cross-reactive IgE binding epitope. This proposal is supported
by the significantly decreased binding of serum IgE to the mutants
Pru av 1 G46P and Pru av 1 Immunoblot Inhibition Experiments--
For IgE immunoblot
inhibition experiments a serum pool of seven patients was tested with
Bet v 1a, Mal d 1, Api g 1, Pru av 1, Cor a 1.0401, and
Pyr c 1 transferred to nitrocellulose. Preincubation of the serum
pool with Bet v 1a showed complete inhibition of IgE binding to the
related major food allergens (Fig. 6).
Hence, all of the IgE binding epitopes presented by these food
allergens exist on the molecular surface of Bet v 1a as well. This
finding is consistent with the experience that sensitization usually
occurs to birch pollen, whereas the related food allergies are a
consequence of the cross-reaction of the resulting pollen-specific IgE
antibodies. To investigate the IgE cross-reactivity with the two major
food allergens from cherry and celery, preincubation of the serum pool was performed with Pru av 1 and Api g 1. Complete inhibition of IgE
binding to the major cherry allergen was obtained with Pru av 1 as
the positive control. By contrast, only a small reduction of IgE
binding to Pru av 1 on the solid phase resulted from using Api g 1
as an inhibitor (Fig. 7). No IgE
inhibition was detected with buffer as control. Serum from a
nonallergic donor was used as negative control. In other words,
Pru av 1 must contain at least one IgE binding epitope that is not
presented by Api g 1. Sequence alignment of Pru av 1 with Api g 1
(Fig. 1) shows that the P-loop region is not conserved in Api g 1;
the P-loop is not only shorter by one residue, but also the negatively
charged Glu45 is substituted by a positively charged
lysine. The proposal that the P-loop region forms one of the
cross-reactive epitopes can therefore provide a simple explanation of
why preincubation with Api g 1 fails to efficiently inhibit IgE
binding to Pru av 1. To verify this hypothesis the preparation and
subsequent immunological as well as structural characterization of
several allergen mutants is currently under way in our
laboratories.
Implications for the Physiological Function--
Bet v 1
and Pru av 1 form a large internal hydrophobic cavity with a volume
of ~1600 Å3 (Fig. 8). This
forked cavity has three openings to the protein surface, one at the
P-loop, one between
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Structure-based sequence alignment with
Pru av 1 of Pyr c 1 (83.5% sequence identity to Pru av 1),
Mal d 1 (82.9%), Cor a 1.0401 (64.4%), Bet v 1 isoform a
(59.1%), Api g 1 (41.2%), and the START domain of MLN64
(8.5%). The sequence positions above and below the sequences
correspond to Pru av 1 and MLN64, respectively. Gaps in the alignment
are indicated by dots. Residues conserved in at least four
of the six allergens are highlighted by gray
boxes and residues conserved in all six allergens by black
boxes. The secondary structure elements of Pru av 1 are shown
below the alignment. The alignment of the allergens with Pru av 1 is
based on homology models created by SWISS-MODEL (55) using the lowest
energy structure of Pru av 1 as a template. The alignment of the
START domain of MLN64 with Pru av 1 is based on a comparison of the
PDB entry of the START domain of MLN64 with the lowest energy structure
of Pru av 1 by the Dali server (53). The 129 MLN64 residues used for
the alignment are printed in uppercase letters and residues
not used for the alignment in lowercase. Formatting was
performed using ALSCRIPT (56).
scalar coupling constants
measured (23) were smaller than 6.0 Hz, indicating
backbone torsion
angles between
80° and
40°. For the 48 3JHNH
coupling constants greater
than 8.0 Hz, the corresponding
angles were restrained to between
160° and
80°. A hydrogen bond was assumed if the acceptor of a
slowly exchanging amide proton could be identified unambiguously from
the results of initial structure calculations. For each of the 34 hydrogen bonds the distance between the amide proton and the acceptor
was restrained to less than 2.3 Å and the distance between the amide
nitrogen and the acceptor to less than 3.3 Å. These experimental
restraints served as an input for the calculation of 60 structures
using restrained molecular dynamics with X-PLOR 3.851 (35). To this end, a three-stage simulated annealing protocol (36-38) with floating assignment of prochiral groups (39) was carried out as described previously (11, 40, 41), with the following modifications. For
conformational space sampling, 160 ps with a time step of 2 fs were
simulated at a temperature of 2000 K, followed by 120 ps of slow
cooling to 1000 K and 90 ps of cooling to 100 K, both with a time step
of 1 fs. Simulation times longer than these were tested, but no
significant improvement of the results could be observed. A
conformational data base term for both backbone and side-chain dihedral
angles (42) was included in the target function to improve the
stereochemical properties of the structures. After simulated annealing
the structures were subjected to 250 steps of Powell minimization (43)
of the full target function followed by 1000 steps without recourse to
the conformational data base potential. The 22 structures showing the
lowest energy values (excluding conformational data base potential)
were selected for further characterization using X-PLOR 3.851 (35) and
PROCHECK 3.4 (44). Together with the experimental restraints, the
atomic coordinates of this set of 22 structures have been deposited
with the Protein Data Bank (PDB accession code 1E09).
atoms of Bet v 1
lining the cavity was introduced for each castasterone molecule, which
effectively restrained C-8 to within 7.5 Å of a point near the center
of the cavity. The resulting models were refined by 1000 steps of
Powell minimization (43) of a modified target function where the van der Waals interaction was represented by a Lennard-Jones-type potential. The results were transferred to Pru av 1 by placing the
castasterone molecules into equivalent positions of the lowest energy
structure of Pru av 1, which was followed by 1000 steps of Powell
minimization of the original target function (including all
experimental restraints and the modeling distance restraints) to remove
steric clashes and subsequent refinement by 1000 steps of Powell
minimization of the modified target function (again including all restraints).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
scalar
coupling constants and 68 distance restraints for the observed hydrogen
bonds complete the set of experimental restraints used for the
structure calculation (Table I). The 22 accepted structures showed no single distance restraint violation of
more than 0.30 Å and no systematic violation of more than 0.15 Å. An
analysis with PROCHECK 3.4 (44) revealed that 82.4% of the non-glycine
and non-proline residues adopted a conformation within the most favored
regions of the Ramachandran plot, and 16.6% adopted a conformation in
the additional allowed regions. No residues with a conformation in the
disallowed regions were observed. Even though Kuszewski and Clore (51)
recently reported that the roughness of their conformational data base
potential (42) as used in our calculation may affect convergence and
conformational sampling in structure calculations with very few
experimental restraints, no such problems occurred in the structure
calculation of Pru av 1, probably because of the high number of
experimental restraints. Artifacts arising from the conformational data
base potential can be ruled out because a control calculation without recourse to this potential yielded an almost identical set of structures (atomic root mean square deviations (r.m.s.d.) between the
two average structures were 0.65 Å for the backbone and 0.85 Å for
all heavy atoms). The quality of the structure determination was also
assessed by calculating a total of 600 structures for complete
cross-validation (45). The value of 0.27 ± 0.07 Å for the
cross-validated r.m.s.d. from the test set distance restraints of the
set of 160 accepted structures indicates the high quality of the
solution structure of this comparatively large protein.
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Fig. 2.
Atomic r.m.s.d. from the average structure
(top), distribution of NOEs (center),
and {1H}15N NOE values
(bottom). Backbone r.m.s.d. are indicated by
filled circles and side-chain heavy atom r.m.s.d. by
filled triangles. Error bars for the
{1H}15N NOEs are shown on top of each
{1H}15N NOE bar. The low number
of NOE distance restraints found for the loop from Glu60 to
Tyr64 leads to very high atomic r.m.s.d. As far as
Pro14 and Pro15 are concerned, the lack of NOE
distance restraints was compensated for by the identification of two
hydrogen bonds with the slowly exchanging amide protons of
Leu18 and Phe19, respectively. The residues
with a high number of NOEs are predominantly aromatic (e.g.
Phe19, Tyr150, and Phe81),
emphasizing the importance of the 15N-filtered NOESY for
the structure determination. {1H}15N NOE
values for Val2, Phe3, and Thr122
to Lys129 were measured for the natively purified
protein.
Summary of the structure calculation
-sheet (residues 2-11, 41-43, 53-58, 65-75 with a
kink at Asp72, 80-85, 97-104, and 112-122) and two short
-helices arranged in a V-shaped manner (residues 15-22 and 26-33)
wrap around a long COOH-terminal
-helix (residues 130-153) to form
a basket-like structure with the long helix resembling a handle, thus
creating a large hydrophobic cavity. In contrast to the precision of
the overall structure, however, the loop from Glu60 to
Tyr64 is experimentally less well defined because of the
missing resonance assignments for the amide protons of
Ser62 and Gln63 (23), leading to a marked
increase in atomic r.m.s.d. (Fig. 2). This lack of experimental data
might in fact reflect an actually existing increased flexibility. One
indication for this is the rapid solvent exchange of the amide protons
in this region, resulting in exceptionally weak NMR signals. In
addition, this loop is poorly defined in both sets of solution
structures of Bet v 1 (10, 11), and the determination of the crystal
structure of Bet v 1 yielded two conformers for this loop
(i.e. Asp60 to Lys65), which still
do not fit the electron density well (10). To obtain initial
experimental data concerning the dynamic behavior of Pru av 1
in solution we measured {1H}15N NOE values
(Fig. 2). The low {1H}15N NOEs of
0.489 ± 0.018, 0.359 ± 0.017, and 0.501 ± 0.018 for the amide protons of Glu60, Gly61, and
Tyr64, respectively, strongly support the notion that this
loop shows significantly increased internal flexibility. Surprisingly,
one of the lowest values (0.414 ± 0.017) was measured for the
amide proton of Glu8, which is located in a slight bend in
the middle of the first
-strand.
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Fig. 3.
Backbone overlay of the 22 accepted
structures. The NH2 terminus on the left is
hidden by the loop from Ile86 to Glu96, and the
COOH terminus can be seen on the right. Except for the loop
from Glu60 to Tyr64, which is indicated by an
arrow, the structures are in excellent agreement, especially
as far as the -strands are concerned. The side-chain of
Glu45 shown at the bottom is clearly
solvent-exposed in all structures. The overlay was performed using
Sybyl 6.5 (Tripos Inc., St. Louis, MO).
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Fig. 4.
Schematic representation of the secondary
structure elements based on the lowest energy structure. The same
view is shown as in Fig. 3. Despite being hidden by 3,
the kink in
4 at Asp72 is clearly visible.
The figure was drawn with MolScript 1.4 (57) and rendered with Raster3D
2.2a (58).
T52 observed for some patients
(20).
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Fig. 5.
Backbone overlay of the lowest energy
solution structure of Pru av 1 (green) and the
crystal structure of Bet v 1 (orange) in complex
with one (top) and two (bottom)
castasterone molecules (representative models). The same view is
shown as in Figs. 3 and 4. The loop from Glu60 to
Tyr64 is indicated by an arrow. The overlay was
performed using Sybyl 6.5 (Tripos Inc.).
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Fig. 6.
Immunoblot inhibition of IgE binding to
Bet v 1a (column 1), Api g 1
(column 2), Mal d 1 (column 3),
Pru av 1 (column 4), Pyr c 1 (column
5), and Cor a 1.0401 (column 6) on the
solid phase. A serum pool from birch pollinotic patients with
associated food allergy was preincubated with Bet v 1a (lanes
3) as an inhibitor. Serum from a nonallergic subject
(lanes 1) and samples without inhibitor
(lanes 2) were used as controls.
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Fig. 7.
Immunoblot inhibition of IgE binding to
Pru av 1 on the solid phase with Pru av 1 (lane 1) and
Api g 1 (lane 2) as inhibitors. A sample
without inhibitor (lane 3) and serum from a nonallergic
subject (lane 4) were used as controls.
3 and
1, and one
between
3 and the loop from Glu60 to
Tyr64. The latter is the largest opening, but its size
depends strongly on the conformation of the flexible loop acting as a
flap. Such a large cavity constitutes a very unusual feature for a
protein structure and can therefore be expected to be important for its physiological function. An obvious possibility for the physiological purpose of the cavity is the binding of a hydrophobic ligand. An
indication of what this hypothetical ligand might be was provided by
the recently determined crystal structure of the START domain of the
human protein MLN64 (52), part of which revealed a striking structural
homology to Bet v 1 and Pru av 1 (Fig.
9). Based on a comparison of the PDB
entry of the START domain of MLN64 with the lowest energy structure of
Pru av 1 by the Dali server (53), an alignment of the average
solution structure of Pru av 1 with the crystal structure of the
START domain of MLN64 yielded a backbone atomic r.m.s.d. of 2.89 Å over as many as 129 residues, even though the sequence identity over
these 129 residues is only 8.5% (Fig. 1). Bet v 1 and the START
domain of MLN64 are the only proteins with significant structural
homology to the lowest energy structure of Pru av 1 that were found
by a Dali server data base search. The fact that START domains are
associated with the transfer of lipids, especially of steroids,
suggests phytosteroids as possible ligands for Bet v 1 and
Pru av 1. It should be noted, however, that there are also
significant structural differences between Bet v 1 and Pru av 1 on
one hand and the START domain of MLN64 on the other hand. In addition
to the existence of an additional
-helix and two additional
-strands at the NH2 terminus, the cavity of the START
domain of MLN64 with a volume of about 1000 Å, which is approximately
the volume required for the accommodation of a single steroid molecule,
is much smaller than that of the allergens. Furthermore, the cavity of
the START domain of MLN64 resembles a tunnel, with only two openings to
the protein surface, which correspond to the opening at the P-loop and
to the opening between
3 and the loop from Glu60 to
Tyr64 of Pru av 1. Unfortunately, a quantitative investigation of the
binding of phytosteroids to Pru av 1 by means of NMR titration
experiments is very difficult because of the hydrophobicity of
virtually all the physiologically relevant steroids, but we were able
to gather first qualitative experimental evidence that Pru av 1 does
indeed interact with a particular phytosteroid. Upon the addition of
homocastasterone, a brassinosteroid that is different from the most
widely distributed brassinosteroid, castasterone, only by the
replacement of the methyl group at C-24 with an ethyl group (54),
several amide proton resonances of Pru av 1 disappeared from the
[1H,15N] HSQC spectrum (Fig.
10). This is probably because of severe line broadening as a consequence of exchange processes that are intermediate on the NMR time scale. Interestingly, the affected residues (Leu18, Lys20, Ala21,
Phe22, Val23, Leu24,
Asp25, Ala26, Asn28,
Val30, Ile38, Lys54,
Lys55, Ile56, Lys68,
Lys70, Ile71, Tyr81,
Leu85, Asp89, Lys103, and
Ile128) surround the lower part of the cavity like a funnel
(Fig. 8), thus supporting the expectation that homocastasterone binding takes place inside this cavity. Molecular modeling was used to investigate the steric constraints that are imposed on the orientation and the position of bound steroid molecules by the size and shape of
the cavity. Because the opening between
3 and the loop
from Glu60 to Tyr64 of the lowest energy
solution structure of Pru av 1 is larger than the
corresponding opening of the crystal structure of Bet v 1, we decided
to model the more constrained Bet v 1 complexes first and then
transfer the results to Pru av 1. The cavity of Bet v 1 and
Pru av 1 is so large that it can accommodate one or two castasterone
molecules in several different positions and orientations without
significant structural changes (Figs. 5 and 8). In conclusion, in light
of the above evidence, the physiological function of Bet v 1 and
Pru av 1 most likely involves phytosteroid binding. The striking
structural homology observed between the plant proteins Bet v 1 and
Pru av 1 on one hand and the corresponding domain of the human
protein MLN64 on the other hand, despite a low sequence identity and
despite considerable structural differences, indicates that we might be
dealing with a widely distributed tertiary fold designed to bind
steroids or other lipids for a variety of purposes. Although our
results mark a first step toward the elucidation of the physiological
function of these proteins, a series of questions remains to be
answered by future investigations; for example, is the binding specific
for particular steroids, and if so, what features determine the
specificity? Do these allergens bind two steroid molecules
simultaneously, or is there any additional ligand to occupy the extra
space in the cavity? What exactly is the physiological purpose of their
interaction with steroids?
View larger version (38K):
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Fig. 8.
Visualization of the hydrophobic cavity.
A similar view is shown as in Figs. 3-5. A stick
representation of the backbone heavy atoms of the lowest energy
structure of Pru av 1 is shown in red, and the cavity is
indicated by blue lines. Also shown are the side-chains of
those residues whose amide proton resonances disappeared upon the
presence of homocastasterone (top). These residues are
colored yellow. The locations of one (center) and
two (bottom) castasterone molecules modeled into the cavity
are shown in green. The figure was prepared with InsightII
98.0 (Molecular Simulations Inc., San Diego).
View larger version (45K):
[in a new window]
Fig. 9.
Backbone overlay of the lowest energy
solution structure of Pru av 1 (green) and the
crystal structure of the START domain of MLN64
(red). The same view is shown as in Figs. 3-5.
The overlay was performed using Sybyl 6.5 (Tripos Inc.).
View larger version (40K):
[in a new window]
Fig. 10.
Overlay of the
[1H,15N] HSQC spectra of uniformly
15N-labeled Pru av 1 with (positive signals in
red, negative signals in green) and
without (positive signals in black, negative signals
in blue) homocastasterone in
H2O/Me2SO-d6
(9:1). Amide proton resonances are labeled according to
their residue numbers. Negative resonances are aliased in the indirect
15N dimension F1.
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ACKNOWLEDGEMENTS |
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We thank P. Deuerling, U. Herzing, and R. Hofmann for expert technical assistance.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ro617/11-1) and the Bundesministerium für Bildung und Forschung (BMBF).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1E09) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Recipient of fellowships from the Freistaat Bayern and the Fonds des Verbandes der Chemischen Industrie in cooperation with the BMBF.
To whom correspondence should be addressed: Lehrstuhl
für Biopolymere, Universität Bayreuth,
Universitätsstra
e 30, D-95447 Bayreuth, Germany. Tel.:
+49-921-553540; Fax: +49-921-553544; E-mail:
paul.roesch@uni-bayreuth.de.
Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M101657200
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ABBREVIATIONS |
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The abbreviations used are: NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; r.m.s.d., root mean square deviation(s).
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