From the
National Centre for Biomolecular Research
and Department of Biochemistry, Masaryk University, 611 37 Brno, Czech
Republic, ||European Synchroton Radiation Facility
Experiments Division, BP 220, F-38043 Grenoble cedex, France, and
**Centre de Recherches sur les Macromolécules
Végétales-CNRS (affiliated with Université Joseph
Fourier), BP 53, F-38041 Grenoble cedex 09, France
Received for publication, March 14, 2003 , and in revised form, April 28, 2003.
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ABSTRACT |
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INTRODUCTION |
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Due to the importance of their biological role, there is increasing interest in fungal lectins. However, there is only limited information about them, and although several crystals have been obtained, including the lectins from Flammulina veltipes (9), Pleurotus cornicopiae (10), Pleurotus ostreatus (11), Sclerotium rosfii (12), and Aleuria aurantia (13, 14), no crystal structure has yet been determined.
The lectin from the orange peel mushroom, A. aurantia
(AAL),1 has been
purified from the fruiting bodies of the fungus as a 72-kDa protein composed
of two identical subunits and has been shown to exhibit millimolar range
affinity (Kd = 1.6 x
104 M) for fucose
(15). Later, the primary
sequence was determined and demonstrated the presence of six internal repeats
of about 50 amino acids (16).
Cloning of the gene allowed production of the recombinant lectin in
Escherichia coli
(17). Further characterization
of the lectin specificity demonstrated that all fucose-containing
disaccharides present on glycoconjugates (Fuc12Gal,
Fuc13GlcNAc,
Fuc14GlcNAc, and
Fuc16GlcNAc) displayed similar binding to the lectin, higher
than that shown for highly branched oligosaccharides such as the determinants
of Lewis histo-blood groups
(15,
18,
19). Since AAL is the only
available lectin with high affinity for the
Fuc16GlcNAc present
in the core of complex N-glycans, it is widely used in the
fractionation of glycoproteins.
L-Fucose, as a component of cell surface complex oligosaccharides, is a key participant for cell surface recognition. Nevertheless, until very recently, no characterization of any fucoselectin crystal structure was attained. In the last year, the structure of fucose binding lectins from legume plant Ulex europaeus (lectin I) (20), from animal Anguilla anguilla (eel) (agglutinin) (21), and from bacterium Pseudomonas aeruginosa (lectin II) (22) have been solved with ligated fucose. Interestingly, these three different proteins display different binding modes toward the same monosaccharide.
The crystal structure of AAL-fucose complex reported here has no similarity to any other described fucose-binding lectin. It represents a new fold present in a lectin family common to several pathogenic bacteria and fungi.
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EXPERIMENTAL PROCEDURES |
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Crystallization and Data CollectionCrystallization trials
were performed with Hampton crystallization screens I and II (Hampton
Research, Laguna Niguel, CA) using the hanging drop technique. Crystals were
obtained using the following conditions: 2 µl of precipitant (0.1
M sodium cacodylate buffer, pH 6.5, 0.2 M magnesium
acetate tetrahydrate, 20% polyethylene glycol 8000) mixed with a solution of 2
µl of AAL at a concentration of 10 mg/ml and L-fucose at a
concentration of 137 µg/ml. The drops were allowed to equilibrate over a
reservoir of 1 ml of the precipitating solution at 20 °C. Crystals grew as
platelets to maximum dimensions of 0.3 x 0.3 x 0.05 mm3
after 1 week. They belong to space group P21 with unit cell
dimensions of a = 45.97 Å, b = 86.41 Å,
c = 77.85 Å, and = 90.62° at 100 K. The asymmetric
unit accommodates two monomers, corresponding to a
Vm of 2.20 Å3
Da1 and a solvent content of 46% solvent. A
mercury derivative was prepared by soaking a crystal in 1 mM sodium
ethylmercurithiosalicylate (thimerosal) (Hampton Research) for 24 h.
Crystals were cryo-cooled at 100 K after soaking them in either paraffin oil or 30% (v/v) glycerol in precipitant solution for the native and mercury derivative, respectively. All data images were recorded on an ADSC Q4R CCD detector (Quantum Corp.) on the fixed energy beamline ID141 at the ESRF (Grenoble, France). Single wavelength highly redundant anomalous diffraction data to 2.0-Å resolution were collected from the thimerosal-soaked crystal and native data to 1.5-Å resolution. Measurements were made at a single x-ray wavelength of 0.934 Å. Diffraction images were processed using MOSFLM (23) and scaled and truncated to structure factors using the CCP4 (24) programs SCALA and TRUNCATE. Data processing statistics are presented in Table I.
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Structure DeterminationThe crystal structure was solved using the single wavelength highly redundant anomalous diffraction technique with data from the mercury derivative. Harker sections of the anomalous difference Patterson map showed two peaks corresponding to one mercury per monomer in the asymmetric unit. Initial mercury site coordinates, phasing, and solvent flattening were carried out with autoSHARP (25),2 which located the two mercury sites. autoSHARP was also directed to search for noncrystallographic symmetry, and the resulting matrix was further refined, together with averaging and phase extension, using DM (27) to give an electron density map of excellent quality. An initial structure was built automatically using ARP/wARP (28), and side chains were docked to give 474 residues out of a total of 624 for the asymmetric unit cell. Manual building using O (29) gave a more complete model, which was then used as a search probe for AMORE (30) molecular replacement using the nonisomorphous high resolution native data.
AMORE gave two clear solutions, which were used to generate a new noncrystallographic symmetry averaging matrix. Phase extension, averaging, and solvent flattening with DM generated new phases and figures of merit for the native data, which was followed by a complete automatic construction, side chain docking, and an initial water molecule construction with ARP/wARP, which gave a model of 588 residues out of 624 with an Rcrys of 20.3% R and Rfree of 23.7%. The remainder of the residues and the fucose molecules clearly defined in density were positioned manually using O. Further refinement cycles with REF-MAC, including automatic water molecule placement using ARP/wARP, manual rebuilding with O, and construction of alternative conformations, where necessary (with occupancies estimated from the refined relative B-factors of the conformations), resulted in a final model of all 624 residues with 10 fucose molecules (5 bound per monomer) and 1166 water molecules with an Rcrys of 14.4% and Rfree of 17.9% to 1.5-Å resolution. Side chain atoms not defined in electron density were retained in the model but with an occupancy set to zero (Table II).
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Surface Plasmon Resonance MeasurementsAll surface plasmon resonance experiments were performed with a Biacore 3000 (Biacore AB, Uppsala, Sweden) at 25 °C using HBS buffer (10 mM Hepes, 150 mM NaCl, pH 7.4) and a flow rate of 5 µl/min. Measurements were carried out simultaneously on all four measuring channels using three different concentrations of immobilized AAL, whereas the fourth channel was used as the control flow cell. A research grade CM5 sensor chip was activated with a 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide solution for 10 min, and 50 µl of AAL in 5 mM maleate buffer, pH 7.0, at concentrations of 50, 10, and 2 µg/ml respectively, was injected to a particular flow cell. The unreacted species on the sensor surface were blocked by 1 M ethanolamine. The blank channel was treated identically except for the lectin injection.
30 µl of carbohydrate solutions (concentrations between 0.39 and 200 µM) in running buffer were injected into the flow cells using the kinject mode. The equilibrium response (after subtraction from the response of the reference surface) of each experiment was used to create curves of analyte binding, which were fitted to a 1:1 steady-state affinity model using Origin version 6.1 software (OriginLab Corp.).
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RESULTS |
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Several amino acids at the N and C termini of the peptide chains protrude
from the base of the -propeller cylinder, associating in a small
antiparallel two-stranded
-sheet that forms a separated domain.
The inner cavity of AAL has a tunnel shape with a diameter of about 8
Å in its middle part and almost closed off on the N terminus side of the
first inner -strands (Fig.
1B). This cavity has a strong hydrophobic character,
being formed mostly by the conserved alanine residues of the first strands of
each propeller blade. The core is filled with a set of about 50 water
molecules forming a well ordered hydrogen bond network (average
B-factor value of 12.5).
Oligomeric StateAAL has been described as a dimer in
solution (15) and is also
observed as a dimer in the crystal structure. The two monomers are very
similar, and superimposition of their backbones gives an r.m.s. value of 0.26
Å. A pseudo-2-fold axis of symmetry generates this dimer in the crystal
(Fig. 1C). The small
domain created by the antiparallel association of the N-terminal and
C-terminal peptides plays a key role in the dimerization, additional contact
being mediated by four loops (those interconnecting blades I and II and blades
II and III and the loops between strands 2-I and
3-I and between strands
2-II and
3-II). Hydrophobic contacts involve the C terminus
Trp312 from each monomer with Lys83 from the other. In
addition, one tyrosine residue, Tyr6, interacts via aromatic ring
stacking with its counterpart on the other monomer through the 2-fold axis.
Four main hydrogen bonds are also established between the side chains of
Asp263 and Ser283 and between the Trp312
nitrogen side chain and backbone carbonyl backbone of Leu59.
This dimerization mode creates a "back to back" association of the two cylinder-shaped monomers. It closes the internal cavities on the N terminus side of the inner strand but leaves the other side of the cavity (i.e. the most open one) accessible to solvent. The fucose binding sites are exposed on each side of the dimer, at distances ranging from 50 to 70 Å from each other.
Fucose Binding SitesThe crystal structure of the complex between AAL and fucose reveals five fucose residues bound per monomer (Fig. 1, A and B). These are located between consecutive blades, in binding sites consisting of pockets at the external face of the cylinder (Fig. 1E). For simplicity, the site located between blades I and II will be named site I, and the following ones will be named consecutively. The site between blades VI and I, which would have been referred as site VI, does not contain any electron density corresponding to a bound fucose molecule. The contacts observed in the five sites of monomer A and monomer B are listed in Table III. The two monomers are almost equivalent, with the exception of marginal contact with symmetry-derived monomers in site IV of A and site V of B. Therefore, only the sites of monomer A are described more lengthily and shown in Fig. 2.
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The five fucose binding sites are not equivalent, but they have the same
architecture and present invariant features. They are made up in the crevasse
between two adjacent blades, and it is mostly amino acids of the four strands
(rather than the loops) in each blade that participate in binding. As
displayed in the alignment of sequence repeats in
Fig. 1D, amino acids
of blade i can therefore participate in site i or site
i 1. Conserved features of the binding sites consist
of five hydrogen bonds between fucose and protein
(Fig. 2); for fucose in site
i, they involve the side chain of three highly conserved residues,
Arg of strand 2 i, Glu of strand
3 i and Trp of strand
4
(i + 1). These five hydrogen bonds make a compact network,
the geometry of which is strictly conserved in the five binding sites. In
addition to NH2 of Arg donating a hydrogen bond to the fucose ring
oxygen, two cooperative sets of bonds are created; NE1 of the site Trp gives a
hydrogen bond to O-3 that in turn donates a hydrogen bond to one acidic oxygen
of the site Glu, whereas NE of Arg is bridged to the other oxygen of Glu via
OH-4 of the fucose.
The second part of the binding site is characterized by a hydrophobic
region. The closer and most conserved hydrophobic contact involves a Trp/Tyr
residue at the extremity of 4 (i + 1). The
aromatic ring stacks against the flat nonpolar face of fucose, with contacts
with the methine and methyl groups at C-4, C-5, and C-6. The conserved
isoleucine residue of strand
2 (i + 1)
establishes additional hydrophobic contact with the methyl group at C-6 of
fucose. Longer distance contact involves the conserved Trp of strand
1 i.
The interactions described above leave hydroxyl groups O-1 and O-2 exposed
to solvent. In all sites, fucose interacts with a number of water molecules
that varies between two and five. The mean hydration number of fucose is four,
which is an unusually high value for a monosaccharide in a protein binding
site. Analysis of the water molecules hydrogen-bound to the sugar indicates
that they are not randomly scattered but can be clustered in different sites.
Fig. 3 illustrates the seven
sites that have been identified. W4 occupies roughly the position of backbone
NH in the sites where the connecting loop between blades does not come into
contact with the fucose. Interestingly, almost none of these water molecules
establish hydrogen bond directly to the protein but are rather in contact with
other water molecules from the solvent. The only exception, W7, stabilizes the
-anomer in site IV (see below), by bridging the O-1a to the OH of
Tyr241 in both A and B monomers.
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Since the six -propeller blades are not identical, there are some
differences between the five binding sites
(Fig. 2). First, fucose is not
bound in the same configuration in the different sites. In both monomers, the
sugar in binding sites I, III, and V adopts the
-configuration
(equatorial O-1), whereas it is bound in the
-configuration (axial O-1)
in binding site IV. In binding site II, both anomers can be identified in the
electron density with an
/
population of 35/65 and 55/45 in
monomers A and B, respectively. This selection of anomeric configuration can
be correlated to differences in the architecture of the binding sites. The
number of hydrogen bonds between fucose and protein varies from five to seven:
five in sites I, III, and V; six in site IV; and seven in site II. When
looking at the differences between individual binding sites, it appears that
I, III, and V are very similar. In these three sites, hydroxyl groups O-1 and
O-2 of the fucose ligand are exposed to the solvent and do not participate in
binding to the protein. On the other hand, in binding sites II and IV, the
outermost external
-strand of the blade is oriented slightly
differentially, and the beginning of the interblade connecting loops is in
contact with fucose. This results in hydrogen bonds between the hydroxyl group
at O-2 and the backbone amide nitrogen of Gln101 and
Gly203 in sites II and IV, respectively. In site II,
Gln101 also interacts via its side chain, resulting in a deeper
binding site.
In aqueous solution, the two configurations of fucose exchange freely by
tautomerization. In the crystal, the -anomer is fixed in three binding
sites, and the
-anomer is fixed in one. It has been checked that this
selection does not arise from steric hindrance and that both anomers could fit
in all sites. It seems more likely that the complex network of water molecules
and the presence or absence of the contacting loop at O-2 influence the
selection.
Analysis of the crevasse between blade VI and blade I, where fucose does not bind, indicates that there is indeed a pocket between the two blades. Nevertheless, two of the polar amino acids responsible for hydrogen bonding of fucose are missing: Ser277 instead of Arg and Gln299 instead of Glu in blade VI. Furthermore, the aromatic amino acid that should stack against the apolar face of fucose is replaced by Arg39 in blade I (Fig. 1D).
Our crystallographic evidence of five fucoses bound on each AAL monomer is in contradiction to previous biochemical studies performed by equilibrium dialysis that concluded that only one carbohydrate binding site existed per monomer (15). The discrepancy between stoichiometry values obtained by equilibrium dialysis and those determined by crystallography may be due to the nonequivalence of the five sites. It seems likely that one of them, most certainly site II that establishes seven hydrogen bonds with fucose, has a higher affinity for fucose than the other ones.
AAL Oligosaccharide SpecificityThe particularity of AAL,
when compared with other fucose binding lectins, is its large range of
affinity. Contrary to U. europaeus agglutinin isolectin 1 or A.
anguilla agglutinin that have a strong preference for
Fuc12Gal terminal disaccharides, AAL binds oligosaccharides or
glycoconjugates bearing
Fuc linked in the 13, 14, and
16 positions all equally well
(15,
18). In fact, the relatively
high affinity for fucose measured by equilibrium dialysis
(Kd = 16 µM)
(15) or by surface plasmon
resonance experiments (Kd = 33 µM)
(33) is not further increased
when various fucose-containing disaccharides are tested
(19). A high resolution NMR
study of free and AAL-bound
Fuc16
GlcNAc-O-Me demonstrated
that only the fucose is bound in the protein binding site, whereas the GlcNAc
moiety rotates freely in the bulk solvent
(34).
It therefore appears that for disaccharides, only the terminal fucose is establishing contact with the proteins. Since A. anguilla agglutinin shows higher affinity to large fucose-containing glycoproteins such as human lactotransferrin (18), it is of interest to test which of the fucose-containing oligosaccharides could be recognized.
A surface plasmon resonance binding assay was used to determine equilibrium
dissociation constants (KD) for AAL binding of
some fucose-containing saccharides. Fig.
4 shows typical sensorgrams obtained after the injection of
analytes over the lectin-covered surface. Since association and dissociation
phases were rapid, binding curves for all substrates were calculated using the
steady-state parts of experimental curves. KD
values were investigated using Scatchard plot analysis and by fitting the data
to a saturation curves. The results are summarized in
Table IV. Final values were
obtained by curves fitting using Origin software version 6.1, which enabled
simultaneous evaluation of all three curves for each substrate with shared
KD. The calculated constants were in agreement
with values obtained by linearization methods. A comparison of
KD values derived from surface plasmon resonance
experiments reveals that AAL lectin shows a slightly higher affinity to
disaccharide -L-Fuc-
-D-Gal than to
L-fucose and Lewis trisaccharides. These results are in agreement
with previously published data
(19), and equilibrium
dissociation constants for all measured sugars are within 1 order of
magnitude, demonstrating that AAL preferentially recognizes a fucose
moiety.
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The presence of an aromatic group at position O-1, either in the or
configuration, lowers the affinity, which is unusual for
protein-carbohydrate interactions, which are often characterized by a
hydrophobic patch close to the binding site. When looking in detail at all of
the
Fuc12Gal-containing oligosaccharides tested, it appears that
the presence of another sugar at position 3 of Gal (i.e. blood group
A and B trisaccharides) does not affect the affinity, whereas substitution at
position 1 of Gal (i.e. fucosyllactose, blood group H type II, and
Lewis oligosaccharides) results in a decrease of the affinity, suggesting a
steric hindrance in this region.
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DISCUSSION |
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The DALI program (41) was
used to identify proteins with close structural similarities available in the
Protein Data Bank (42). The
highest scoring hits were then structurally aligned with the structure
comparison tool of the Proceryon software (ProCeryon Biosciences). Structures
most similar to AAL are sialidases of various origins. The bacterial
Salmonella typhimurium LT2 neuraminidase
(43) (Protein Data Bank code
2SIM
[PDB]
) and Micromonospora viridifaciens sialidase
(44) (Protein Data Bank code
1EUT
[PDB]
) superimpose on the AAL main chain C- coordinates with r.m.s.
values of 2.35 Å for 212 amino acids and 2.38 Å for 206 amino
acids, respectively. An almost identical superimposition (2.49 Å for 208
amino acids) was obtained for the eukaryotic trans-sialidase from
leech (45) (Protein Data Bank
code 2SLI
[PDB]
). The comparison of AAL with bacterial sialidase is shown in
Fig. 5. Structural sequence
alignment only confirms the conservation of hydrophobic amino acids that line
the junction zone of the blades. No clear sequence similarities could be
detected, and of the nine catalytic amino acids responsible for cleaving the
sialic acid from glycoconjugates, only two are conserved in the AAL sequence.
At the present stage, it is difficult to conclude whether a phylogenetic link
exists between sialidases and AAL.
Most of the different propeller superfamilies have evolved in rigidifying the structure by a "Velcro" closure that brings together the C- and N-terminal moiety as part of the same blade (i.e. the C-terminal peptide in the position of strand 1 of the first blade (or alternatively the N-terminal peptide as strand 4 of the last blade)). AAL does bring the two extremities of the chain together in antiparallel association but in a different domain, independent from the blade, looking therefore more like a "zipper" than Velcro.
AAL Repeats in Pathogenic MicroorganismsAAL-like lectins have been purified using fucose affinity columns from the fruiting bodies of other mushrooms. The sequence identity of 12 amino acids of 20 has been demonstrated between the N-terminal sequence of a lectin from another ascomycete mushroom, Melastiza chateri, and AAL (46).
Ascomycetes fungi also include species that can be pathogenic to plants or animals. Two proteins presenting six sequence repeats highly similar to the ones of AAL have been recently identified in two Aspergillus species: Aspergillus oryzae (47), a plant pathogen used for fermentation of rice in sake production, and A. fumigatus,3 a saprophytic fungal that can turn into a dangerous human pathogen in hospital environments. These two lectin sequences have 82% identity and display about 30% identity with the AAL sequence. The 310-amino acid protein from A. oryzae has hemagglutinin activity that is inhibited by L-fucose, whereas D-mannose and neuraminic acids are only weak inhibitors (47). The 314-amino acid protein from A. fumigatus is described as fucose-lectin in the sequence deposition but also seems to correspond to a recently described 32-kDa protein specific for sialic acid (48). This discrepancy is hypothesized to result from differences in strains or culture conditions.
The AAL repeat has also been identified in a different organism, the plant
pathogenic bacterium Ralstonia solanacearum
(49), which, like
Aspergillus and Aleuria, is a soil inhabitant. The R.
solanacearum lectin shares the same specificity profile as AAL; it is
specific for L-fucose and interacts with all fucose-bearing blood
group oligosaccharides. Interestingly, the 91-amino acid sequence only
contains two of the characteristic repeats. The alignment displayed in
Fig. 6 demonstrates that the
amino acids needed to establish the five conserved hydrogen bonds and the two
strong hydrophobic contacts are conserved in the two repeats of R.
solanacearum lectin and in four repeats of the Aspergillus
lectins. In two binding sites of A. fumigatus lectin and A.
oryzae lectin, the Trp of the last -strand that is hydrogen-bonded
to fucose is not conserved, and Glu is replaced by Gln. It can be predicted
that the affinity for fucose would be somewhat decreased in these two
sites.
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Interestingly, from the high similarity between AAL and R.
solanacearum lectin repeats, it could be predicted that R.
solanacearum lectin associates as a trimer, thus reforming a six-bladed
-propeller. No such structure has yet been observed. In such a case,
this bacterial protein could be an example of a "primitive"
propeller, since it is currently hypothesized that the existing
-propellers have been formed by modular duplication of a four-stranded
sheet motif (31).
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FOOTNOTES |
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* Travels and visits between the National Center for Biomolecular Research
and Centre de Recherches sur les Macromolécules Végétales
are supported by a BARRANDE exchange program. The costs of publication of this
article were defrayed in part by the payment of page charges. This article
must therefore be hereby marked "advertisement" in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Stay in Grenoble supported by the French minister program for invited
scientists and partial financial support from the Ministry of Education of the
Czech Republic by Grant LN00A016.
¶ These two authors contributed equally to this work.
Supported by a grant from the French association La Ligue Contre le
Cancer.
To whom correspondence should be addressed. Tel.: 33-476-03-76-36; Fax:
33-476-54-72-03; E-mail:
imberty{at}cermav.cnrs.fr.
1 The abbreviations used are: AAL, A. aurantia lectin; r.m.s., root
mean square.
2 C. Vonrhein, E. Blanc, P. Roversi, and G. Bricogne, manuscript in
preparation.
3 Ishimaru, T., Kubai, S., Bernard, E. M., Tamada, S., Tong, W.,
Soteropuolos, P., Perlin, D. S., and Armstrong, D., SWISS-PROT, deposition
Q8NJT4.
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ACKNOWLEDGMENTS |
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REFERENCES |
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