THE JOURNAL OF BIOLOGICAL CHEMISTRY

2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 278, No. 29, Issue of July 18, pp. 27059 27067, 2003
Printed in U.S.A.

Crystal Structure of Fungal Lectin

SIX-BLADED -PROPELLER FOLD AND NOVEL FUCOSE RECOGNITION MODE FOR
ALEURIA AURANTIA LECTIN*
Received for publication, March 14, 2003, and in revised form, April 28, 2003
Published, JBC Papers in Press, May 5, 2003, DOI 10.1074/jbc.M302642200

Michaela Wimmerova, Edward Mitchell, Jean-Frederic Sanchez**, Catherine Gautier**,

and Anne Imberty**
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 Macromolecules Vegetales-CNRS (affiliated with Universite Joseph Fourier),
BP 53, F-38041 Grenoble cedex 09, France

Lectins are carbohydrate-specific proteins that are key

players in many recognition events at the molecular or cellular level (1). Fungi, either mushrooms or filamentous fungi,
* Travels and visits between the National Center for Biomolecular
Research and Centre de Recherches sur les Macromolecules Vegetales
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.
The atomic coordinates and structure factors (code 1OFZ) have been
deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://
www.rcsb.org/).
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.: 33-476-03-7636; Fax: 33-476-54-72-03; E-mail: imberty@cermav.cnrs.fr.
This paper is available on line at http://www.jbc.org

often depend on host association (symbiosis or parasitism)

and appear to use lectins for host recognition and/or adhesion. One of the first examples of a lectin-mediated interaction between a fungus and its host was discovered in the
nematode-trapping fungus Arthrobotrys oligospora (2). In
higher fungi, lectins are involved in molecular recognition
during the early stage of mycorrhization. An example is their
role in the high specificity of the Lactaria mushroom/tree
symbiotic association (3). A role of lectins in mycoparasitism
has been proposed for a number of human pathogens such as
Candida albicans (4), the agent causing oral candidosis, and
Aspergillus fumigatus (5), which is a major life-threatening
pathogen in hospital environments, responsible for invasive
pulmonary aspergillosis in immunodeficient patients (6).
Lectin-mediated recognition is also involved in plant mycoparasitism (7, 8).
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 1.6 104 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, Fuc1
3GlcNAc, Fuc1 4GlcNAc, and Fuc1 6GlcNAc) 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 Fuc1 6GlcNAc
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
1
The abbreviations used are: AAL, A. aurantia lectin; r.m.s., root
mean square.

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Aleuria aurantia lectin is a fungal protein composed

of two identical 312-amino acid subunits that specifically recognizes fucosylated glycans. The crystal structure of the lectin complexed with fucose reveals that
each monomer consists of a six-bladed -propeller fold
and of a small antiparallel two-stranded -sheet that
plays a role in dimerization. Five fucose residues were
located in binding pockets between the adjacent propeller blades. Due to repeats in the amino acid sequence,
there are strong similarities between the sites. Oxygen
atoms O-3, O-4, and O-5 of fucose are involved in hydrogen bonds with side chains of amino acids conserved in
all repeats, whereas O-1 and O-2 interact with a large
number of water molecules. The nonpolar face of each
fucose residue is stacked against the aromatic ring of a
Trp or Tyr amino acid, and the methyl group is located
in a highly hydrophobic pocket. Depending on the precise binding site geometry, the - or -anomer of the
fucose ligand is observed bound in the crystal. Surface
plasmon resonance experiments conducted on a series
of oligosaccharides confirm the broad specificity of the
lectin, with a slight preference for Fuc12Gal disaccharide. This multivalent carbohydrate recognition fold is a
new prototype of lectins that is proposed to be involved
in the host recognition strategy of several pathogenic
organisms including not only the fungi Aspergillus
but also the phytopathogenic bacterium Ralstonia
solanacearum.

27060

Structural Basis of Fucose Binding by A. aurantia Lectin

TABLE II
Refinement statistics

TABLE I
Data collection and phasing statistics
Values in parenthesis refer to the highest resolution shell. Rmerge I
I/I, where I observed intensity. Rcullis(anom) r.m.s. (DANOcalc
DANOobs)/r.m.s. (DANOobs). Phasing power(anom) r.m.s. (DANOcalc)/r.m.s.
(DANOcalc DANOobs).
Mercury

Amino acids
Protein atoms
Solvent atoms
Sugar atoms
Resolution limits ()
Working set R (observation)
Test set R (observation)
Highest resolution shell
Working set R
Test set R
Cruickshanks DPI
Average Biso (2)
All atoms
Protein atoms
Solvent
Residues with incomplete
chain density

Native

ID141:ESRF
P21
2.0
2.072.00
0.934

ID141:ESRF
P21
1.5
1.551.50
0.934

46.9
86.2
78.2
90.0
94.2
90.0
615086
41982
14.7 (15.0)
8.6 (20.0)
6.9 (3.3)
99.9 (99.9)
99.9 (99.9)

46.0
86.4
77.8
90.0
90.6
90.0
534572
94102
3.6 (2.8)
4.9 (37.4)
8.4 (1.9)
96.8 (96.8)

11.9
1.67
0.77
0.36

14.4

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.
EXPERIMENTAL PROCEDURES

MaterialsAAL was purchased form from Vector Laboratories, Inc.

(Burlingame, CA). The sample was dissolved in water, and salts were
removed by ultracentrifugation using Nanosep 3K Omega (Pall Corp.,
Ann Arbor, MI) with washing by water. Lewis a (-L-Fuc-(134)[-DGal-(133)]-D-GlcNAc), Lewis X (-l-Fuc-(133)[-D-Gal-(134)]-D-GlcNAc), blood group A (-L-Fuc-(132)-[-D-GalNac-(133)]-D-Gal), blood
group B (-L-Fuc-(132)-[-D-Gal-(133)]-D-Gal), and blood group H type
II (-L-Fuc-(132)--D-Gal-(134)-D-GlcNAc) trisaccharides were purchased from Dextra Laboratories (Reading, UK). L-fucose, p-nitrophenyl--L-fucoside, p-nitrophenyl--L-fucoside, -L-Fuc-(132)-D-Gal, and
-L-Fuc-(132)--D-Gal-(134)-D-Glc were purchased from Sigma.
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 l 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 l of
AAL at a concentration of 10 mg/ml and L-fucose at a concentration of
137 g/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 0.3 0.05 mm3 after 1 week. They
belong to space group P21 with unit cell dimensions of a 45.97 , b
86.41 , c 77.85 , and 90.62 at 100 K. The asymmetric unit
accommodates two monomers, corresponding to a Vm of 2.20 3 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

Residues in alternative
conformations

Number of outliers on
Ramachandran plota
Overall G factora
Distance deviations
Bond distances ()
Bond angles (degrees)
Planar groups ()
Chiral volume
deviation (3)
a

Values

2 312
2436 A; 2457B
1166
2 5 11
19.96-1.50
0.144 (92,218)
0.179 (1884)
0.263 (5239)
0.273 (107)
0.067
12.8
A: 12.1, B: 16.7
32.8
A: Ala254, Lys296. B: Asp42,
Gln51, Glu56, Lys69, Gln101,
Lys108, Lys136, Lys152,
Val163, Lys188
A: Arg21, Leu59, Val81, Ser88,
Val109, Ser150, Ser207,
FUC1002.
B: Ser14, Val25, Leu59, Ser95,
Thr154, Asn197, Ser205,
Ser207, Ser283, Gln289,
Gln291, FUC1002.
0
0.0
0.013
1.556
0.008
0.165

Analyses were determined by PROCHECK (26).

beamline ID14 1 at the ESRF (Grenoble, France). Single wavelength

highly redundant anomalous diffraction data to 2.0- resolution were
collected from the thimerosal-soaked crystal and native data to 1.5-
resolution. Measurements were made at a single x-ray wavelength of
0.934 . 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.
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 REFMAC, including automatic water molecule placement using ARP/wARP,
manual rebuilding with O, and construction of alternative conforma2
C. Vonrhein, E. Blanc, P. Roversi, and G. Bricogne, manuscript in
preparation.

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Beamline
Space group
Resolution ()
Highest resolution shell ()
Wavelength ()
Cell dimensions
a ()
b ()
c ()
(degrees)
(degrees)
(degrees)
Total number of hkl
Number of unique hkl
Average multiplicity
Rmerge (%)
Average I/(I)
Completeness (%)
Completeness for anomalous
data (%)
Wilson B-factor (2)
Phasing power (anom) to 2.0
Rcullis (anom) to 2.0
Average figure of merit to 2.0

Parameters

Structural Basis of Fucose Binding by A. aurantia Lectin

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FIG. 1. A, ribbon diagram of monomer A of AAL complexed with fucose shown as sticks. B, Connolly surface of AAL color-coded according to the
hydrophobicity potential (from brown for hydrophobic to blue for hydrophilic) displayed with the MOLCAD program (50). The red balls represent
the water molecules present in the tunnel. C, dimer of AAL with stick representation of the amino acids involved in the interaction of monomers.
D, amino acid sequence of AAL shown as six aligned repeats. The four -strands are indicated by arrows. Residues with a colored background are
involved in the fucose binding sites. They are color-coded according to their blade as in A and are underlined when they participate in binding of
fucose in site i 1. Residues in red participate in the dimer interface. Residues in italic type line the central cavity of the monomer. Boxed residues
are hydrophobic amino acids that make van der Waals contacts between adjacent blades. E, stereoview of site 1 in monomer A with the final
-weighted 2Fo Fc electron density map around the fucose molecule. The density is contoured at 1.0 . All molecular drawings in all figures were
prepared with MOLSCRIPT (51) and RASTER-3D (52) unless otherwise indicated.

27062

Structural Basis of Fucose Binding by A. aurantia Lectin

TABLE III
Contacts (distances in ) between fucose and monomer A (roman characters) and monomer B (italic characters)
Conserved hydrogen bonds and hydrophobic contacts are in boldface type. The maximal error on non bonding distances is 0.13 as evaluated
from Cruickshanks DPI value based on the R factor (Table II).
Ligand atom

O-1

Site 1

W1 (2.7/2.9)
W2 (2.7/2.5)

O-1

Site 2

W1 (2.6/3.0)
W3 (3.1/2.9)

Site 3

Gln101.OE1 (3.2/2.6)
W1 (3.1/3.1)

O-2
W5 (2.8) W4 (2.8)

Gln101.N (3.0/3.1)c
Gln101.OE1 (2.8/3.3)c
Wat2 (2.7/2.9)c

Site 4

W1 (2.7/3.0)
W3 (3.1) W2 (2.8)

Site 5

W2 (2.7)
(Ser104.O - 3.1)a
Wat1 (2.7/2.7)
Wat3 (3.1)
Wat7 (3.2/3.0) (bridge)b

W3 (2.6/3.1)
W5 (2.8/3.0)
W6 (2.8/3.0)

Gly203.N (2.8/2.9)
W3 (2.7/2.7)
W5 (2.9)
(Gln72.OE12.75)d

W4 (2.7/2.9)
W5 (2.6)

Glu36.OE1 (2.7/2.7)
Trp97.NE1 (3.0/3.0)
W6 (3.0/2.9)

Glu89.OE1 (2.6/2.7)c
Trp153.NE1 (2.8/2.7)c

Glu146.OE1 (2.6/2.6)
Trp199.NE1 (2.9/3.0)
W6 (2.9/3.1)

Glu191.OE1 (2.6/2.6)
Trp245.NE1 (2.9/2.9)
W6 (2.8/2.8)

Glu238.OE1 (2.6/2.6)
Trp298.NE1 (2.9/2.9)
W6 (2.8/2.9)

O-4

Arg24.NE (2.8/2.8)
Glu36.OE2 (2.6/2.7)

Arg77.NE (3.0/3.0)c
Glu89.OE2 (2.7/2.7)c

Arg131.NE (2.8/2.8)
Glu146.OE2 (2.7/2.7)

Arg179.NE (2.8/2.9)
Glu191.OE2 (2.7/2.6)

Arg226.NE (2.8/2.8
Glu238.OE2 (2.6/2.5)

O-5

Arg24.NH2 (2.9/2.9)

Arg77.NH2 (2.9/3.1)c

Arg131.NH2 (2.9/2.9)

Arg179.NH2 (2.8/2.9)

Arg226.NH2 (2.9/2.9)

Hydrophobic
(C-4, C-5,
C-6)

Trp15

Trp68,

Trp120

Ile173

Trp219

Ile74
Ile76
Tyr92

Pro128
Ile130
Trp149

Leu178
Trp194

Pro223
Ile225
Tyr241

Ile274
Ile276
Trp292

Val91

C-1, C-2

Cys193

In monomer A only: hydrogen bond with A chain related by symmetry 1 x, y 1/2, 2 z.

Values averaged over the -Fuc and -Fuc in the site (less than 0.02- deviation).
c
In monomer B only: hydrogen bond with B chain related by symmetry x, y, z 1.
d
Water molecule bridging to Tyr241.OH.

a
b

tions, 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-
resolution. Side chain atoms not defined in electron density were retained in the model but with an occupancy set to zero (Table II).
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 l/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/Nhydroxysuccinimide solution for 10 min, and 50 l of AAL in 5 mM
maleate buffer, pH 7.0, at concentrations of 50, 10, and 2 g/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 l of carbohydrate solutions (concentrations between 0.39 and 200
M) 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.).
RESULTS

AAL Overall FoldThe six tandem repeats of the AAL

amino acid sequence are organized around a six-fold
pseudoaxis of symmetry (Fig. 1A). Each repeat consists of a
twisted four-stranded antiparallel -sheet, and the overall arrangement corresponds to the fold previously described as a
six-bladed -propeller (31, 32). The global shape is a short
cylinder, or tore, with an approximate diameter of 45 and a
height of 35 . In the -propeller fold, each consecutive -sheet
has its first strand (number 1) lining the central cavity of the
protein and the last one (number 4) most exposed to solvent at

the cylinder surface. Loops connecting the strands within each

module are rather short, with the exception of blade III (amino
acids 108 162) that displays longer loops (Fig. 1D). The consecutive blades are connected by long segments that run from
the outside of the protein to the central tunnel. Superposition of
the main chain -strand repeats gives r.m.s. values between
1.3 and 1.4 , indicative of their high spatial similarity.
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 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 Bfactor 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 . 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 mon-

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O-3

Structural Basis of Fucose Binding by A. aurantia Lectin

27063

omer 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 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.
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

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FIG. 2. Stereoscopic representation

of the fucose binding sites I, II, and
IV of monomer A. Hydrogen bonds are
displayed as dashed lines.

27064

Structural Basis of Fucose Binding by A. aurantia Lectin

FIG. 3. Superposition of all 10 binding sites, with only one

fucose represented. The seven clusters of water molecules have been
indicated.

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, 1 4,
and 1 6 positions all equally well (15, 18). In fact, the relatively
high affinity for fucose measured by equilibrium dialysis (Kd
16 M) (15) or by surface plasmon resonance experiments (Kd
33 M) (33) is not further increased when various fucose-containing disaccharides are tested (19). A high resolution NMR
study of free and AAL-bound Fuc1 6GlcNAc-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

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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.
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.

Structural Basis of Fucose Binding by A. aurantia Lectin

27065

FIG. 4. Sensorgram of the interaction of increasing amounts of -LFuc-(132)- -D-Gal to the immobilized AAL at 25 C. The sugar
concentration ranges from 0.39 M (lowest
curve) to 200 M (top curve). Each injection was of 360-s duration at a flow rate of
5 l/ml. Inset, Scatchard plot of the
sensorgram.

Ligand

KD

M
L-Fucose

-L-Fuc(13 2)-D-Gal
-L-Fuc(1 3 2)-D-Gal(1 4)-D-Glc
Blood group A trisaccharide: -L-Fuc-(1 3 2)-[-D-GalNAc-(1 3 3)]-D-Gal
Blood group B trisaccharide: -L-Fuc-(1 3 2)-[-D-Gal-(133)]-D-Gal
Blood group H type II trisaccharide: -L-Fuc-(1 3 2)--D-Gal-(1 34)-D-GlcNAc
Lewis a trisaccharide: -L-Fuc-(1 34)[-D-Gal-(1 33)]-D-GlcNAc
Lewis X trisaccharide: -L-Fuc-(1 33)[-D-Gal-(1 34)]-D-GlcNAc
p-Nitrophenyl--L-fucoside
p-Nitrophenyl--L-fucoside

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.
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.
DISCUSSION

Comparison with Other -Propeller ProteinsThe modular

organization of -propeller proteins can create symmetry ranging from 4- to 8-fold. They have a cylindrical/disk shape in
common and a high structural rigidity. The -propellers have
extreme diversity in function and are found in many organisms
such as viruses, bacteria, and eukaryotes (31, 32, 35). For a
given number of blades, there are no sequence similarities
between the different members of the family. Interestingly, a

24.1 1.7
11.7 1.0
102.9 4.3
20.5 1.4
23.4 2.3
59.0 4.7
89.5 6.3
103.9 4.4
43.1 3.1
93.0 8.3

KA
104

4.15
8.55
0.97
4.88
4.27
1.69
1.12
0.97
2.32
1.08

rather high number of -propellers with known crystal structures are carbohydrate-active enzymes, such as the five-fold
-L-arabinanase (36), the six-fold sialidase/neuraminidase (37)
and glucose dehydrogenase (38), and the seven-fold galactose
oxidase (39). The only lectin that has been identified as a
-propeller is tachylectin-2 (40). It consists of five blades with
highly similar repeats and five GlcNAc-binding sites located
between the blades. When comparing overall structures and
carbohydrate binding sites, there are no similarities between
AAL and tachylectin-2 (Fig. 5). The overall shape of tachylectin-2 is disklike, and the amino acids involved in the carbohydrate binding sites are located in long loops and not in
-strands as in AAL. It therefore seems that there is no phylogenic relationship between these two lectins.
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) and Micromonospora viridifaciens sialidase (44) (Protein Data Bank code 1EUT) superimpose on the AAL main
chain C- coordinates with r.m.s. values of 2.35 for 212 amino
acids and 2.38 for 206 amino acids, respectively. An almost
identical superimposition (2.49 for 208 amino acids) was
obtained for the eukaryotic trans-sialidase from leech (45) (Protein Data Bank code 2SLI). 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

Downloaded from http://www.jbc.org/ at University of Connecticut Health Center Library on June 22, 2015

TABLE IV
Equilibrium dissociation and association constants for the interaction between fucose and AAL measured by SPR experiments

27066

Structural Basis of Fucose Binding by A. aurantia Lectin

3
Ishimaru, T., Kubai, S., Bernard, E. M., Tamada, S., Tong, W.,
Soteropuolos, P., Perlin, D. S., and Armstrong, D., SWISS-PROT, deposition Q8NJT4.

(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 fucosebearing blood group oligosaccharides. Interestingly, the 91amino 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.
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

FIG. 5. Orthographic representation of different -propellers

with ligand represented as sticks. A, AAL-fucose complex. B, S.
thyphimurium sialidase-sialic acid derivative complex (Protein Data
Bank code 2SIM). C, tachylectin-2-GlcNAc complex (Protein Data Bank
code 1TL2).

FIG. 6. Alignment of amino acid sequence repeats from AAL, N-terminal sequence of M. chateri lectin (MCL), A. fumigatus lectin
(AFL), A. oryzae lectin (AOL), and R. solanacearum lectin (RSL). Amino acids that are conserved in fucose binding sites and could therefore
be interacting with the ligand are displayed on black and gray background for establishing hydrogen bonds or hydrophobic contacts, respectively.

Downloaded from http://www.jbc.org/ at University of Connecticut Health Center Library on June 22, 2015

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 310amino 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

Structural Basis of Fucose Binding by A. aurantia Lectin

that the existing -propellers have been formed by modular
duplication of a four-stranded sheet motif (31).
AcknowledgmentsWe thank the ESRF, Grenoble, for access to synchrotron data collection facilities. H. Lortat-Jacob (IBS, Grenoble) is
acknowledged for access and help with the use of Biacore. We thank
Prof. N. Gilboa-Garber (Bar-Ilan University, Israel) for bringing our
attention to AAL and for helpful scientific discussion and correction of
the manuscript.
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27067

Michaela Wimmerova, Edward Mitchell,

Jean-Frederic Sanchez, Catherine Gautier and
Anne Imberty
J. Biol. Chem. 2003, 278:27059-27067.
doi: 10.1074/jbc.M302642200 originally published online May 5, 2003

Access the most updated version of this article at doi: 10.1074/jbc.M302642200

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Glycobiology and Extracellular Matrices:

Crystal Structure of Fungal Lectin:
SIX-BLADED -PROPELLER FOLD
AND NOVEL FUCOSE RECOGNITION
MODE FOR ALEURIA AURANTIA
LECTIN