Báo cáo khoa học: Structural analysis of the jacalin-related lectin MornigaM from the black mulberry (Morus nigra) in complex with mannose ppt

Structural analysis of the jacalin-related lectin MornigaM
from the black mulberry (Morus nigra) in complex with
mannose
Anja Rabijns
1
, Annick Barre
2
, Els J. M. Van Damme
3
, Willy J. Peumans
3
, Camiel J. De Ranter
1
and Pierre Rouge
´
2
1 Laboratory of Analytical Chemistry and Medicinal Physicochemistry, Faculty of Pharmaceutical Sciences, K. U. Leuven, Belgium
2 Surfaces Cellulaires et Signalisation chez les Ve
´
ge
´
taux, UMR-CNRS 5546, Po
ˆ
le de Biotechnologie ve
´
ge
´
tale, Castanet-Tolosan, France
3 Department of Molecular Biotechnology, Ghent University, Belgium
During the last decade numerous lectins that are struc-
turally related to jacalin, the Gal ⁄ GalNAc-specific lec-

tin from jackfruit (Artocarpus integrifolia) seeds [1],
have been isolated and characterized from plants
belonging to taxonomically distant families [2–13]. At
present, jacalin-related lectins are subdivided on the
basis of their apparent monosaccharide-binding speci-
ficity in Gal-specific (gJRL) and Man-specific jacalin-
related lectins (mJRL). Hitherto, gJRL have been
identified in only a few species of the family Moraceae
such as Osage orange (Maclura pomifera) (MPA), jack
fruit (Artocarpus integrefolia) (jacalin) and a few other
Artocarpus species, and in black mulberry (Morus
nigra) (MornigaG). In contrast to the gJRL, mJRL are
not confined to the Moraceae family but are wide-
spread among plants as is illustrated by the isolation
and characterization of such lectins from jack fruit
(artocarpin) and mulberry (MornigaM), banana (Musa
acuminata; Musaceae), hedge bindweed (Calystegia
sepium; Convolvulaceae), bindweed (Convolvulus arven-
sis; Convolvulaceae), Jerusalem artichoke (Helianthus
tuberosus; Asteraceae), Parkia platycephala (Fabaceae:
Mimosoideae subfamily), Japanese chestnut (Castanea
crenata; Fagaceae), rice (Oryza sativa; Poaceae) and
Keywords
carbohydrate-binding site; jacalin-related
lectin; mannose; Morus nigra; quaternary
association
Correspondence
P. Rouge
´
, Surfaces Cellulaires et

Signalisation chez les Ve
´
ge
´
taux, UMR-CNRS
5546, Po
ˆ
le de Biotechnologie ve
´
ge
´
tale, 24
Chemin de Borde Rouge, 31326 Castanet-
Tolosan, France
Fax: +33 05 62 19 35 02
E-mail:
(Received 22 March 2005, revised 25 April
2005, accepted 31 May 2005)
doi:10.1111/j.1742-4658.2005.04801.x
The structures of MornigaM and the MornigaM–mannose complex have
been determined at 1.8 A
˚
and 2.0 A
˚
resolution, respectively. Both struc-
tures adopt the typical b-prism motif found in other jacalin-related lectins
and their tetrameric assembly closely resembles that of jacalin. The carbo-
hydrate-binding cavity of MornigaM readily binds mannose. No major
structural rearrangements can be observed in MornigaM upon binding of
mannose. These results allow corroboration of the structure–function rela-

tionships within the small group of Moraceae lectins.
Abbreviations
BanLec, banana (Musa acuminata) lectin; Calsepa, Calystegia sepium (hedge bindweed) agglutinin; Conarva, Convolvulus arvensis
(bindweed) agglutinin; Heltuba, Helianthus tuberosus (Jerusalem artichoke) agglutinin; JRL, jacalin-related lectin; gJRL, galactose-specific
jacalin-related lectin; mJRL, mannose-specific jacalin-related lectin; MPA, Maclura pomifera (Osage orange) agglutinin; MornigaG, Gal-specific
Morus nigra (black mulberry) agglutinin; MornigaM, Man-specific Morus nigra (black mulberry) agglutinin; Orysata, rice (Oryza sativa)
agglutinin.
FEBS Journal 272 (2005) 3725–3732 ª 2005 FEBS 3725
the true fern Phlebodium aureum (Polypodiaceae). The
subdivision in gJRL and mJRL was initially proposed
on the basis of the nominal specificity towards mono-
saccharides but is also in perfect agreement with a
classification of the JRL according to the overall struc-
ture of their corresponding genes. More recent specific-
ity studies indicated that at least within the family
Moraceae the differences in specificity are not so a
clear-cut. It has been demonstrated, indeed, that the
presumed T-antigen ⁄ GalNac-specific jacalin behaves as
a polyspecific lectin capable of interacting ) albeit with
a (much) lower affinity ) with many other sugars
including Man, Gluc, Neu5Ac or MurNAc [14,15]. In
this respect, the mJRL isolated from other plants
families such as, e.g. Calsepa from Calystegia sepium
(Convolvulaceae) and Heltuba from Helianthus tubero-
sus (Asteraceae), which exhibit an exclusive specificity
towards mannose and do not interact with unrelated
sugars like Gal or GalNAc [7,16] differ from jacalin.
However, these available data on the specificity of JRL
offer no explanation for the dramatic differences in the
agglutination activity between the mJRL of different

origin. All mJRL studied thus far are very weak agglu-
tinins as compared to the gJRL (more than three
orders of magnitude less potent) except MornigaM
which is nearly as potent as jacalin. Because one can
reasonably assume that the exceptionally high agglu-
tinating activity of MornigaM is intimately linked to
its sugar binding properties the three-dimensional
structure of MornigaM in complex with a simple sugar
was determined to decipher the structural features
responsible for the unusual enhanced activity of this
Moraceae lectin.
Results
Quality and overall view of the structure
The final model obtained for uncomplexed MornigaM
converged to an R-factor of 19.0% and an R-free
value of 21.0% (Table 1). It comprises four MornigaM
subunits with 154 out of the total 161 amino acid resi-
dues, 575 water molecules, three acetate molecules,
four glycerol molecules and one sulfate molecule. The
first seven N-terminal residues are not seen in the elec-
tron density map and are not included in the model.
The model has a good geometry and has r.m.s. devia-
tions from ideal bond lengths and angles of 0.005 A
˚
and 1.45°, respectively. All residues were found in the
most favoured (85.7%) and generously allowed regions
(14.3%) of the Ramachandran plot. None of the resi-
dues was found in the disallowed regions. The average
temperature factor for the main-chain and side-chain
atoms are 17.6 A

˚
2
and 19.9 A
˚
2
, respectively. In total
12 cis-peptide bonds were found in the structure (three
per monomer: Leu21, Pro85 and Pro123). Further
refinement statistics are given in Table 1 for both the
uncomplexed and Man-complexed MornigaM.
The MornigaM protomer exhibits the b-prism fold
typically found in the JRL family (Fig. 1). It consists
of three four-stranded b-sheets forming three Greek
keys motifs: 1 (b1,b2,b11,b12), 2 (b3, b4, b5,b6) and
3(b7,b8,b9b10) (Fig. 2A,B). Although the single-chain
MornigaM protomer definitely differs from jacalin by
the lack of a proteolytic cleavage of its protomer into
an a and a b chain, it nicely superimposes on the simi-
larly folded two-chain jacalin protomer and other
single-chain Man-specific protomers of Heltuba or
artocarpin.
MornigaM adopts a homotetrameric quaternary
arrangement very similar to that of many other JRL
like jacalin [1], MPA [17] and artocarpin [18]
(Fig. 2C,D). All four monomers within the tetramer
superimpose well with r.m.s. differences ranging
between 0.20 A
˚
and 0.47 A
˚

. The largest structural dif-
ference observed between the four protomers is the
different conformation of loop 99–107 (L2) in the A
protomer as compared to its conformation in the B, C
and D protomers of the tetramer. This loop forms
the roof of the sugar binding pocket of MornigaM
and hence its different orientation might be related
to the observed lack of binding of mannose in the A
Table 1. Crystallographic data for MornigaM and the MornigaM–
Man complex.
Parameter MornigaM
MornigaM–
Man complex
Data collection:
Space group P65 P65
Used wavelength (A
˚
) 0.912 0.910
Used beam line X11 X11
Resolution limit (A
˚
) 1.8 (1.83–1.80) 2.0 (2.03–2.00)
Total observations 250868 211071
Unique reflections 100939 (5072) 74409 (3763)
Completeness of all data (%) 98.4 (99.7) 99.1 (100.0)
Completeness of
data (%) (I >2r)
90.6 (70.4) 89.8 (71.7)
Mean I ⁄ r 20.8 (5.0) 21.3 (4.9)
R

sym
value (%) 4.2 (16.0) 4.5 (18.9)
Refinement:
R-work (%) 19.0 19.1
R-free (%) 21.0 21.0
Atoms in protein ⁄ solvent 4640 ⁄ 616 4640 ⁄ 583
Mean B in protein ⁄ solvent (A
˚
2
) 18.7 ⁄ 27.6 20.4 ⁄ 29.0
r.m.s.d. bond lengths (A
˚
) 0.005 0.005
r.m.s.d. bond angles (°) 1.45 1.42
Structure of a jacalin-related lectin complexed to mannose A. Rabijns et al.
3726 FEBS Journal 272 (2005) 3725–3732 ª 2005 FEBS
protomer (see below). Furthermore, a minor structural
heterogeneity can be observed in the N-terminal region
of the different protomers. Like jacalin, the MornigaM
homotetramer exhibits a strong electronegative surface
(Fig. 2E,F). The carbohydrate-binding cavities are
electronegatively charged due to the Asp153 residue
that occupies the centre of the cavity.
The carbohydrate-binding site
Comparison of the backbone atoms of the uncom-
plexed and Man-complexed MornigaM structures
shows that no significant structural rearrangements
occur upon mannose binding. The r.m.s. deviation
between both structures is only 0.11 A
˚

, based on the
superposition of the Ca atoms. Except for protomer
A, a clear density is seen for the bound mannose mole-
cule in the carbohydrate-binding site of protomers B,
C and D which is composed of three convergent loops
connecting strands b1tob1 (loop 1), b7tob8 (loop 2)
and b11 to b12 (loop 3), located at the top of the pro-
tomers (Fig. 3A). Man is specifically anchored to resi-
dues Gly27 (loop 1), Phe150-Val151-Asp153 (loop 3)
by a network of 8 hydrogen bonds (Table 2) with oxy-
gens O3, O4, O5 and O6, respectively (Fig. 3B).
Depending on the protomer, Gly27N interacts (pro-
tomers C and D) or does not interact (protomer B)
with O4 of Man. An additional stacking between the
aromatic ring of Phe150 and the pyranose ring of Man
reinforces the interaction of MornigaM with the sugar.
A very similar H-bond network was observed in the
previously X-ray solved MeMan–artocarpin [18] and
Man–Heltuba [19] complexes. Superposing the Ca of
the amino acid residues forming the monosaccharide-
binding site of MornigaM with those of Heltuba and
artocarpin yielded r.m.s. values of 0.263 A
˚
and
0.142 A
˚
, respectively, thus indicating that all these resi-
dues occupy very similar positions in all the mJRL. In
fact, rather close r.m.s. of 0.432 A
˚

and 0.362 A
˚
were
measured with the corresponding amino acid residues
forming the Gal ⁄ GalNAc-binding site of jacalin (PDB
code 1JAC) and MPA (PDB code 1JOT), suggesting a
similar topology for the monosaccharide-binding site
of the gJRL that apparently accounts for the promis-
cuous character of this gJRL.
The size and shape of the carbohydrate-binding site
of MornigaM and other JRL essentially depends on
both the conformation of the three loops that delineate
the binding cavity and the presence therein of amino
acid residues with bulky side chains. The carbohy-
drate-binding cavity of MornigaM is largely widened
between loops 2 and 3 but its opening on the other
side, between loops 1 and 2, is strongly restricted by
the side chain of Lys106 which protrudes from loop 2
to close up the cavity. These structural discrepancies
of the carbohydrate-binding cavity should have a pro-
found influence on the type of oligosaccharides that fit
in the site of the JRL.
Discussion
Resolution of the three-dimensional structure of apo
MornigM X-ray at 1.8 A
˚
revealed a homotetrameric
organization similar to those found for jacalin and all
Moraceae JRL studied thus far but not for any other
JRL. Resolution of the structure of a MornigaM ⁄ Man

Fig. 1. Sequence and structure comparison of MornigaM to other jacalin-related lectins. (A) Sequence alignment of MornigaM with jacalin as
a member of the gJRL group and Heltuba and artocarpin as members of the mJRL group. Identical residues are coloured white with a black
background and similar residues are coloured black and open boxed. The amino acid residues forming the monosaccharide-binding site are
indicated by stars. b-Strands forming the Greek keys 1 (b1,b2,b11,b12), 2 (b3-b6) and 3 (b7-b10) of jacalin (upper arrows) and MornigaM
(lower arrows) protomers are indicated.
A. Rabijns et al. Structure of a jacalin-related lectin complexed to mannose
FEBS Journal 272 (2005) 3725–3732 ª 2005 FEBS 3727
complex at 2.0 A
˚
further demonstrated that a network
of seven or eight hydrogen bonds anchors O3, O4, O5
and O6 of Man to residues Gly27, Phe150, Val151 and
Asp153 (all of which protrude from loops 1 and 2 that
delineate the carbohydrate-binding cavity of the lectin
protomer). A closer examination of the structure of
the binding sites indicates that specificity of the Mora-
ceae lectins is primarily determined by the orientation
of the side chains of the four amino acid residues resi-
dues that form the monosaccharide-binding site of the
lectins. An r.m.s. of only 0.2–0.4 A
˚
was measured
when superimposing the Ca of these four residues
from different Moraceae JRL, which implies that there
is very little structural tolerance at the level of their
respective monosaccharide-binding sites. This holds
true particularly for the Gly residue, which is either
free at the N-terminus of the large polypeptide of the
two-chain gJRL (in casu jacalin, MPA and MornigaG)
or located around position 10–20 in the uncleaved pro-

tomer of the mJRL (in casu MornigaM and artocar-
pin). Due to this strictly conserved orientation the Gly
residue of both types of Moraceae JRL can interact
with both the equatorial (Gal ⁄ GalNAc) and axial
(Man ⁄ Glc) O4 of monosaccharides. It should be
emphasized, however, that in spite of this particular
structural feature jacalin exhibits a preferential specific-
ity for Gal, GalNAc and the T antigen, and binds
other sugars with a much lower affinity.
As with most other plant lectins the reactivity of
MornigaM and other JRL is not limited to simple
sugars but also extends to disaccharides and more
complex oligosaccharides [20–22]. Structural analyses
indicated that the carbohydrate-binding cavity of
MornigaM is sufficiently extended to accommodate
more bulky saccharides and that the atomic structure
of this cavity accounts for the oligosaccharide-bind-
ing specificity of each of these lectins. It is worth
mentioning in this context that loop 2, which forms
the roof of the carbohydrate-binding cavity in all the
N
N
β5
β4
β2
β12
β11
β8
β3
β6

β9
β7
β10
β1
AB
CD
EF
Fig. 2. Structure and surface analysis of MornigaM and jacalin. (A,
B) Ribbon diagrams showing the arrangement of the 12 b-strands
of the MornigaM protomer in three Greek keys 1, 2 and 3 coloured
orange (strands b1, b2, b11, b12), blue (strands b3-b6) and pink
(strands b7-b10), respectively. N and C indicate the N- and C-ter-
mini of the MornigaM polypeptide, respectively, and the stars indi-
cate the location of the monosaccharide-binding site. (C, D)
Tetrameric arrangement of the protomers of MornigaM (C) and jac-
alin (D). The b-chains of the jacalin protomers are coloured cyan.
The carbohydrate-binding sites are indicated by stars. (E, F) Mole-
cular surface of the MornigaM (E) and jacalin (F) tetramers showing
the distribution of the electrostatic potentials. The negative poten-
tial is coloured red and displayed at )5 kT level, the positive poten-
tial is colored blue and displayed at +5 kT level (1 kT ¼ 0.6 kcals).
Neutral surfaces are white. The electrostatic potentials were calcu-
lated and displayed with
GRASP [37].
AB
Fig. 3. The carbohydrate-binding site of MornigaM and other jac-
alin-related lectins. (A) Ribbon diagram of the carbohydrate-binding
site of MornigaM showing the three loops L1 (grey), L2 (pale grey)
and L3 (mid grey) forming the carbohydrate-binding cavity. Man-
nose (in grey ball-and-sticks) occupies the monosaccharide-binding

site. (B) Network of hydrogen bonds (dashes) anchoring Man to
the amino acid residues of the monosaccharide-binding site of
MornigaM.
Structure of a jacalin-related lectin complexed to mannose A. Rabijns et al.
3728 FEBS Journal 272 (2005) 3725–3732 ª 2005 FEBS
Moraceae JRL plays a key role in the delineation of
the size and shape of the binding site. The presence
in loop 2 of bulky amino acid residues has appar-
ently no effect on the accessibility of monosaccha-
rides but could dramatically reduce the accessibility
of the carbohydrate-binding cavity for oligosaccha-
rides. This observation is in good agreement with
the recent suggestion (based on a database analysis
of jacalin-like lectins) that loop 2 is a very important
structural feature in determining the oligosaccharide-
binding specificity of JRL [23].
Experimental procedures
Isolation of the lectin
MornigaM was isolated from mulberry (Morus nigra) bark
by affinity chromatography on Man-Sepharose 4B, as pre-
viously described [11]. Three successive rounds of affinity
chromatography were performed to ensure the purity of the
Man-specific lectin. The lectin preparation gave a single
protein band when checked by SDS ⁄ PAGE.
Crystallization and structure resolution
Crystals suitable for diffraction analysis were grown from a
55% saturated ammonium sulfate solution containing 0.1 m
imidazole buffer pH 7.0 as described elsewhere [24]. Subse-
quently, crystals of the complex between MornigaM and
Man were prepared by soaking an uncomplexed MornigaM

crystal with a 50 mm Man solution for approximately 48 h.
Both the uncomplexed and complexed crystals could be
cryoprotected by soaking the crystals in the original crystal-
lization condition with 25% glycerol for  30 s. Soaking
experiments performed under similar conditions with Gal
remained unsuccessful.
Data collection with cryo-cooling at 100 K was carried
out on the native and soaked crystals at the X11 beam line
of the DESY synchrotron in Hamburg. All data processing
was done using denzo and scalepack [25]. The space
group was assigned to be P6
5
with a ¼ b ¼ 110.7, c ¼
159.2 A
˚
. Checking of the diffraction data at the twin server
[26] showed that all measured crystals were partially mero-
hedrally twinned with variable twin fractions ranging from
11% to 35%. The twin fractions for the data sets used
for final structure determination of MornigaM and of
MornigaM–Man were 14.5% and 18.7%, respectively. Both
data sets were detwinned using the algorithm described by
Yeates [26], yielding data sets in which all reflections had
their twin component removed. Data collection statistics
for both data sets are given in Table 1.
The phase problem for the MornigaM structure was
solved by the molecular replacement technique in x-plor
[27] using the detwinned data. The coordinates of half a
jacalin molecule (chains A and C, PDB code 1JAC) were
used as a search model for the uncomplexed MornigaM

structure. Two dimers were easily found and combining
them into a tetramer gave an R factor of 46% (R-free
47%) after initial rigid body refinement to 3 A
˚
. Based on a
calculated Matthews coefficient of 2.35 A
˚
3
ÆDa
)1
[28], it was
assumed that the asymmetric unit consisted of eight mono-
mers. However, in the molecular replacement search only a
single tetramer was found. The corresponding solvent con-
tent for the crystals is 73%.
Refinement was performed by the cns package [29] using
torsional angle dynamics and individual B-factor refine-
ment. A randomly selected 10% of the data sets were set
aside for cross-validation using the R-free value. Bulk sol-
vent correction was used. Solvent molecules were progres-
sively added when they met the following requirements: (a)
a minimum 3 r peak had to be present in the |F
obs
|-|F
calc
|
difference map; (b) a peak had to be visible in the 2|F
obs
|-
|F

calc
| map; (c) during refinement the B-value for the water
molecule should not exceed 60 A
˚
2
; and (d) the water mole-
cule had to be stabilized by hydrogen bonding. Refinement
was always performed against detwinned data; different
twin fractions were tested and used to optimize the refine-
ment. Eventually twin fractions of 11.60% and 14.74%
Table 2. List of hydrogen bonds connecting the mannose to the different residues of the MornigaM tetramer.
Mannose A B C D
O1 – – H
2
O21: 2.84 A
˚
H
2
O572: 2.67 A
˚
O2 – – – –
O3 – Gly27N: 3.17 A
˚
Gly27N: 2.84 A
˚
Gly27N: 2.85 A
˚
O3 – – H
2
O475: 2.76 A

˚
–
O4 – H
2
O257: 2.57 A
˚
H
2
O174: 2.57 A
˚
H
2
O325 : 2.55 A
˚
O4 – Asp153Od1: 2.47 A
˚
Asp153Od1: 2.58 A
˚
Asp153Od1: 2.51 A
˚
O5 – Phe150N: 3.00 A
˚
Phe150N:3.06 A
˚
Phe150N: 2.99 A
˚
O6 – Asp153Od2: 2.57 A
˚
Asp153Od2 : 2.70 A
˚

Asp153Od2: 2.72 A
˚
O6 – Val151O: 3.14 A
˚
Val151O:3.19 A
˚
Val151O: 3.19 A
˚
O6 – Val151N: 3.05 A
˚
Val151N:3.11 A
˚
Val151N: 2.97 A
˚
O6 – Phe150N: 2.90 A
˚
Phe150N:2.90 A
˚
Phe150N:2.79 A
˚
A. Rabijns et al. Structure of a jacalin-related lectin complexed to mannose
FEBS Journal 272 (2005) 3725–3732 ª 2005 FEBS 3729
gave the best result for the MornigaM and the MornigaM–
Man structure refinement, respectively. Visual inspection
and model building were done in O [30]. Electron and dif-
ference density maps were used to confirm the presence of
the mannose molecule in the structure of the MornigaM–
Man complex. To accurately fit the electron density map
several amino acids in primary sequence deposited in the
SWISS-PROT database (accession number Q8LGR3) had

to be changed. These changes include V17I, P100A and
V157F. The observed differences are probably due to the
(documented) heterogeneity of MornigaM. The presence of
the replaced amino acid residues, the water molecules and
the ions were all checked in simulated annealing omit maps.
Assessment of the quality of the coordinates was done with
the programs procheck [31] and moleman [32]. The
coordinates and structure factors of the MornigaM and
the MornigaM–Man structure have been deposited in the
Protein Data Bank [33] with the codes 1XXQ and 1XXR,
respectively. Ribbon diagrams of MornigaM and other
JRL were drawn with molscript [34] and rendered with
bobscript [35] and raster3d [36].
Molecular surface analysis
Molecular surface and electrostatic potentials were calcula-
ted and displayed with grasp using the parse3 parameters
[37]. The solvent probe radius used for molecular surfaces
was 1.4 A
˚
and a standard 2.0 A
˚
Stern layer was used to
exclude ions from the molecular surface [38]. The inner and
outer dielectric constants applied to the protein and the sol-
vent were, respectively, fixed at 4.0 and 80.0, and the cal-
culations were performed keeping a salt concentration of
0.145 m. No even distribution of the net negative charge of
the carboxylic group of negatively charged residues was
performed between their two oxygen atoms prior to the cal-
culations. Surface topology of the carbohydrate-binding

sites was rendered and analysed with pymol (W.L. DeLano,
).
Sequence comparison and alignment
The program espript [39] was used to compare the amino
acid sequence of MornigaM to other JRL sequences
(Fig. 1A). Multiple amino acid sequence alignments were
based on clustal x [40].
Acknowledgements
A.R. is a Postdoctoral Research Fellows of the Fund
for Scientific Research-Flanders (Belgium). The finan-
cial support of CNRS is gratefully acknowledged (A.B.
and P.R.). We thank the beam line scientists at DESY
for technical support and the European Union for sup-
port of the work at EMBL Hamburg through the
HCMP to Large Installations Project, contract no.
CHGE-CT93-0040.
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