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INTRODUCTION |
The disaccharide structure Gal
1,3GalNAc
occurs on the
surface of tumor cells as a mucin-associated antigenic marker, termed the Thomson-Friedenreich or T-antigen. It is one of the few chemically well defined tumor antigens with a proven association with malignancy (1). The disaccharide is also the central element in
O-linked glycopeptide structures on other mammalian
glycoproteins. This structure can be recognized by several seed lectins
from different plant families, including two that have been
structurally characterized in complexes with the disaccharide, peanut
agglutinin (2) and amaranthin (3). For a lectin to be useful as a
detection agent for the T-antigen, it must show low affinity for
possible cross-reactive structures on cells. These include the
Gal
1,3GalNAc
moiety of gangliosides,
N-acetyllactosamine and sialylated forms of the T- and
Tn-antigens. The peanut agglutinin has recently been engineered to
improve its specificity (4).
Particularly high specificity for the T-antigen is shown by two lectins
from the Moraceae plant family, jacalin (from Artocarpus integrifolia) and Maclura pomifera agglutinin
(MPA).1 They show low
affinity for
-galactopyranosides (5, 6), including
N-acetyllactosamine, and a >1000-fold preference for Gal
1,3GalNAc
Me (Ka for jacalin, 4 × 105 M
1) compared with the
-glycoside (6). They also bind strongly the methyl and
p-nitrophenyl
-glycosides of Gal and GalNAc, with Ka values of of 0.2-1 × 105
M
1 (5-7). MPA and jacalin are homotetrameric
proteins comprising subunits with 133 residue
-chains and remarkably
small
-chains of 20-21 residues (7-9). For jacalin, it has been
shown that the chains arise from a precursor protein by cleavage of an
N-terminal 39-residue piece and excision of a four-residue linker that
joins the
-chain to the
-chain (10). These lectins show no
sequence homology to any other plant lectin families, such as the
legume lectins, wheat germ agglutinin, or ricin. Two other members of the MPA/jacalin family have recently been reported, one being a
mannose-specific lectin also from A. integrifolia (11). The second is a lectin from a taxonomically distinct source, the rhizomes of Calystegia sepium (12), with specificity for mannose and maltose. Both these lectins are single-chain proteins.
Overall, MPA and jacalin are 85% homologous (8), but each displays
both genetic and post-translational heterogeneity (9). Despite this,
both have been crystallized without fractionation of the isoforms
(13-14). However, MPA has fewer genetic forms, two (as compared with
at least four for jacalin), no glycopeptide, and a dominant
-chain
isoform (9). It therefore offers better prospects for structure
determination at high resolution. The structure of jacalin complexed
with the monosaccharide ligand methyl
-D-galactopyranoside has recently been reported (15). The basic architecture of the jacalin monomer units was a
-prism with internal three-fold pseudo-symmetry. We now report the structure of MPA complexed with the biologically more important ligand, the
T-antigen disaccharide. This structure was solved by a combination of
multiwavelength anomalous diffraction (MAD) and molecular replacement techniques, as heavy atom derivatives proved difficult to obtain.
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EXPERIMENTAL PROCEDURES |
Crystallization of MPA with Gal
1,3GalNAc--
The complex was
crystallized by the sitting drop method of vapor diffusion as reported
previously (7). The data set used for the structure determination was
collected using a San Diego Multiwire type area detector (Table I).
Derivative Search--
Soaking the above crystals with heavy
atom compounds or co-crystallization all failed to yield heavy atom
derivatives. Co-crystallization of the native protein with heavy atom
compounds was then attempted. A new crystal form of C222 was obtained
with p-hydroxymercurisulfonic acid. Hanging drops were used
and the reservoir contained a mixture of 0.5 M
Li2SO4, 12% polyethyleneglycol 8000, and 1%
octyl-
-D-glucopyranoside buffered to pH 7.0 with 0.1 M Hepes. The concentration of the protein was 28 mg/ml, and
it was mixed with twice the stoichiometric amount of
p-hydroxymercuriphenylsulfonic acid. However, the phasing power of the mercury based on a MAD data set was very low, probably due
to disorder of the mercury atoms in the crystals. These crystals were
then used in soaking experiments, and a successful MAD candidate was
obtained with 1 mM trimethyl-lead acetate solution (Table I). The crystals showed a weak absorption edge of lead when checked with synchrotron radiation in National Synchrotron Light Source, Brookhaven National Laboratory. They were flash-frozen in liquid nitrogen with buffer containing 15% ethylene glycol.
MAD Data Collection and Data Processing--
A flash-frozen
crystal was used for the data collection on BM14 at European
Synchrotron Radiation Facilities (ESRF), Grenoble, France. Six data
sets were collected at six wavelengths, three around the absorption
edge of mercury and three at that of lead. These wavelengths were
chosen to minimize the f' and maximize the f"
differences at the mercury and lead edges and to give both high energy
and low energy "remote" wavelengths. No evidence of mercury
anomalous scattering was seen, but the lead anomalous signal was very
clear. A 1° range was used for each oscillation, and a total of
180° was collected at each wavelength. A 2.9-Å data set was
collected using the ESRF Image Intensified CCD (II/CCD) detector (3.4-s
readout time, 1242 × 1152 pixels). The data were corrected for
spacial distortion and nonuniform response using the program FIT2D (16)
and integrated using DENZO (17). Scaling was performed with SCALEPACK
(17), keeping Bijvoet mates separate. The data were of very high
quality with highest Rsymm of 2.6% overall to
2.9 Å, and under 10% for the highest shells (3.0-2.9 Å). The
Rsymm clearly varied slightly with the presence
of anomalous signal. The completeness and redundancy of this MAD data
set were high (Table I), with the most
complete data set having 96.7% completeness and the least 95.6%. An
average redundancy is 6.5 (Bijvoet pairs included, i.e. a
redundancy of 3, counting Bijvoets as separate).
MAD Phasing--
The CCP4 program suite (18) was used for
further processing; interwavelength scaling was performed with SCALEIT,
and the anomalous and dispersive Pattersons were calculated with FFT. The anomalous Patterson maps were easily interpreted by hand
calculation, yielding two lead sites, which were refined using the
program VECREF (18). The dispersive Patterson showed exactly the same sites. The peaks corresponding to the two sites (Pb1 = 0.145, 0.214, and 0.217; Pb2 = 0.250, 0.180, 0.107) in the anomalous Patterson were 11.9 and 10.0
above the background. MLPHARE was used
for the phasing using initial input f' and f"
values extracted from the measured absorption spectra using the
programs CROMER,2
XASFIT,3 and
KRAMIG.3 The point of inflection was chosen as the
reference wavelength. Phasing was attempted at 2.9-Å resolution and
yielded an electron density map with a figure of merit of 73%,
excellent phasing power, and low Cullis R factor (Table
II). DM was used for solvent flattening and histogram matching to improve the electron density. The figure of
merit after density modification improved to 84%. The map was displayed with the program O (19) and was of high quality and readily
interpretable. A difference Fourier calculated using the phases
obtained from the lead derivative did not display other significant
sites.
Tracing of the Molecular Backbone--
The program O (19) was
used to trace the C
backbone of the molecule with the command BATON.
Since the map was of excellent quality and there is a single Trp in
each of the two chains, tracing of the whole molecule was readily
achieved. Two 
subunits of MPA were found in the asymmetric unit.
The side chains of the molecules were fitted in subsequently. The model
used for the molecular replacement search was based on the better of
the two molecules.
Molecular Replacement to Determine the MPA-Disaccharide
Structure--
Since the quality of the above preliminary model was
exceptionally good when judged by its secondary structure, it was tried directly for molecular replacement to determine the structure of MPA
complexed with the disaccharide. The program XPLOR (20) was used for a
molecular replacement search, with only the 133 residues of the
subunit as model, and for subsequent refinement. The resolution range
used for the search was 4.0-8.0 Å, and only reflections higher than 2
were used. The PC refinement method following a rotation search in
X-PLOR was used to find the orientation of the search model. The
orientation was
1 = 133.48°,
2 = 2.41°, and
3 = 280.54°. It corresponded to the
fourth highest peak in the rotation search. This peak was the highest
peak in the PC refinement (height 5.9085-0.2) by only a
small amount. However, the translation search provided confirmation as
the maximum of the T-function was more than 12
high for the space
group P6422 as compared with 7
for P6222. The R value was 50.91% versus 59.41%. Therefore
the correct space group is P6422. The fractional
coordinates of the translation function are 0.137, 0.325, and 0.094. The height of the T-function also justified that this solution was a
correct one.
Refinement of the Structure--
The model was refined by
simulated annealing with X-PLOR (20) (version 3.854) to 2.2-Å
resolution using data with an F cutoff of 2
. As in the
molecular replacement search, only the
subunit was used in the
first two cycles. The first cycle started with rigid body refinement,
followed by steps of slow cool refinement from 3000 to 330 K. The
overall temperature used was kept at 15 Å2. From the third
cycle onward, 16 residues (positions 3-18) of the
subunit and the
disaccharide were added in as the density became visible. Individual
temperature factor refinement was included from the third cycle on, and
manual correction of the model with graphics was performed between
cycles. The R factor dropped to 0.229 after three cycles,
with a free R factor of 0.291. At this stage, electron
density for water molecules was clearly visible, and these were added
with the program Waterpick (20) and the resolution range was changed to
6.0-2.2 Å. Water molecules with refined B factors above 60 Å2 and density less than 1
in the
2Fo
Fc map were subsequently
removed. The final model contains 91 solvent molecules. Only positional
and individual temperature factor refinements were performed from the
fifth cycle on. Details of the refinement statistics are given in Table
III.
 |
RESULTS |
Structure Determination--
The complex of MPA and the T-antigen
disaccharide Gal
1,3GalNAc was crystallized as reported previously
(13), but the space group was redetermined as P6422 or
P6222. There is one 
monomer in the asymmetric unit,
and the Vm was 2.83 Å3/Da. Extensive
searches for heavy atom derivatives by soaking or cocrystallization
with the complex were unsuccessful, possibly because of the low pH of
the crystallization and because the primary sequence has no cysteine
residues and only one histidine. In addition, MPA has no exchangeable
Ca2+ or other metal ions. Co-crystallization trials of the
ligand-free protein with heavy atoms led to a new crystal form in the
space group C222 (Table I). Soaking experiments then produced a heavy atom derivative with trimethyl lead acetate, an excellent candidate for
MAD. This diffracted beyond 1.7 Å and the lead absorption edge was
detected at the National Synchrotron Light Source, Brookhaven National
Laboratory. MAD was therefore used to determine the MPA structure based
on this single derivative. Data sets for four different wavelengths in
the vicinity of the lead absorption edge were collected at the ESRF,
Grenoble, using an Image Intensified CCD detector (Table I). Two lead
sites were easily identified in the anomalous and dispersive Patterson
maps, and a high quality electron density map was produced based on the
MAD phasing. A model of MPA was built from this map and it was used
without refinement to determine the structure of the complex of MPA
with the disaccharide by molecular replacement. The model of the
complex was refined with the program X-PLOR, and the statistics for the
final model are given in Table III. The
sequences used for the model were those of the dominant
-chain and
the
2a isoform (8, 9). The root mean square deviation
between the initial MAD model of the
-chain and the refined
structure was only 0.8 Å. An Fo
Fc "omit" map of the disaccharide is shown in
Fig. 1. The lead atoms in the ligand-free
crystal are located in the disaccharide binding site, interacting with
the amino group of the terminal Gly. This offers a further explanation
for the difficulties in obtaining heavy atom derivatives. Its location
also prevents meaningful comparison of the native and ligand-filled
binding sites.

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Fig. 1.
Quality of the final electron density map in
the disaccharide region. The Fo Fc omit map is contoured at 2.5 . The
dashed lines show the experimental electron density in the
binding site, and the disaccharide structure is shown by solid
lines.
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Oligomeric Structure and Crystal Packing--
Fig.
2 shows the crystal packing in the unit
cell. There is one 
subunit per asymmetric unit and 12 (
)
monomers in the unit cell. The contacts between the tetramers are in
the region of the T-antigen binding site, and include intermolecular
disaccharide-protein contacts. Fig. 3
shows the overall structure of the MPA tetramer and T-antigen
complex.

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Fig. 2.
Crystal packing of the
MPA-tetramer-disaccharide complexes. One unit cell is shown edge
on in stereo, with the x and y axes in the plane
and the z axis vertical. The unit cell contains two
half-tetramers (pink) and monomers from eight other
tetramers (two green, two yellow, and four
red). The disaccharide ligands (white or
yellow) are at the corners of the tetramers and mediate the
contacts between them. These interactions result in the formation of a
lattice-like structure, as described under "Discussion."
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Fig. 3.
Backbone structure of the tetramer with the
bound ligand. The Greek key subdomains of each monomer are shown
in red, green, and purple with the
-chain in blue (see Fig. 4). This figure and Figs. 4 and
5 were prepared with MOLSCRIPT (21) and RASTER 3D (22).
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Structure of the MPA Monomer--
The overall MPA structure is
very similar to that of jacalin in its folding. As predicted from the
CD spectra (7) and sequence (8), MPA is a
-sheet protein. In the
refined model of the monomer, there are 12
-strands that form three
subdomains (Fig. 4). The four
-strands
of each subdomain form a Greek key motif, i.e. two
anti-parallel pairs. The overall structure is a three-fold pseudo-symmetric
-prism. Interestingly, the three-fold nature was
also predicted from the protein sequencing based on a possible internal
repeat in jacalin (8). The
-prism's core is stabilized partly by
hydrophobic interactions and partly by hydrogen bonding, including
interactions of five water molecules deeply buried among the three
subdomains. The interaction between the
- and
-chains is through
six interstrand hydrogen bonds between the
-chain and the last
segment of the
-chain, from residue 125 to residue 131. Together,
these strands form half of the third Greek key subdomain. No electron
density corresponding to the first two residues of the
-chain was
visible. There was electron density for the last two residues of the
-chain, but it was too weak to uniquely locate the side chain atoms.
Therefore, these four residues were not included in the final model.
The N terminus of the
-chain projects out of the globular structure
of the 
monomer into the center of the tetramer (Fig. 3).

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Fig. 4.
Backbone structure of the  monomer with
the ligand. Three subdomains together form the faces of a
-prism structure for the  monomer. The -strands of the
Greek key subdomains are colored as follows. Strands 2-5 form the
first subdomain in red, strands 6-9 form the second
subdomain in green, and strands 1, 10, and 11 in
purple plus part of the -chain in blue form the third subdomain.
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Interaction of MPA with T-antigen--
The connecting loops
between
-strands in the distal region of the monomer plus the
N-terminal residue of the
-chain form the ligand-binding site (Fig.
4). The GalNAc moiety of the disaccharide fits in a groove-shaped
subsite formed by the N-terminal residue (Gly-1), and the turns of
residues 46-49, 76-82, and 122-125. There are no water molecules
directly involved in the GalNAc-protein interactions. Fig.
5 shows the interactions of the GalNAc
with some of the MPA residues. This sugar has the expected chair
conformation with one side facing the amino group of Gly-1, Tyr-122,
Trp-123, and Asp-125. Its hydrogen bonding interactions are summarized in Table IV, and further details are
given in Fig. 6. The O-3, O-4, and the
carbonyl oxygen of the N-acetyl group (O-7) all form hydrogen bonds with the Gly-1 amino group. The O-4 also hydrogen bonds
to both carboxylate oxygens of Asp-125. The ring oxygen O-5 forms an
H-bond with the peptide nitrogen of Tyr-122. The O-6 is at the bottom
of the pocket in a network of hydrogen bonds with the Tyr-122 peptide
nitrogen, the peptide nitrogen of the Trp-123, and one carboxylate
oxygen of the Asp-125 side chain, which are in a tetrahedral array with
C-6. The carbonyl oxygen of Trp-123 is slightly beyond H-bonding
distance of the O-6 at 3.35 Å, and interacts with the peptide nitrogen
of Gly-121 (3.10-Å). The carboxylate oxygens of Asp-125 also interact
with a water molecule, W-23, and the hydroxyl of Ser-119. The distance
between the Gly amino group and the carboxylate OD2 of
Asp-125 is 3.44 Å (Fig. 6). On its other face, the GalNAc is
surrounded by the side chains of Phe-48, Tyr-78, Tyr-122, and Trp-123
with the phenol ring of the Tyr-78 parallel to the GalNAc ring (Fig.
5). The distance between this phenol ring and the GalNAc ring is
approximately 4.0 Å. These hydrophobic interactions contribute
significantly to the overall interaction between GalNAc and MPA.

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Fig. 5.
Binding of the GalNAc moiety to MPA.
H-bonds from backbone and side chain atoms and aromatic interactions in
a pocket around the GalNAc contribute to the strong interaction between the GalNAc moiety of the disaccharide and the MPA binding site.
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Fig. 6.
Hydrogen-bonding scheme for the
MPA-disaccharide complex. The two charged groups of Gly-1 and
Asp-125 form most of the H-bonds, to the GalNAc. The remaining H-bonds
are from backbone atoms plus two water molecules, one of which forms
the only H-bond to the Gal.
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The Gal moiety of the disaccharide protrudes from the end of the GalNAc
pocket and is located between symmetrically related tetramers in the
crystal (Fig. 7). There are no direct
H-bonds to the protein, but O-6 interacts with a water molecule W-40, which in turn H-bonds to the peptide NH of Thr-79 and to another water
molecule, W-23. The latter water molecule in turn H-bonds to the OD1 of
Asp-125. Although there is no subsite for the Gal, its interaction with
the protein through these water molecules accounts for the higher
affinity of the disaccharide compared with monosaccharides. The Gal is
in close contact with the neighboring tetramer through its C-2 and C-3
hydroxyls. Its O-3 forms a hydrogen bond with the Glu-76 peptide oxygen
(3.15 Å) of the symmetrically related molecule, whereas water molecule
W-59 forms hydrogen bonds with O-2 (2.82 Å) and the same Glu-76
peptide oxygen (3.08 Å). The Gal ring is also parallel to the phenol
ring of Tyr-78 of the symmetrically related molecule at a distance of
approximately 4.0 Å. There is also one protein-protein interaction, a
H-bond between the two Tyr-78 hydroxyls (3.18 Å).

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Fig. 7.
Intertetramer interactions around the Gal
moieties. The Gal O-6 binds to one tetramer through a pair of
water molecules. The Gal O-2 and O-3 interact with the carbonyl oxygen
of Glu-76 of the second tetramer, both by direct H-bonding and through
a third water molecule. The two Tyr-78 hydroxyls form a H-bond in the
center of the interfacial structure.
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The overall conformation of the disaccharide is within the minimum
energy area calculated for the disaccharide with the GalNAc as the
anomer.4 The interglycosidic
angles are
80° and
128°. This suggests that the
intertetramer interactions between the galactoses in the crystal do not
alter the disaccharide conformation from its bound state with MPA in
solution.
 |
DISCUSSION |
Structure and Processing of MPA--
As expected from their
sequence homology, MPA and jacalin have a common tertiary and
quaternary structure. All of the MPA
-chain residues could be
positioned in the electron density, and all except the two N-terminal
and the two C-terminal residues of the
-chain. In the jacalin case,
the three N-terminal and two C-terminal residues of the
-chain could
not be placed. The jacalin
-chain is one residue shorter as well, so
two more residues were seen in the MPA
-chain, i.e. 17 of
21 residues compared with 15 of 20 for jacalin. The C-terminal end is
created by the post-translational proteolytic cleavage that removes
four linking residues between the
- and
-chains in the jacalin
precursor (10), so its mobility in MPA is not surprising. The jacalin processing also removes 39 residues that precede the
-chain, and the
rather hollow center of the tetramer occupied only by the four crossing
-chains suggests these precursor segments could fit within the core
region. The processing also creates a key ligand binding group, the
amino group of the terminal Gly residue of the
-chain. However,
whether the precursor protein has activity, i.e. if the
post-translational proteolytic processing is essential for activity,
cannot be predicted with certainty from the importance of this
-NH2 group. The recently described MPA homologs, artocarpin and Calsepin, that bind mannose and maltose do not undergo internal cleavage (11, 12) and Calsepin has a deletion at the position of the
four linker residues in jacalin.
Carbohydrate Specificity of Moraceae Lectins--
The overall
H-bonding scheme for the MPA-disaccharide complex (Fig. 6) shows that
the ligand is bound by MPA predominantly through the GalNAc unit, both
through hydrogen bonds, mainly to atoms in the peptide backbone, and
van der Waals interactions with a cluster of Tyr, Phe, and Trp
residues. These interactions mainly account for the strong affinity and
specificity of MPA for the T-antigen disaccharide. In contrast, the Gal
projects from the protein and H-bonds only through two water molecules, while also interacting with the neighboring tetramer in the crystal. The dominance of the GalNAc in the overall recognition of the disaccharide is in marked contrast to the interactions found between peanut agglutinin and the same disaccharide (2). In the latter structure, the Gal O-3, O-4, and O-6 and the GalNAc O-4 all form H-bonds to side chains or Gly backbone atoms, plus there are a series
of H-bonds through two pairs of water molecules with Gal O-2 and the
carbonyl oxygen of the GalNAc, respectively. The strength of the
disaccharide recognition by MPA despite its fewer bonding partners may
arise from so many of the H-bonds being to charged groups,
i.e. the Asp-125 carboxylate side chain and the terminal -NH3+ of Gly-1. Such H-bonds have been
shown to be stronger than ones with neutral partners (27). The van der
Waals interactions of the sugars with aromatic residues will also
contribute not only to the binding of the disaccharide to the site but
also to the intertetramer interactions of the Gal.
The structure satisfactorily explains the specificity characteristics
of MPA. Although the Gal
1,3GalNAc ligand would be present as a
mixture of
- and
-anomers, only the
-anomer is bound. The
positioning of the GalNAc against the protein surface would make it
impossible for any
glycoside of GalNAc or Gal to bind in the same
orientation, explaining the strong anomeric preference of the Moraceae
lectins (5, 6). The preference of MPA for the T-antigen compared with
the Gal
1,3GalNAc
element of gangliosides and lactosamine
structures is also attributable to this feature. However, there is
clearly an open area above the GalNAc O-1 for an
-linked aglycone
(Fig. 5). NMR studies of an O-linked glycopeptide (25)
suggest that the disaccharide when O-linked to Ser or Thr will be perpendicular to the peptide backbone. This arrangement would
be easily accommodated in MPA. The higher affinity for
-nitrophenyl glycosides can be attributed to van der Waals interactions of the
nitrophenyl ring with the nearby aromatic side chains. For the Gal,
several of its hydroxyls are exposed and apparently could be
substituted without impairing binding to MPA. There have been reports
that jacalin can accept the 2,3'-sialylated glycopeptide (26). The
natural ligand for the Moraceae lectins is of course unknown, but the
binding site should be capable of accepting a polysaccharide with a
Gal
1,3GalNAc
internal element as readily as it does the short
O-linked glycopeptides.
The GalNAc moiety of the Gal
1,3GalNAc disaccharide is bound by MPA
in the same position and orientation as the Me
-D-Gal in
the jacalin structure (15). This supports the Asp-125 carboxylate being
in the charged form despite the lower pH required for the MPA
crystallization, 4.5 compared with 7.3 for jacalin (15). The
spectroscopic and binding properties of jacalin were interpreted as
suggesting independent subsites for
and
methyl galactosides (6). However, the lack of H-bonds between the Gal and neighboring residues in the same monomer and the few van der Waals contacts suggest
that there is no true subsite for the Gal moiety. It remains possible
that
-galactosides could be bound in the
-Gal/GalNAc site but in
an orientation that produces different spectroscopic effects.
Further x-ray studies should resolve this point. The hydrogen
bonding interactions of the jacalin with Me
-D-Gal and MPA with the GalNAc unit of the disaccharide are generally similar. The
additional energy of binding of GalNAc compared with Gal by MPA comes
in part from the H-bond from the carbonyl oxygen of the NAc to the
terminal amino group (Gly-1), and this adds to the importance of this
group in the overall recognition process. The methyl moiety of the
acetyl group projects away from any side chains, unlike the prediction
from jacalin-ligand modeling (15).
All of the ligand-contacting residues in the two proteins are
completely conserved. However, the two proteins differ in two aspects
of their binding. Jacalin has a three-fold higher affinity for Me
-D-Gal and shows a smaller increase in intrinsic
fluorescence on binding this ligand than does MPA (7). This points to
some minor conformational difference in the ligand-free structures, possibly due to residue 2 in the
-chain being Lys in jacalin and Val
in MPA. These residues are in the neighborhood of Trp-123.
Comparison to Other Lectin Structures--
The internal three-fold
pseudo-symmetry seen in MPA also occurs in ricin (28), amaranthin (3),
and snowdrop lectins (29), each of which have, however, different
overall architectures to MPA. The higher internal sequence homology of
the snowdrop lectin leads to a valance of 3 for its protomer, and in
the ricin case the original assignment of one Gal-binding site
per protomer has been challenged by recent site-directed mutagenesis
experiments (30). Unlike these plant lectins, the MPA binding site is
formed primarily from contiguous loops joining
-strands 1 and 2 in
each Greek key subdomain; hence, it cannot have further binding
sites.
The recently reported new member of the Moraceae lectin structural
family, Calsepa, shows approximately 30% sequence homology to the
jacalin and MPA
-chains (11). The homology is particularly strong in
the second and third subdomains. More importantly, the conserved
residues are mainly in the
-strands and include two of the three
internal Ile residues that fill the domain interior, the third being
Leu in Calsepa. Of the residues that form the binding site in MPA,
Calsepa has identical residues for Asp-125 and Tyr-122, whereas Phe-47
in MPA is Ser and Trp-123 is Tyr. The N-terminal Gly can be aligned
with an internal Gly in Calsepa, but there is a deletion at the
equivalent position to the four-residue linker segment of jacalin (11).
The conservation of the key Asp-125, which forms H-bonds with the O-4
and O-6 of the GalNAc, in a lectin with Man/maltose specificity is
reminiscent of the conserved Asp/Asn feature of the binding sites of
legume lectins with similar specificity differences (31). The change of
Phe-47 and the extension on the N-terminal Gly will create a quite
different structure in this half of the site in Calsepa, consistent
with the differences at two hydroxyls of its ligands from the Gal ones of MPA. This protein also shows that the post-translational processing that appears to be essential in jacalin and MPA is not obligatory in
all members of this family. Calsepa also lacks a pro-segment equivalent
to the 39-residue portion preceding the
-chain in jacalin (11).
The role of the Gal interactions in the tetramer-tetramer association
in the crystal packing is remarkable. It has some significant parallels
in the formation of lattices by legume lectins, recently reviewed by
Brewer (32), S- and C-type animal lectins (33), and the snowdrop lectin
(29) when they interact with branched oligosaccharides. These lattices
have been observed in both electron microscopy and x-ray
crystallographic experiments with divalent, trivalent, and tetravalent
lectins interacting with divalent ligands. This phenomenon is thought
to be of considerable biological significance for lectin function,
where cross-linking of cell-surface glycoconjugates leads to signal
transduction effects. Crystals of the lattice type lectin complexes
have higher solvent contents than usual and open channels, a feature
also seen in the MPA case, although the solvent content is only 57%.
In MPA, the placement of the sites at the corners of the tetramer is
ideal for lattice formation of the kind seen with the other animal and
plant lectins. However, its ability to form lattices with a small
"monovalent" ligand is novel. Whether the interactions are
sufficiently strong to mediate tetramer-tetramer interactions by the
disaccharide in solution is presently being investigated.
In conclusion, the present MPA-disaccharide structure has shown that
the recognition of the T-antigen on tumor mucins will be predominantly
through the GalNAc moiety, but the geometry of the interaction may be
considerably more complex if lattice formation can occur under the
conditions of cell-lectin binding. The ability of MPA to differentiate
lung cell tumor types (34, 35), to preferentially label human
translational cell carcinomas (36) and its toxicity toward insect
larvae (37) may well rest upon this unusual association mechanism.
We thank Dr. Bryan G. Williams for his
continued support of this work, Dr. J.-R. Brisson for providing the
minimum energy plot for the disaccharide, Rebecca To for protein
purification, Dr. Stephen Evans for assistance with graphics, Dr. Focco
van den Akker for initial T-antigen coordinates, Dr. A. P. Hammersley for correction of the II/CCD data, Dr. Eldon Walker for
computer support, Drs. Eric Fançon and Michel Roth for beamtime
at ESRF, and EMBL and ESRF for their support.