(Received for publication, June 13, 1995; and in revised form, August 9, 1995)
From the
The conformational features of sucrose in the combining site of lentil lectin have been characterized through elucidation of a crystalline complex at 1.9-Å resolution, transferred nuclear Overhauser effect experiments performed at 600 Mhz, and molecular modeling. In the crystal, the lentil lectin dimer binds one sucrose molecule per monomer. The locations of 229 water molecules have been identified. NMR experiments have provided 11 transferred NOEs. In parallel, the docking study and conformational analysis of sucrose in the combining site of lentil lectin indicate that three different conformations can be accommodated. Of these, the orientation with lowest energy is identical with the one observed in the crystalline complex and provides good agreement with the observed transferred NOEs. These structural investigations indicate that the bound sucrose has a unique conformation for the glycosidic linkage, close to the one observed in crystalline sucrose, whereas the fructofuranose ring remains relatively flexible and does not exhibit any strong interaction with the protein. Major differences in the hydrogen bonding network of sucrose are found. None of the two inter-residue hydrogen bonds in crystalline sucrose are conserved in the complex with the lectin. Instead, a water molecule bridges hydroxyl groups O2-g and O3-f of sucrose.
Specific recognition of carbohydrates by protein receptors is of growing interest in biology. In addition to immunoglobulins, carriers, and toxins, carbohydrates can interact with lectins, a class of multivalent and ubiquitous carbohydrate binding proteins (Sharon and Lis, 1989). Animal lectins have been shown to be implicated in the social life of the cells, mediating cell recognition and cell adhesion (Drickamer and Taylor, 1993). Plant lectins are also involved in recognition processes and play a role in the interaction with symbiots or pathogens (Diaz et al., 1989; Chrispeels and Raikhel, 1991). They can be purified easily and their specificities make them useful as components of affinity columns for the separation of glycoconjugates or as markers of blood groups and tumor cell lines (Lis and Sharon, 1986).
Legume lectins are a large family of homologous proteins displaying a broad range of different carbohydrate specificities. Several x-ray structures of legume lectins complexed with carbohydrates have been determined recently: concanavalin A with mannose (Derewenda et al., 1989), Lathyrus ochrus isolectins with mannose and glucose (Bourne et al., 1990a), with a trisaccharide (Bourne et al., 1990b), with a biantennary octasaccharide (Bourne et al., 1992) and with a biantennary glycopeptide (Bourne et al., 1994), Erythrina corallodendron lectin with lactose (Shaanan et al., 1991), pea lectin with trimannose (Rini et al., 1993), and Griffonia simplicifolia isolectin IV with the Lewis b determinant (Delbaere et al., 1993).
To study recognition
processes at the atomic level, different experimental and theoretical
methods can be used. Many carbohydrate-protein complex structures have
been elucidated recently by x-ray crystallography (Vyas, 1991; Bourne et al., 1993) at high resolution. Detailed information derived
from crystallographic data allows us to estimate the driving forces
behind carbohydrate-protein interactions (Vyas, 1991; Imberty et
al., 1993). However, whereas x-ray crystallography provides us
with a static picture, NMR experiments can provide information about
the conformation as well as about the movement of the ligand in the
binding site. In the special case of weakly bound ligands, with a high
rate of exchange between the bound and the free states, transferred
nuclear Overhauser effects (TRNOEs) ()can be observed,
giving information about the conformation of the carbohydrate in the
binding site (Clore and Gronenborn, 1982; Ni, 1994). This method
allowed the determination of the conformational changes undergone by
oligosaccharides upon binding to ricin (Bevilacqua et al.,
1990, 1992), to antibodies (Glaudemans et al., 1990, Bundle et al., 1994, Arepalli et al., 1995), and to a human
lectin (Cooke et al., 1994). Molecular modeling is a
complementary tool for both x-ray and NMR methods. Several methods of
docking monosaccharides to protein receptors have been developed using
molecular mechanics. Such methods allow for a systematic exploration of
all possible positions and orientations of the ligand in the binding
site (Rao et al., 1989; Imberty et al., 1991). In
addition, it is possible, using computer simulations, to evaluate the
conformational space accessible for flexible ligands such as
oligosaccharides (Reddy and Rao, 1992; Imberty and
Pérez, 1994).
The aim of this work is to study the conformation of sucrose in interaction with lentil lectin, using three different and complementary approaches: x-ray crystallography, NMR, and molecular modeling. Lentil lectin specifically recognizes mannose and glucose. The three-dimensional structure of this lectin was refined at 1.8-Å resolution in the uncomplexed form (Loris et al., 1993, 1994a) and at 3.0-Å resolution for the complex with glucose (Loris et al., 1994b). Sucrose is an abundant carbohydrate of interest to the food industry for its sweet taste. The molecular conformation of sucrose has been studied extensively, and many experimental and theoretical data are available (see review by Pérez(1994)). Sucrose has been shown to be flexible in dilute solution (Hervé du Penhoat et al., 1991; Poppe and Van Halbeek, 1992), but its interactions with protein receptors have never been investigated. We report here the first x-ray structure of sucrose complexed with a protein. In addition, transferred NOEs have also been obtained for lentil lectin complexed with sucrose. Both sets of experimental data can be rationalized by comparison with the conformational behavior of sucrose in the binding site as predicted by molecular modeling.
Figure 1: Perspective view of sucrose. Hydrogen atoms of interest for the NMR study are labeled. Arrows indicate the torsion angles of interest.
The orientation of the
three hydroxymethyl groups is given by the torsion angles g,
f, and
f.
g =
(O5-g-C5-g-C6-g-O6-g);
f =
(O5-f-C5-f-C6-f-O6-f);
f =
(O5-f-C2-f-C1-f-O1-f).
The signs of the torsion
angles are in agreement with the recommendations given by the IUPAC-IUB
Commission of Biochemical Nomenclature (IUPAC-IUB, 1971). The three
preferred orientations of the primary hydroxyl groups are referred to
as either gauche-gauche (GG), gauche-trans (GT), or trans-gauche (TG), with respective values of -60°,
60°, and 180° (Marchessault and Pérez,
1979). The distortion of the fructofuranose ring is described by the
Cremer-Pople puckering parameters Q and (Cremer and
Pople, 1975). Q is the puckering amplitude which measures the
deviation from the planarity, and
is the phase angle of
puckering.
From the TRNOE experiments, build-up curves were drawn by plotting the NOE cross-peak volumes against the mixing times. Inter-proton distances and their standard deviations were calculated from the NOE build up curves by normalizing on a H2-g/H4-g theoretical distance of 2.47 Å and extrapolating to zero mixing time using a least square fit (Baleja et al., 1990).
The same procedure was used to calculate the relaxed map of isolated sucrose with the TRIPOS force field.
Figure 2:
Stereoscopic representation of a dimer of
the lentil lectin complexed with sucrose as determined by
crystallography. -Sheets are shown as arrows. The two
sucrose molecules, which are bound at opposite ends of the lectin
dimer, are drawn as ball and stick models. This
figure was prepared using MOLSCRIPT (Kraulis,
1991).
The final electron density of the sucrose moiety
is represented in Fig. 3. The conformations of the two bound
sucrose molecules are almost identical for the two subunits of the
dimer and are characterized by torsion angles of =
107° and 105° and
= -58° and
-59°, respectively. The torsion angles for the primary
hydroxyl groups
g,
f, and
f have a GG, GT, and GG
orientation, respectively. The puckering parameters of both fructose
residues have values of Q = 0.47 and
=
260°, which corresponds to a shape between an E
envelope and a
T
twist, both which are
observed in sucrose-containing crystalline compounds
(Hervé du Penhoat et al., 1991). Since
both sucrose moieties are so similar, only the first one will be
described in the following parts of the paper.
Figure 3: Two different stereo views of the electron density of the sucrose molecule as determined by crystallography.
The hydrogen bond
network between the sucrose molecule and the lentil lectin binding site
has been investigated thoroughly (Table 2). Starting from the
crystal structure, hydrogen atoms were added, and their positions were
optimized using the TRIPOS force field. The resulting hydrogen bond
network is shown in Fig. 4. The glucose residue of sucrose
displays the same hydrogen bonding scheme and the same stacking
interaction between the hydrophobic face of glucose and the aromatic
ring of Phe-123 as observed in the lentil lectin-glucose complex
(Loris et al., 1994b). The fructose residue displays some
direct interactions or contacts with the amino acids from the binding
site: hydrophobic contacts exist between the side chain of Phe-123
and the protons and H4-f and H6-f of the fructose moiety. One of the
H6-f hydrogen atoms is located just between the aromatic ring of
Phe-123
and the methyl group of Ala-30
, creating a continuous
hydrophobic cluster. In addition, oxygen O6-f is almost at hydrogen
bonding distance from the acid group of Glu-31
. In fact, many
hydrogen bonds are mediated through one or two water molecules as shown
in Fig. 5. Such water bridges are observed for O2-f and O3-f
with Tyr-124
, Ala-126
, Ala-127
, and Asn-125
. No
direct inter-residue hydrogen bonds are observed within the sucrose
molecule, but one strong interaction is mediated by a water molecule
between O3-f and O2-g.
Figure 4: Stereoscopic representation of the sucrose binding site in the crystal structure of lentil lectin-sucrose complex. For the sake of clarity, only amino acids involved in intermolecular hydrogen bonding are shown. The sucrose molecule is displayed in bold. Hydrogen atoms have been generated on sucrose and on amino acids from the binding site in order to determine the hydrogen bond network (represented with dotted lines).
Figure 5: Hydrogen bond network of sucrose in the lentil lectin binding site with the water molecules. The symmetry-related sucrose is also shown.
As shown in Fig. 6, both carbohydrate
binding sites interact with each other via crystal contacts. A large
nearly symmetric water network connects the sucrose molecule bound to
the first lectin monomer to a symmetry mate of the sucrose bound in the
second monomer. A water bridge is present between the O2 atoms of both
glucose residues, while the oxygen O1-f of each fructose residue is
hydrogen bonded to Gly-97 of a symmetry-related lectin molecule.
Other interactions of the sucrose molecules with symmetry-related
lectin monomers are mediated via water bridges and involve Ser-39
,
Gln-95
, Thr-96
, Gly-99
.
Figure 6: Stereoscopic representation of the sucrose-mediated crystal lattice interaction between both lectin monomers. The two sucrose molecules in the center of this figure form a lattice contact via the water network that is drawn schematically in Fig. 5. The figure was prepared using MOLSCRIPT (Kraulis, 1991).
Figure 7:
H NMR spectra for the free and
the bound ligand. a, one-dimensional spectrum of sucrose. b, one-dimensional NOESY of H1-g for the free ligand where
positive NOEs are observed. c, one-dimensional TRNOE of H1-g
for the complex of lentil lectin with sucrose, showing the negative
NOEs. d, build-up curves of some observed transferred
NOEs.
Figure 8:
a,
adiabatic ``relaxed'' energy map of sucrose calculated with
MM3. Energy was calculated as a function of and
torsion
angles with 10° increments (adapted from Casset et
al.).
Isoenergy contours are drawn by interpolation of
1 kcal
mol
above the absolute minimum in an
energy window of 10 kcal
mol
. Conformation of
crystalline sucrose is indicated by a diamond shape. b,
adiabatic ``relaxed'' energy map of sucrose calculated with
TRIPOS. Energy was calculated as a function of
and
torsion angles with 20° increments. c, four
``relaxed'' potential energy surfaces of sucrose in lentil
lectin binding site calculated with TRIPOS. Energy was calculated as a
function of
and
torsion angles with 20° increments.
Conformation of sucrose in the crystal structure of the complex is
indicated by a star on the corresponding energy map, and the
lowest energy conformations of the three main low energy regions have
been indicated by A, B, and C.
Several low energy conformers from the three low energy domains of
each map were selected and then fully energy minimized without
constraints on the glycosidic torsion angles. Releasing these
constraints yields some minor adjustments in the and
torsion angles (2° to 4°). The lowest energy conformer was
obtained for the conformation GG-GT-GG with torsion angles
= 84° and
= -46° (conformer A). The
hydrogen bond network of this global minimum is listed in Table 2. The fructose residue is not involved in hydrogen bonding
with the protein, but hydrophobic interactions exist between the
fructose moiety (H4-f and H6-f) and the side chains of amino acids
Phe-123
and Ala-30
. In contrast, conformer B (
=
82° and
= -161°) has hydrophobic contacts
only between Ala-30
and H6-f while conformer C (
=
138° and
= 37°) has interactions only between
Ala-30
and H4-f. Fig. 9shows these three different
conformers, selected from the map GG-GT-GG, in the binding site of
lentil lectin. For the three minima, details of the energy of
interaction are listed in Table 4.
Figure 9: Stereoscopic representation of the main low energy conformers of sucrose in the binding site as found by molecular modeling. A, B, and C refer to the conformations of sucrose as located on the energy maps of Fig. 8b.
Even if these
differences in conformation are not very important, they result in
major differences in the hydrogen bond network of sucrose. None of the
two inter-residue hydrogen bonds in crystalline sucrose (O1-f
O2-g and O6-f
O5-g) are conserved in the complex with the
lectin. A new feature in this complex is the presence of one water
molecule located between the glucose and fructose residues. This water
creates a strong water bridge between O2-g and O3-f. In a recent
molecular dynamics simulation with explicit solvent, this given site is
occupied by a water molecule during 25% of the trajectory (Engelsen et al., 1995).
For sake of comparison, the conformation of both crystalline sucrose (Brown and Levy, 1973; Hanson et al., 1973) and sucrose complexed with lentil lectin are indicated on the potential energy maps of Fig. 8. For the solid state sucrose, the crystal conformation belongs to the main energy well, but is about 2 kcal/mol higher in energy than the calculated global minimum (Pérez et al., 1993). This small disagreement between solid state conformation and computation can be rationalized by either distortion due to packing effects or by the inadequacy of the force field for a molecule in which overlapping exoanomeric effects are present (French et al., 1993). On the other hand, the conformation of sucrose complexed with lentil lectin corresponds well to the global energy minimum predicted by computer simulations.
Figure 10: Stereoscopic representation of the sucrose molecule in the binding site of lentil lectin determined from the crystallographic study (black lines) and from the molecular modeling study (gray lines).
However, when looking at the data obtained from the transferred NOE study, the situation is quite different. Table 5shows a comparison of inter-protons distance calculated using the three different approaches. The NMR distances are in good agreement with solid state and theoretical conformations for both the glucose ring and the interglycosidic distances. This good agreement confirms that the glycosidic linkage is immobilized in the lectin-sucrose complex. The situation is quite different for the fructose ring, where the agreement is not so good. This is related to the fact that, for a furanose ring, inter-proton distances vary widely for small distortions of the pucker (Olson and Sussman, 1982). Therefore, it seems quite likely that the fructose ring, which is not involved in a strong interaction with the protein, is still relatively mobile.
Both the crystallographic and NMR studies point toward a unique conformation for the glycosidic linkage in the bound sucrose molecule. Whereas the glycosidic linkage conformation is close to the one observed in crystalline sucrose, the orientations of the hydroxymethyl groups differ significantly. Therefore, the scheme of intramolecular hydrogen bonds within the sucrose molecule is completely different. Our NMR study provides some insight into the residual conformational flexibility of the sucrose in the protein binding site. This flexibility is mainly confined to fluctuations of the fructofuranose ring.
The global energy minimum predicted by the molecular modeling study has the same glycosidic linkage conformation as the conformation determined by experimental methods. The shape of the energy map also indicates that no large movements are allowed once the molecule is bound by the lentil lectin in this conformation. However, from the theoretical point of view, two other conformations of sucrose could also be bound by the lectin. The energies calculated for these complexes are only slightly higher than the global energy minimum (which is also the one observed in the crystal). It seems to us that the protein selects the most stable solution conformation of the ligand, as is also observed in most other lectin-carbohydrate and antibody-carbohydrate interactions.
The atomic coordinates and structure factors (code 1LES) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.