(Received for publication, September 21, 1995)
From the
Despite the fact that complex saccharides play an important role in many biological recognition processes, molecular level descriptions of protein-carbohydrate interactions are sparse. The legume lectin concanavalin A (con A), from Canavalia ensiformis, specifically recognizes the trimannoside core of many complex glycans. We have determined the crystal structure of a con A-trimannoside complex at 2.3-Å resolution and now describe the trimannoside interaction with con A. All three sugar residues are in well defined difference electron density. The 1,6-linked mannose residue is bound at the previously reported monosaccharide binding site; the other two sugars bind in an extended cleft formed by residues Tyr-12, Pro-13, Asn-14, Thr-15, and Asp-16. Hydrogen bonds are formed between the protein and all three sugar residues. In particular, the 1,3-linked mannose residue makes a strong hydrogen bond with the main chain of the protein. In addition, a water molecule, which is conserved in other con A structures, plays an important role in anchoring the reducing sugar unit to the protein. The complex is further stabilized by van der Waals interactions. The structure provides a rationale for the high affinity of con A for N-linked glycans.
It is well established that carbohydrates play a role in a myriad of important biological recognition processes; infection, the immune response, cell differentiation, and neuronal development may all be regulated to some extent by protein-carbohydrate interactions(1, 2, 3, 4) . One area of therapeutic interest in carbohydrate recognition has relied on the their role as cell surface receptors enabling adherence of bacteria, parasites, and viruses in the early stages of infection(5, 6) . The abnormal structure and levels of certain tumor cell surface glycans may also present opportunities for therapeutic intervention (7) . The notion of using oligosaccharide analogues to disrupt cell-cell recognition is an appealing one, and is the focus of considerable current activity in relation to the development of anti-inflammatory agents(8) . However, the ubiquitous use of carbohydrates in nature potentially poses serious specificity problems. Understanding the molecular basis of carbohydrate recognition might provide the necessary basis on which to rationally design biologically active saccharide analogues.
Although highly homologous, and often sharing monosaccharide selectivity, plant lectins exhibit exquisite oligosaccharide specificity(9) . While the function of these proteins is unknown, their interaction with saccharides has proved a valuable source of fundamental information. Structures of protein-saccharide complexes have been reported for lectins from Erythina coroallodendron (EcorL)(10) , Griffonia simplicifolia (GS4)(11) , Lathyrus ochrus (LOL1)(12, 42, 43, 44) , pea(13) , and lentil (preliminary data only)(14) . The GS4 and EcorL lectins are galactose-specific, while LOL1, pea, and con A are mannose-specific. Interestingly, the overall organization of the monosaccharide binding site is conserved among the lectins(9) . In GS4 and EcorL, the galactose residue is rotated relative to mannose in the binding site with only subtle changes in the precise side chain organization observed. Oligosaccharide complexes of EcorL, GS4, and LOL1 have provided structural insights into carbohydrate recognition, and, in particular, these structures have provided experimental evidence for the importance of water molecules in mediating carbohydrate recognition (12) .
Concanavalin A (con A) ()is the most extensively studied member of the lectin
family, and was first isolated and crystallized in 1919(15) .
Although the structure of the protein was determined in the early
1970s(16) , it was not until 1989 that the 2.9-Å
structure of con A-methyl
-D-mannopyranoside complex was
determined (17) . This represented the first structure of any
lectin carbohydrate complex, and it explained the so-called
``Goldstein rules'' for con A monosaccharide
specificity(18) . The sugar was reported to be anchored to the
protein by several direct hydrogen bonds and by van der Waals
interactions. Subsequent extension of the resolution to 2.0 Å (19) permitted a more detailed description of the contacts
between the protein and the monosaccharide. However, the same depth of
understanding is not available to explain the oligosaccharide
specificity of lectins. The precise contributions of hydrogen bonding,
van der Waals interactions, and rearrangement of bound and bulk water
to the specificity of the lectin-oligosaccharide interactions continues
to be a subject of interest(20) .
The oligosaccharide
specificity of con A is well documented(9) . Interactions are
centered on the so-called trimannoside core (Fig. 1) found in
all N-linked glycans, and it is this specific interaction that
forms the basis of con A's use as a tool in histochemical
staining(21) . Although there are several reports of modeling
studies on the con A-oligosaccharide
interaction(22, 45, 46) , no crystal
structure has been reported for con A complexed to any oligosaccharide.
We now report the 2.3-Å resolution structure of con A bound to
the N-linked glycan core trimannoside
Man1-6(Man
1-3)Man.
Figure 1: Biantennary N-linked glycan. The trimannoside core is shown boxed.
Con A and Man1-6(Man
1-3)Man were
purchased from Sigma (Poole, United Kingdom) and Dextra Laboratories
(Reading, United Kingdom), respectively.
Crystals (dimensions: 0.3
mm 0.4 mm
1.2 mm) of the protein-carbohydrate complex
were obtained, after 2 weeks, from a hanging drop of 8 mg/ml protein
and 7 mM trimannoside equilibrated against 20% polyethylene
glycol (M
6000), pH 9.0. All diffraction data were
collected at room temperature on a crystal mounted in a glass capillary
using the Enraf-Nonius/MacScience DIP2000 dual image plate. X-rays were
generated using an Enraf-Nonius FR591 rotating anode generator and
focused using the MacScience mirror system. The fresh crystal
diffracted to 2.1 Å; however, crystal decay limited the effective
resolution to 2.3 Å. Data were recorded as 252 non-overlapping
12.5-min 0.5° oscillations and processed using DENZO and
SCALEPACK(23) . The crystal had a primitive unit cell of
dimensions a = 81.65 Å, b =
66.68, c = 108.32,
=
=
90.0°,
= 97.79°. The asymmetric unit contains a
tetramer; Matthew's number 2.9 Da Å
,
approximately 52% solvent. A k
2n systematic
absence was visible, and the space group was assigned as
P2
. A summary of the data is given in Table 1. The
structure was determined using the molecular replacement procedure
AMORE (24) as implemented in the CCP4 package (25) .
The 2-Å structure of the methyl
-D-mannopyranoside-con A complex (Protein Data Bank code
5CNA) was used as the search model (with metal ions, sugars, and waters
removed). A random subset of data (10%) was omitted from all refinement
calculations in order to provide an unbiased assessment of the
refinement(26) . The raw molecular replacement solution in
P2
was rigid body refined to 2.7 Å and gave a free R-factor of 34.0% and an R-factor of 34.6%. An F
- F
electron density map was generated with phases calculated
from the rigid refined model, strong density (>5
) was observed
for all 8 metal ions and for 8 of the 12 possible sugar units, with
weak but convincing density for the remaining 4. This was taken as
confirmation of the correctness of the space group assignment and the
molecular replacement solution. Refinement proceeded with X-PLOR (27) (restrained positional and thermal factor) alternating
with manual intervention using O(28) . Non-crystallographic
positional and thermal factor restraints were maintained throughout the
refinement. The metal ions were included in the refinement with zero
electrostatic charge, and the trimannoside molecules were included when
a difference F
- F
map showed 3
density for all 12
mannose residues (Fig. 2). Water molecules were added to the
model, if (a) they corresponded to peaks with magnitudes
greater than 3.5
in the F
- F
map, (b) they made physically
reasonable hydrogen bonds with the protein (or other ordered water
molecules), (c) they subsequently reappeared in peaks of
1.0
in the 2F
- F
map, and (d) a drop in the
free R-factor was observed. The final model comprises 948
residues and has an R-factor of 20.5% and a free R-factor of 25.5% for data between 8 and 2.3 Å with F > 2.5
F, for all data in the range from 8
to 2.3 Å, the R-factor is 21.7%. Table 2gives a
summary of refinement results. A Ramachandran plot (not shown) revealed
1 residue in a generously allowed region and no residues in disallowed
regions, 86.5% of the residues were in the most favored regions and the
remaining 13.5% in additionally allowed regions. All stereochemical
parameters measured by PROCHECK version 3.3 (29) were better
than average for a structure at 2.3 Å (data not shown).
Co-ordinates (code 1CVN) and structure factors (code R1CVNSF) have been
deposited with Protein Data Bank(30) . Co-ordinates will be
available 6 months after the date of this publication, and structure
factors after 1 year.
Figure 2:
Difference electron density F - F
map (contoured at 3
) shown with the refined position
of the trimannoside molecule superimposed. The map phases were derived
from the simulated anneal omit procedure (27) and weighted with
SIGMAA(25, 40) . All the sugar atoms are in well
defined electron density. (Figure was generated by
O(28) .)
Figure 3:
The con A tetramer with bound
trimannosides. Con A is shown in a ribbon representation, trimannoside
as a stick model and metal ions as space-filling spheres
(Ca, white; Mn
, red). The trimannoside molecules are located at the extreme
vertices of the tetramer and as a consequence are involved in lattice
contacts, which stabilize the crystal. The precise arrangement of the
crystal contacts are different for each subunit. (Figure was generated
by RASTER3D(41, 47) .)
Since O4 of the reducing sugar unit is in contact with Tyr-12, substitution at this position is expected to abolish ligand binding in the mode observed for the unsubstituted trimannoside. Such a steric clash accounts for the reduced binding (34) of con A by N-linked glycans possessing a bisecting GlcNAc residue at O-4 of the trimannoside unit.
Figure 4: The extended trimannoside binding site of con A. Panel A, a close-up view of the saccharide in the binding site. The amino acids are shown as space-filling spheres with side chains colored by residue type; the trisaccharide is shown in stick format. (Figure was generated by RASTER3D(41, 47) .). Panel B, a schematic representation of the hydrogen bonds between the sugar and protein. The distances for these hydrogen bonds are given in Table 3.
The reducing mannose residue makes good hydrogen bonds to both Tyr-12 and a bridging water molecule, which is in turn ligated by Asn-14, Asp-16, and Arg-228. In both pea lectin and LOL1 (42) the con A Tyr-12 is replaced by Phe, which is incapable of making the hydrogen bond to the reducing mannose O-4 observed in the con A-trimannoside complex. In addition, two of the three residues that act as ligands to the structural water are replaced by non-polar residues: Asp-16 by Ala (37) and Arg-228 by Gly(42) . Although a water is found in the same position in LOL1 (but not in pea lectin), it is only ligated by one Asn residue. The two key recognition elements for the reducing unit of trimannoside are therefore substantially different in pea lectin and LOL1. The 1,3-linked mannose residue forms a strong hydrogen bond to the main chain of the protein, with additional hydrogen bonds to O-3 and O-4 from Thr-15. This residue is conserved in LOL1, but is replaced by Ala in pea lectin(37) . We have already mentioned that the contribution of Thr-15 to trimannoside recognition warrants further investigation.
We note that the amino acids we have identified as being important for recognition of trimannoside by con A ( Table 3and Table 4, Fig. 4B) are conserved in the Dioclea grandiflora lectin(38) , which also has a high affinity for trimannoside(36) .
A comparison between the native and mannoside complexes of con A at 2.0-Å resolution shows that trimannoside binds to what appears to be a relatively rigid and preformed extended binding site from which water molecules are displaced. Specific direct and indirect interactions with amino acids, which are not conserved in LOL1 and pea lectin, account for the high affinity and specificity of con A for the trimannosyl core of N-linked glycans.
The atomic coordinates and structure factors (codes 5CNA, 1 CVN, and R1CVNSF) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.