Lisa M. Wright, Stephen D. Wood and Colin D. Reynolds
School of Biomolecular Sciences, Liverpool John Moores University, Max Perutz Building, Byrom Street, Liverpool, L3 3AF, England.
Pierre Rizkallah
CCLRC Daresbury Laboratory, Warrington, WA4 4AD, England.
Anthony K. Allen
Imperial College, School of Medicine, London, W6 8RF, England.
Els J.M. Van Damme and Willy J. Peumans
Catholic University of Leuven, Laboratory for Phytopathology and Plant Protection, Willem de Croylaan 42, 3001 Leuven, Belgium.
Received 2 December 1997, accepted 27 March 1998
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http://plab.ku.dk/tcbh/Lectins12/Wright/paper.htm Lectins, Biology, Biochemistry, Clinical Biochemistry, Volume 12, including Proceedings from the 17th International Lectin Meeting in Würzburg, 1997,edited by Edilbert van Driessche, Sonia Beeckmans and Thorkild C. Bøg-Hansen, published by TEXTOP, Lemchesvej 11, DK-2900 Hellerup, Denmark, ISBN number 87-984583-0-2 |
Introduction
Lectins have the capability to serve as recognition molecules, binding specifically and reversibly to carbohydrates. These proteins have been isolated from all classes of living organisms but most is known about the lectins found in plants. Although our understanding of the biological functions of plant lectins is not entirely clear, there is growing evidence that the most important physiological role of lectins is in the plant's defense against pests, such as bacteria, fungi, viruses and predators (1).
Over the last decade, lectins have been isolated from the bulbs, tubers, rhizomes and corms of the monocotyledonous families Amaryllidaceae, Orchidaceae and Alliaceae. These lectins exhibit varying degrees of antiretroviral activity, particularly against HIV (2). Their activity against HIV has been attributed to the binding of the sugar moieties on the HIV coat protein GP120 to the lectin. Structural studies of lectin complexes with mannosides will assist in understanding the molecular basis of this process.
The crystal structure of the first representative of this family of lectins, the snowdrop lectin (3) showed a novel fold, a 12-stranded beta-barrel (beta-prism). Here we report the structure determination of the first representatives from the Liliaceae family, the native lectin from bluebell (Scilla campanulata) bulbs (having a subunit molecular mass of 13.1 kDa) and its complexes with alpha-D-mannose, alpha1,6-dimannoside and alpha1,3-alpha1,6-trimannoside.
Materials and Methods
Protein Purification
The 13.1 kDa bluebell bulb lectin (SCA-MAN) was purified using a mannose-Sepharose 4B affinity column, as described previously (4).
Crystallization
Crystallization conditions were surveyed using the vapour-diffusion hanging-drop method (5) over 1ml reservoirs on siliconized cover slips in Linbro trays at 293K.
X-ray Diffraction
Data were collected using the rotation method at the Synchrotron Radiation Source (SRS), Daresbury Laboratory, U.K., with a MAR Research image plate detector system.
Results
Crystallization
Droplets of solution contained SCA-MAN at a concentration of more than 5 mg ml-1, 10 mM diaminopropane (DAP) and 600 mM phosphate buffered saline (PBS). These were equilibrated against varying concentrations of ammonium sulphate (AS) in water. For crystals of the complexes, the appropriate saccharide was added to the droplets at a concentration of 5 to 10 mM. All of the SCA-MAN crystals grew over a wide range of pH values. The best crystals grew at pH 4.7 to 4.8, in equilibrium against 70% concentrated AS (Figure 1).
Figure 1. A native SCA-MAN crystal
Data Collection
For the native SCA-MAN crystals and the three saccharide-bound complexes, four full data sets were collected at the SRS, Daresbury Laboratory, U.K., on stations 7.2, 9.5 and 9.6. Large MAR image plate detectors were used, and data reduced using the MOSFLM (6) and CCP4 (7) packages. The trimannoside-complex crystals were mounted on hairloops and cryocooled to 100K. The three other crystal types were mounted in thin walled capillaries and cooled to 277K. Native SCA-MAN and the mono- and dimannoside complexes crystallized in space group P21212, whereas the trimannoside-complex crystals were monoclinic, space group C2. Crystallographic data on these and all of the other monocot lectins characterized to date are given in Table 1.
| Unit Cell Parameters |
| Monocot Lectins | Space Group | a(Å) | b(Å) | c(Å) | b (o) | No. Monomers/ Asymmetric Unit | Resolution (Å) | References |
|
SCA-MAN (Native) |
P21212 |
70.8 |
93.7 |
46.9 |
- |
2 |
1.7 |
4 |
| SCA-MAN - alphaMan | P21212 | 70.7 | 93.0 | 47.1 | - | 2 | 1.6 | 12 |
| SCA-MAN - Man-alpha1,6-Man | P21212 | 70.6 | 92.8 | 47.3 | - | 2 | 1.6 | 13 |
| SCA-MAN - Man alpha1,6(Man-alpha1,3)Man | C2 | 100.5 | 121.0 | 89.0 | 105.6 | 6 | 2.5 | 14 |
| GNA - Me-alphaMan | P212121 | 138.8 | 64.1 | 62.1 | - | 4 | 2.3 | 3 |
| GNA - Man-alpha1,3-Man | I213 | 138.2 | 138.2 | 138.2 | - | 2 | 3.0 | 15 |
| GNA - Man-alpha1,6- (alpha1,3-Man)Man- alpha1,6(alpha1,3-Man) | I4122 | 96.3 | 96.3 | 68.4 | - | 1 | 2.0 | 16 |
| HHA - alphaMan | C2 | 73.4 | 100.3 | 62.2 | 137.3 | 2 | 2.3 | 8 |
| NPA - Man-alpha1,3-Man | C222 | 73.1 | 100.9 | 37.0 | - | 1 | 2.0 | 17 |
Table 1. Crystallographic data of all monocot lectins characterized to date
Structure Determination
The co-ordinates of the partially refined amaryllis structure (8) which has 54% sequence identity and 81% similarity to SCA-MAN were used in the molecular replacement (MR) calculations with AMoRe (9) to solve the native SCA-MAN structure. The model obtained from the MR studies of the bluebell lectin SCA-MAN was corrected for sequence differences and refined (XPLOR (10) and REFMAC (11)), for both the native and complex structures. Difference density was clearly visible for the 7- and 4-residue inserts after the first strand of beta-sheet, close to the N-terminus. The insertion was modelled and refined for all four SCA-MAN structures. The native lectin structure was refined further by the insertion of solvent molecules, and the saccharide units for the three complexes.
Discussion
Structure of the Molecule
Recent crystallographic studies on the monocot bulb lectins have resulted in the structure determinations of snowdrop (Galanthus nivalis) lectin (3) and amaryllis (Hippeastrum) lectin (8). Galanthus nivalis agglutinin (GNA), Hippeastrum hybrid agglutinin (HHA) and SCA-MAN all contain a tertiary structure consisting of three sequential beta-sheet subdomains (I, II and III) related by a pseudo 3-fold axis of symmetry. Each subdomain comprises a flat, 4-stranded, antiparallel beta-sheet so that the monomer structure forms a triangular-shaped 12-stranded beta-barrel-like fold (Figure 2). Two monomers form stable dimers in which their C-terminal strands are exchanged. Two dimers aggregate to give a loose tetramer.
Figure 2. SCA-MAN Monomer. Each monomer has three four-stranded -sheets which overall form a twelve-stranded beta-barrel-like fold. Each beta-sheet subdomain contains a saccharide-binding site. Figure prepared using MOLSCRIPT (18).
Subdomain I is a hybrid beta-sheet made up of both the N-terminal and C-terminal regions with the outer strand donated by the second monomer that forms the dimer.Saccharide Binding SitesLike GNA and HHA, each subdomain of SCA-MAN contains a saccharide-binding site (Figure 2). Sequence alignment of corresponding subdomains of SCA-MAN clearly illustrates the high degree of internal sequence identity (Figure 3).
Figure 3a. The SCA-MAN monomer: Alignment of the amino acid sequences of SCA-MAN, HHA and GNA. The % identity of HHA to SCA-MAN is 54% while the % identity of GNA to SCA-MAN is 52%. The similarity of HHA to SCA-MAN is 81% and the % similarity of GNA to SCA-MAN is 75%.
Figure 3b. The SCA-MAN monomer: Sequence alignment of corresponding subdomains in the SCA-MAN monomer. Subdomain I is on the left-hand side and subdomain III is on the right-hand side.
Figure 3c. The SCA-MAN monomer: Stereo plot of the SCA-MAN monomer.
The binding of alpha-D-mannose at the subdomain I site is illustrated in Figures 4 and 5.
Figure 4. Subdomain I saccharide-binding siteThe electron density clearly shows the alpha-D-mannose residue position. Figure prepared using SETOR (19).
Figure 5. A schematic diagram to show hydrogen bonding contacts between the alpha-D-mannose residue and residues of the saccharide-binding site in subdomain I. Diagram generated by LIGPLOT (20).
Several hydrogen-bond contacts are made with the hydroxyl groups of mannose. The 2-OH group interacts with the side chains of Asp 102 and Asn 104 and a water molecule; the 3-OH group with Gln 100 and Gln 8; the 4-OH group with Tyr 108 and the 6-OH group with three water molecules. The 1-OH group is also involved in making a direct H-bond to Asp 10 and makes van der Waals contacts with His 94. In the SCA-MAN - Man- 1,6-Man complex the disaccharide is positioned with its reducing end in the binding pocket. The reducing sugar forms hydrogen bond contacts with the protein residues in this region in a similar way to that found for the monosaccharide - SCA-MAN complex, whereas the non-reducing mannose residue does not make any hydrogen bonds with residues in the protein, only with solvent molecules (Figures 6 and 7)
Figure 6 Subdomain I saccharide-binding site in the SCA-MAN - Man-alpha1,6-Man complex. Figure generated using SETOR (19).
Figure 7. A schematic diagram to show the hydrogen bonding contacts between the dimannose residue and residues of the saccharide-binding site in subdomain I. Diagram generated by LIGPLOT (20).
Refinement of our SCA-MAN - trimannose complex (14) is currently in progress. Preliminary results suggest that the Man alpha1-6 (Man alpha1-3) Man does not bind at subdomain I as found in the mono- and dimannose SCA-MAN complexes. There is, however, clear electron density for all three mannose rings at subdomain III (Figure 8), with a binding mode in which the second (or internal) mannose unit of the trisaccharide is bound at the conserved monosaccharide site (Figure 9) in a similar fashion to that found for the GNA - mannopentose complex (16).
Figure 8. Subdomain III saccharide-binding site in the SCA-MAN - trimannoside complex. Figure prepared using SETOR (19).
Figure 9. A schematic diagram to show the hydrogen bonding contacts between the trimannoside and residues of the saccharide-binding site in subdomain III. Diagram generated by LIGPLOT (20).
Further studies of SCA-MAN in complex with other, more-complicated mannosides are underway. It is hoped that by analysing the structures of the native SCA-MAN with a variety of carbohydrate complexes the mechanism by which lectins bind specifically and reversibly to carbohydrates will be better understood. Full publications of each of the structures described above will be given elsewhere in more detail, at a later date.
Acknowledgements
The authors gratefully acknowledge financial support for this research from The Leverhulme Trust (F/754/A) and the Mitzutani Foundation for Glycoscience, CCLRC Daresbury Laboratory for the provision of beam-time and other facilities, and Liverpool John Moores University for general support. We thank the staff of stations 7.2, 9.5 and 9.6 at Daresbury Laboratory for their assistance and Professor E.H. Evans for her encouragement and interest in this project.
References
| THE COMPLETE REFERENCE TO THIS PAPER IS:
http://plab.ku.dk/tcbh/Lectins12/Wright/paper.htm Lectins, Biology, Biochemistry, Clinical Biochemistry, Volume 12, including Proceedings from the 17th International Lectin Meeting in Würzburg, 1997,edited by Edilbert van Driessche, Sonia Beeckmans and Thorkild C. Bøg-Hansen, published by TEXTOP, Lemchesvej 11, DK-2900 Hellerup, Denmark, ISBN number 87-984583-0-2 |