Starting with the structure analysis of ConA in the 70s, the crystal structures of hundreds of different lectins and their carbohydrate complexes have been determined. Lectins, multivalent carbohydrate-binding proteins which specifically bind different sugar structures, have received considerable attention in recent times on account of the realization of the importance of protein−sugar interactions, especially at the cell surface, in biological recognition. They occur in plants, animals, fungi, bacteria and viruses. Plant lectins constitute about 40% of the lectins of known structure. They can be classified into five structural groups, each characterized by a
specific fold. Among them, legume lectins constitute the most extensively investigated group. Peanut lectin is a legume lectin which has been studied thoroughly in this laboratory. These studies have provided a wealth of structural and functional information. However, some gaps still exist in our understanding of the structure, interactions and multivalency of peanut
lectin. The work presented here addresses these gaps.
The hanging drop method was used for crystallizing PNA and its complexes. Intensity data were collected on Mar Research imaging plates mounted on Rigaku RU-200 or ULTRAX-18 X-ray generators. The Oxford
cryosystem was used when collecting data at low temperature. The data were processed using DENZO and SCALEPACK of HKL suite of
programs. The structure factors from the processed data were calculated using TRUCATE of CCP4 suite of programs. The molecular replacement program AMoRe was used for structure solutions. Structure refinements
were carried out using the CNS software package and REFMAC of CCP4. Model building was done using the molecular graphics program FRODO. INSIGHT II, ALIGN, CONTACT and PROCHECK of CCP4 were used for
the analysis and validation of the refined structure. Dynamic light scattering
experiments were carried out using a Dyanpro Molecular Sizing Instrument, and the collected data were analyzed using Dynamic V6 software.
Until recently, it has been possible to grow crystals of peanut lectin only when complexed with sugar ligands. It has now been possible to grow them at acidic pH in the presence of oligopeptides corresponding to a loop in the lectin molecule. Crystals have also been prepared in the presence of
the peptides as well as lactose. Low pH crystal forms of the lectin−lactose complex similar to those obtained at neutral pH could also been grown. Thus, crystals of peanut lectin grown in different environmental conditions, at two pHs with and without sugars bound to the lectin, are now available. They have been used to explore the plasticity and hydration of the molecule. A detailed comparison among different structures shows that the lectin
molecule is sturdy and the effect of changes in pH, ligand-binding and environment on it is small. The region involving the curved front β-sheet and loops around the second hydrophobic core is comparatively rigid. The
back β-sheet involved in quaternary association, which exhibits considerable
variability, is substantially flexible. So is the sugar-binding region. The numbers of invariant water molecules in the hydration shell are small and they are mainly involved in metal coordination or in stabilizing rare
structural features. Small, consistent movements occur in the combining site on sugar-binding, although the site is essentially preformed.
Crystal structures of peanut lectin complexed with Galβ1-3Gal, methyl-T-antigen, Galβ1-6GalNAc, Galα1-3Gal and Galα1-6Glc and that of a crystal grown in the presence of Galα1-3Galβ1-4Gal have been determined using data collected at 100 K. Use of water bridges as a strategy for generating carbohydrate specificity was earlier deduced from the complexes of the lectin with lactose (Galβ1-4Glc) and T-antigen (Galβ1-
3GalNAc). This has been confirmed through the analysis of the complexes with Galβ1-3Gal and methyl-T-antigen (Galβ1-3GalNAc-α-OMe). A detailed analysis of lectin−sugar interactions in the complexes shows that
they are more extensive when β-anomer is involved in the linkage. As expected, the second sugar residue is ill defined when the linkage is 1-6. There are more than two-dozen water molecules, which occur in the
hydration shells of all structures determined at resolutions better than 2.5 Å.
Most of them are involved in stabilizing the structure, particularly loops. Water molecules involved in lectin−sugar interactions are also substantially
conserved. The lectin molecule is robust and does not appear to be affected by change in temperature.
Multivalency is believed to be important in the activity of lectins, although definitive structural studies on it have been few and far between. A study has been carried out on the complexation of tetravalent peanut lectin
with a synthetic compound containing two terminal lactose moieties, using a combination of crystallography, dynamic light scattering and modelling. Light scattering indicates the formation of an apparent dimeric species and also larger aggregates of the tetrameric lectin in the presence of the bivalent ligand. The crystals of presumably crosslinked lectin molecules could be obtained. They diffract very poorly, but the X-ray data from them are good enough to define the positions of the lectin molecules. Extensive modelling
on possible crosslinking modes of protein molecules by the ligand indicated that systematic crosslinking could lead to crystalline arrays. The studies also
provided a rationale for the crosslinking in the observed crystal structure. The results obtained provide further insights into the general problem of multivalency in lectins. They indicate that crosslinking involving
multivalent lectins and multivalent carbohydrates is likely to lead to an
ensemble of a finite number of distinct periodic arrays rather than a unique
array.
PNA is among the most thoroughly studied lectins. Its structure demonstrated that open structures without point group symmetry cannot be ruled out for oligomeric proteins. It also contributed to the identification of
legume lectins as a family of proteins in which small alterations in essentially the same tertiary structure lead to large changes in the quaternary association. Among other things, studies on PNA−sugar complexes led to the identification of water bridges as a strategy for generating carbohydrate
specificity in addition to providing detailed information on PNA−sugar
interactions. The work reported here significantly added to the information
on this important lectin provided by earlier studies. On the basis of a detailed examination of structures of crystals grown under different environmental conditions, the relatively rigid and flexible regions of the molecule could be delineated. The picture that emerges is that of a robust
protein with a substantially preformed combining site. The work also added to the information on the dependence of protein−sugar interactions on the different glycosidic linkages in disaccharides. The investigations reported here also provided further insights into the multivalency of peanut lectin.
| Identifer | oai:union.ndltd.org:IISc/oai:etd.ncsi.iisc.ernet.in:2005/405 |
| Date | 08 1900 |
| Creators | Natchiar, S Kundhavai |
| Contributors | Vijayan, M |
| Source Sets | India Institute of Science |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Relation | G20301 |
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