Structural studies on Peanut Lectin-Carbohydrate complexes

Abstract

Lectins are carbohydrate-binding proteins that specifically recognize diverse sugar structures and mediate a variety of biological processes such as cell-cell and host-pathogen interactions, serum glycoprotein turnover, and innate immune responses. There has been a spurt in lectin research in recent years due to their ability to specifically bind to cell surface carbohydrates and their diverse applications. A rich source of lectins are the seeds of leguminous plants, but they are also found in all classes and families of organisms. Legume lectins have similar tertiary structures but exhibit a large variation in their quaternary structures. The carbohydrate-binding site in all of them is made up of four loops. Three of the loops are substantially conserved in all legume lectins of known structure, while the fourth loop, which is variable, is thought to confer specificity. Legume lectins, which share the same monosaccharide specificity, often exhibit markedly different oligosaccharide specificities. The introductory chapter gives a broad overview of lectins from a structural point of view.

The rest of the thesis is concerned with structural studies on the carbohydrate complexes of peanut (Arachis hypogaea) agglutinin (PNA), a non-glycosylated tetrameric lectin of Mr 110,000 Da with 236 amino acid residues in each subunit. PNA specifically recognizes the tumor-associated Thomsen-Friedenreich antigen (T-antigen; Gal?1-3GalNAc), at the disaccharide level.

Diffraction data from the complexes were collected on a Siemens-Nicolet area detector system mounted on a GX-20 Marconi avionics rotating anode X-ray generator and a MAR imaging plate system mounted on a Rigaku RU200 rotating anode generator. The data were processed using the XENGEN, MAR-XDS, and DENZO/SCALEPACK suites of programs. The molecular replacement calculations were carried out using AMoRe. The refinements were performed using PROLSQ and X-PLOR.

The observed variability in quaternary association in legume lectins was earlier thought to arise mainly due to the presence of covalently bound sugar at the intersubunit interface. The structure of PNA (a non-glycosylated protein) indicates that the observed variability in the quaternary association of legume lectins is caused by factors intrinsic to the protein itself. At physiological pH, PNA exists as an 'open' tetramer with four identical polypeptide chains, which does not exhibit the 222 (D2) or the 4-fold (C4) point group symmetry expected in a tetramer. The structure of its complex with lactose (Gal?1-4Glc) at 2.25 Å, in the final refinement and analysis of which the author participated, highlights the differences in carbohydrate binding between galactose/N-acetylgalactosamine-specific legume lectins as opposed to glucose/mannose-specific legume lectins. The detailed analysis of the tertiary structure brings to focus features of the legume lectin fold not sufficiently appreciated in earlier work. Furthermore, in legume lectins, more than 50% of the tertiary structure exists in loops. An analysis of protein hydration demonstrates, among other things, the role of water molecules in stabilizing the structure of these loops.

The X-ray analysis of the complex with T-antigen shows that the 20 times higher affinity of the protein for T-antigen compared to that for lactose arises primarily due to two additional water bridges. The structures of the N-acetyllactosamine (Gal?1-4GlcNAc) and the methyl-?-galactose complexes, and a comparative study involving these and the other complexes of the lectin, indicate that the role of interacting sugar hydroxyls, when absent, is often mimicked by ordered water molecules, not only at the primary combining site but also at the site of the second sugar ring in the lectin. The similarity of peanut lectin-sugar interactions with those in other Gal/GalNAc-specific lectins extends, to a substantial degree, to water bridges as well. The study provides a structural explanation for the exclusive specificity of peanut lectin for galactose at the monosaccharide level, as opposed to that of other lectins for galactose as well as N-acetylgalactosamine. The complexes also provide a qualitative structural rationale for differences in the strengths of the binding of peanut lectin to different sugars. The complex with C-lactose demonstrates unambiguously that the synthetic sugar mimics its natural counterpart in its interaction with PNA.

The structures of the complexes of PNA with lactose at acidic pH were solved in a monoclinic form and a triclinic form at resolutions of 2.6 and 3.5 Å, respectively. The quaternary structure of the lectin at this pH remains the same as that at physiological pH. The former contains two tetramers in the asymmetric unit. Sugar binding is observed in three subunits in this form. It is prevented in two subunits by interactions with neighboring molecules. Two other subunits have no sugar bound to them, although the combining sites are accessible. Some water molecules at these sites occur close to the positions of carbohydrate hydroxyl groups in sugar-bound subunits. A loop in a neighboring molecule binds to the carbohydrate-binding site in one subunit by partly mimicking the interactions of the glucose moiety in lactose. Furthermore, some of the oxygen atoms of this peptide stretch are topologically equivalent to some of the sugar hydroxyls. This same loop interacts in an analogous manner with the carbohydrate-binding site in two subunits in the triclinic form, which contains a tetramer in the unit cell. It was not possible to interpret the density (possibly for lactose) at the binding site of the other two subunits in this form on account of the inferior resolution. The differences in the crystal packing between the orthorhombic, monoclinic, and triclinic forms have been attempted to be rationalized based on the high affinity of the protein for the peptide loop at acidic pH.

While pursuing structural studies on the PNA complexes, the author has also been involved in the crystal structure determination of the key DNA repair enzyme from E. coli Uracil DNA Glycosylase (UDGase) in its complex with a proteinaceous inhibitor. This work is described in Appendix A. The author has also participated in an ongoing small molecule project in the laboratory, which involves preparation and X-ray structure determination of the complexes of L- and DL-amino acids with carboxylic acids. The work carried out in the project is described in Appendix B.

A part of the results described in this thesis has already been reported in the following publications:

Banerjee, R., Das, K., Ravishankar, R., Suguna, K., Surolia, A. & Vijayan, M. (1996). Conformation, protein-carbohydrate interactions and a novel subunit association in the refined structure of peanut lectin-lactose complex. J. Mol. Biol., 259, 281-296.

Ravishankar, R., Ravindran, M., Suguna, K., Surolia, A. & Vijayan, M. (1996). Crystal structure of the Peanut lectin-T-antigen complex. Prog. Biophys. Mol. Biol. 65 Suppl. 1, 33.

Ravishankar, R., Ravindran, M., Suguna, K., Surolia, A. & Vijayan, M. (1997). Crystal structure of the peanut lectin-T-antigen complex. Carbohydrate specificity generated by water bridges. Current Science, 72, 855-861.

Ravishankar, R., Nagasuma Chandra, R. & Vijayan, M. (1998). X-ray studies on crystalline complexes involving amino acids and peptides XXXIV. Novel mode of aggregation, Interaction patterns and chiral effects in the maleic acid complexes of DL- and L-Arginine. J. Biomol. Struct. Dyn., 15, 1093-1100.

Ravishankar, R., Surolia, A., Vijayan, M., Lim, S. & Kishi, Y. (1998). Preferred conformation of C-lactose at the free and peanut lectin bound states. J. Am. Chem. Soc., 120, 11297-11303.

Ravishankar, R., Bidya Sagar, M., Roy, S., Pumapatre, K., Handa, P., Varshney, U. & Vijayan, M. (1998). X-ray analysis of a complex of Escherichia coli Uracil DNA Glycosylase (EcUDG) with a proteinaceous inhibitor. The structure elucidation of a prokaryotic UDG. Nucl. Acids Res., 26, 4880-4887.

Ravishankar, R., Suguna, K., Surolia, A. & Vijayan, M. (1999). The crystal structures of the complexes of peanut lectin with Methyl-?-galactose and N-acetyllactosamine and a comparative study of carbohydrate binding in Gal/GalNAc specific legume lectins. Acta Cryst. Sect. D (in press).