Structural Studies on Non-toxic Homologues of type II Ribosome Inactivating Proteins (RIPs)
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This thesis is concerned with structural and related studies on two non-toxic homologues of type II RIPs. Type II RIPs, which are more toxic than type I, contain an additional lectin chain (B-chain) covalently linked to the catalytic chain (A-chain). The lectin chain facilitates the attachment of the protein to the cell surface, leading to the internalisation of the protein through endocytosis. The catalytic chain has N-glycosidase activity and is made up of three domains, whereas the lectin chain consists of two homologous β-trefoil domains carrying a sugar binding site each. Interestingly, non-toxic homologues of type II RIPs also exist in nature. The present thesis involves studies on two such non-toxic proteins, namely, snake gourd seed lectin (SGSL) and bitter gourd seed lectin (BGSL), with the primary objective of elucidating the structural basis for the absence of their toxicity. In the case of SGSL, the catalytic chain is cleaved into two, although it does not affect the structural integrity of the molecule. This is the first time a three-chain RIP/RIP homologue is observed. It was observed that the non-toxicity of SGSL results from a combination of changes in the catalytic and the carbohydrate-binding sites. The structure determination of the SGSL in its native and in complex with Me-α-Gal and Lac led to the elucidation of the molecular geometry of the protein and the binding sites. ITC measurements of the SGSL-sugar interactions showed the presence of a single binding site which was in contrast to the two binding sites observed in crystal structures. In an attempt to resolve this anomaly, molecular dynamic simulations were carried out on the native protein as well on its complex with Me-α-Gal and lactose. The simulations again confirmed the robustness of the protein structure and indicated that the binding of sugar at only one site is stable. The structures of nine independent crystals of BGSL and its sugar complexes have been determined. BGSL is a four-chain, two-fold symmetric molecule, made up of two identical two-chain modules, each consisting of a catalytic chain and a lectin chain, connected by a disulphide bridge. Unlike in other type II RIPs, BGSL has a sugar-binding site only on one lectin domain. The adenine-binding site in the catalytic chain is defective. These subtle changes appear to contribute to the non-toxicity of the lectin. The plasticity of the molecule is mainly caused by the presence of two possible well-defined conformations of a surface loop in the lectin chain. One of them, chosen in the sugar complexes, facilitates an additional interaction with the bound sugar. The N-glycosylation of the lectin involves a plant-specific glycan while that in toxic type II RIPs of known structure involves a glycan, which is animal as well as plant-specific. Preliminary investigations on the anti-tumor activity of SGSL and BGSL, indicated their preference for neoplastic cells and their ability to induce apoptosis in them.