Function/structure relationship study of trichosanthin, a Chinese medicinal protein, and its interaction with acidic ribosomal protein, P0

Trichosanthin (TCS) is a Chinese medicinal protein isolated froth the root tuber of Trichosanthes kirilowi Maximowicz. It is a 27kDa protein with multiple pharmacological properties, including abortifacient, anti-tumor and anti-human immunodeficiency virus (HIV). It is believed that the pharmacological properties of TCS are related to ribosome-inactivation, by breaking, the specific glycosidic bond of adenine 4324 from the 28S rRNA.;The first topic of my research is to find the role of Glu-85. The structure of [E85Q]-TCS and AMP complex was obtained. It is deduced that there are two sites for substrate binding in TCS, one is for recognition and another ion hydrolysis. The structure also indicated that protonation of substrate adenine is carried out by a water molecule in the active pocket of TCS during its N-glycosidase action.;Previous research showed that the C-terminal tail of TCS can be deleted to generate a mini-TCS (C7-TCS) with antigenicity. The second topic of my study is to resolve the role of the C-terminal of TCS. Structure of C7-TCS showed that deletion of the C-terminal tail destabilizes the protein structure and makes Trp192 more solvent exposed. The relationship between the C-terminal tail and Trp192 was determined by mutating Trp192 to Phe in wild-type TCS and C7-TCS, generating W192F-TCS and W192F-C7-TCS. The crystal structure of C7-TCS, [W192F]-TCS and [W192F]-C7-TCS were determined and compared. Trp192 was identified as an important residue in stabilizing the conformation of TCS. Besides, the accumulative effect of Trp192 and the C-terminal tail is significant on the ribosome-inactivating activity. By comparing the structures, it was found that, the hydrogen bond formed by amino acids 240 and 35 seems to be essential for the structure and amino acid 240 should be a critical residue for the connection of the N-terminal and C-terminal domains in trichosanthin.;Ribosome-inactivating activity is the most important activity of TCS and RIPs. Therefore, the third topic of my study is to find the important of interaction between TCS and ribosomal proteins. Two ribosomal proteins, P0 and P1, have been identified previously to interact with TCS. By yeast two-hybrid screening, three cut of ten charge residues in TCS were identified to be the interaction sites between TCS and ribosomal protein P0. The interaction region was located on the surface of TCS near the entrance to the active pocket. The interaction with P0 was shown to be carried out by electrostatic interaction between the positively charge residues of TCS. However, the mutation of all the concerned residues in TCS gave only a mild reduction in inhibiting the protein synthesis of an in vitro reticulocyte translation system, showing that the interaction between TCS and P0 only plays a minor role in the ribosomal inactivating activity of TCS.