title: Structural basis for the interaction of [E160A-E189A]-trichosanthin with adenine.
abstract: Trichosanthin is a ribosome-inactivating protein that cleaves specifically the N-glycosidic bond of A-4324 of 28S rRNA. Trichosanthin and its variant [E160A-E189A]-trichosanthin were found to bind an adenine base with a K(d) value of approximately 0.2mM. To determine how this doubly mutated variant of trichosanthin interacts with adenine, the co-crystal structure of [E160A-E189A]-trichosanthin and adenine was resolved to 0.193nm which revealed that the active site conformation of the doubly mutated variant is isomorphous to wild-type trichosanthin. Water molecules were found at locations corresponding to the eliminated side chain of Glu-160 and Glu-189. On the other hand, the adenine base interacted with [E160A-E189A]-trichosanthin in a manner similar to that in wild-type trichosanthin. Our structural analysis illustrates that Glu-160 and Glu-189 in trichosanthin do not play an important role in maintaining the active site conformation and binding adenine, an essential step for substrate-enzyme interaction. On the other hand, removal of two glutamate residues changed a large patch of negatively charged surface to a positive charge, which may account for the destabilization of the oxocarbenium-like transition-state and the significant decrease in ribosome-inactivating activity in [E160A-E189A]-trichosanthin.
authors.affiliation: Department of Biochemistry, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, People's Republic of China. pcshaw@cuhk.edu.hk
authors.forename: Pang-Chui
authors.initials: PC
authors.lastname: Shaw
authors.forename: Kam-Bo
authors.initials: KB
authors.lastname: Wong
authors.forename: Denise S-B
authors.initials: DS
authors.lastname: Chan
authors.forename: Roger L
authors.initials: RL
authors.lastname: Williams
mesh_terms: D000225:Adenine / Q000737:chemistry*; D018360:Crystallography, X-Ray; D005517:Foodborne Diseases / Q000517:prevention & control; D006801:Humans; D006860:Hydrogen Bonding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D015978:Trichosanthin / Q000737:chemistry*
chemical_list: D015978:Trichosanthin; D000225:Adenine