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 0.2 mM. 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 0.193 nm 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.