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Structural basis for the interaction of [E160A-E189A]-trichosanthin with adenine
Pang-Chui Shaw1, Kam-Bo Wong, Denise S-B Chan
1Department of Biochemistry, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, People's Republic of China. pcshaw@cuhk.edu.hk
Summary
Trichosanthin, a ribosome-inactivating protein, binds adenine. Mutating Glu-160 and Glu-189 did not alter adenine binding or active site structure, but reduced activity.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Biology
Background:
- Trichosanthin is a plant-derived ribosome-inactivating protein (RIP) known to cleave 28S rRNA.
- Understanding the structure-function relationship of trichosanthin is crucial for its therapeutic applications.
Purpose of the Study:
- To investigate the structural basis of adenine binding by trichosanthin.
- To elucidate the role of Glu-160 and Glu-189 residues in the enzymatic activity and adenine interaction of trichosanthin.
Main Methods:
- X-ray crystallography was used to determine the co-crystal structure of the [E160A-E189A]-trichosanthin variant with adenine at 0.193nm resolution.
- Binding affinity studies were performed to assess the interaction between trichosanthin variants and adenine.
Main Results:
- The crystal structure revealed that the active site conformation of [E160A-E189A]-trichosanthin is similar to the wild-type, with water molecules occupying the positions of the mutated glutamate residues.
- Adenine binding to [E160A-E189A]-trichosanthin occurred in a manner comparable to the wild-type protein.
- Mutation of Glu-160 and Glu-189 did not significantly affect adenine binding but resulted in a positively charged surface patch, potentially destabilizing the transition state and reducing ribosome-inactivating activity.
Conclusions:
- Glu-160 and Glu-189 are not critical for maintaining the active site conformation or for adenine binding in trichosanthin.
- The observed decrease in ribosome-inactivating activity in the mutated variant is likely due to altered surface charge distribution impacting transition-state stabilization.