Substrate binding and catalysis in trichosanthin occur in different sites as revealed by the complex structures of several E85 mutants.
Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) which possesses rRNA N-glycosidase activity. In recent years, its immunomodulatory, anti-tumor and anti-HIV properties have been revealed. Here we report the crystal structures of several E85 mutant TCS complexes with adenosine-5'-monophosphate (AMP) and adenine. In E85Q TCS/AMP and E85A TCS/AMP, near the active site of the molecule and parallel to the aromatic ring of Tyr70, an AMP molecule is bound to the mutant without being hydrolyzed. In the E85R TCS/adenine complex, the hydrolyzed product adenine is located in the active pocket where it occupies a position similar to that in the TCS/NADPH complex. Significantly, AMP is bound in a position different to that of adenine. In comparison with these structures, we suggest that there are at least two subsites in the active site of TCS, one for initial substrate recognition as revealed by the AMP site and another for catalysis as represented by the NADPH site. Based on these complex structures, the function of residue 85 and the mechanism of catalysis are proposed.
Guo
Qing
Q
Laboratory of Structural Biology and the MOE Laboratory of Protein Science, School of Life Science and Engineering, Tsinghua University, Beijing 100084, China.
Zhou
Weihong
W
Too
Hiu-Mei
HM
Li
Jianhui
J
Liu
Yiwei
Y
Bartlam
Mark
M
Dong
Yicheng
Y
Wong
Kam-Bo
KB
Shaw
Pang-Chui
PC
Rao
Zihe
Z
D000225:Adenine / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism*; D019380:Anti-HIV Agents / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics*; D009249:NADP / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D015978:Trichosanthin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D019380:Anti-HIV Agents; D000249:Adenosine Monophosphate; D009249:NADP; D015978:Trichosanthin; D000225:Adenine