The structural basis of Trp192 and the C-terminal region in trichosanthin for activity and conformational stability.
Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) possessing N-glycosidase activity. TCS has various pharmacological properties, including immunomodulatory, anti-tumor and anti-HIV activities. Up to seven C-terminal residues of TCS (TCS-C7) can be deleted resulting in lower antigenicity with minimal effects on its activity. However, an additional problem is that the minimal effects on activity are higher than the reduction in antigenicity. In the present work, the crystal structure of TCS-C7 was determined. It shows the details of the C-terminal residues of TCS-C7, and in particular the hydrogen bonds between P35 and L240, S196 and L240, and W192 and L239, which play an important role in maintaining the structure of TCS-C7. Further analysis shows that the hydrogen bonds related to Leu240 are key in maintaining the relationship between N- and C-terminal domains. The major role of the C-terminal tail appears to stabilize the structure of TCS. The conformation between helix H7 at the N-terminal domain and the C-terminal tail at the C-terminal domain is also revealed. Two mutants, TCS-W192F and TCS-C7-W192F, were prepared and crystal structures were determined. These variants have greatly reduced ribosome-inactivating activities compared with TCS and TCS-C7, respectively, and TCS-W192F and TCS-C7-W192F have a similar stability in guanidine hydrochloride compared with TCS-C7. This suggests that Trp192 can affect the ribosome-inactivating activity of TCS.
Ding
Yi
Y
Laboratory of Structural Biology and the MOE Laboratory of Protein Science, School of Life Science and Engineering, Tsinghua University, Beijing 100084, China.
Too
Hiumei
H
Wang
Zhilong
Z
Liu
Yiwei
Y
Bartlam
Mark
M
Dong
Yicheng
Y
Wong
Kambo
K
Shaw
Pangchui
P
Rao
Zihe
Z
D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D009154:Mutation; D011487:Protein Conformation; D012270:Ribosomes / Q000378:metabolism; D013329:Structure-Activity Relationship; D015978:Trichosanthin / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism*
D015978:Trichosanthin; D014364:Tryptophan