TY - JOUR
T1 - Incorporation of a glycine within the conserved TCPCP motif of human neuronal growth inhibitory factor significantly reduces its bioactivity
AU - Ding, Zhi-Chun
AU - Chen, Dong
AU - Ni, Feng Yun
AU - Zheng, Qi
AU - Cai, Bin
AU - Yao, Wen Hua
AU - Wang, Yang
AU - Zhou, Guo Ming
AU - Huang, Zhong Xian
N1 - Funding Information:
We thank the National Science Foundation of China (NSFC) for the financial support.
PY - 2008/8/8
Y1 - 2008/8/8
N2 - It has been reported that the 6CPCP9 motif near the N-terminus is pivotal to the inhibitory activity of human neuronal growth inhibitory factor (hGIF). In order to better understand the biological significance of this region on the structure, property and function of hGIF, we introduced a highly flexible residue, Gly, either in front of the 6CPCP9 motif (the IG6 mutant, TGCPCP) or in the middle of it (the IG8 mutant, TCPGCP) and investigated their structural and metal binding properties in detail. The results showed that the overall structure and the stability of the metal-thiolate clusters of the two mutants were comparable to that of hGIF. However, the bioassay results showed that the bioactivity of the IG6 mutant decreased significantly, while the bioactivity of the IG8 mutant was almost abolished. Molecular dynamics simulation results showed that the backbone of the IG6 mutant exhibited high similarity to that of hGIF, and the two prolines could still induce structural constraints on the 6CPCP9 tetrapeptide and form a similar conformation with that of hGIF, however, the conformation of the first five amino acid residues in the N-terminus was quite different. In hGIF, the five residues are twisted and form a restricted conformation, while in the IG6 mutant this peptide extends more naturally and smoothly, which is similar to that of MT2. As to the IG8 mutant, the Gly insertion broke the 6CPCP9 motif, thus probably abolishing the interactions with other molecules and eliminating its inhibitory activity. Based on these results, we suggested that although the structure adopted by the 6CPCP9 motif is the determinant factor of the inhibitory bioactivity of hGIF, other residues within the N-terminal fragment (residue 1-13) may also influence the peptide conformation and contribute to the protein's bioactivity.
AB - It has been reported that the 6CPCP9 motif near the N-terminus is pivotal to the inhibitory activity of human neuronal growth inhibitory factor (hGIF). In order to better understand the biological significance of this region on the structure, property and function of hGIF, we introduced a highly flexible residue, Gly, either in front of the 6CPCP9 motif (the IG6 mutant, TGCPCP) or in the middle of it (the IG8 mutant, TCPGCP) and investigated their structural and metal binding properties in detail. The results showed that the overall structure and the stability of the metal-thiolate clusters of the two mutants were comparable to that of hGIF. However, the bioassay results showed that the bioactivity of the IG6 mutant decreased significantly, while the bioactivity of the IG8 mutant was almost abolished. Molecular dynamics simulation results showed that the backbone of the IG6 mutant exhibited high similarity to that of hGIF, and the two prolines could still induce structural constraints on the 6CPCP9 tetrapeptide and form a similar conformation with that of hGIF, however, the conformation of the first five amino acid residues in the N-terminus was quite different. In hGIF, the five residues are twisted and form a restricted conformation, while in the IG6 mutant this peptide extends more naturally and smoothly, which is similar to that of MT2. As to the IG8 mutant, the Gly insertion broke the 6CPCP9 motif, thus probably abolishing the interactions with other molecules and eliminating its inhibitory activity. Based on these results, we suggested that although the structure adopted by the 6CPCP9 motif is the determinant factor of the inhibitory bioactivity of hGIF, other residues within the N-terminal fragment (residue 1-13) may also influence the peptide conformation and contribute to the protein's bioactivity.
KW - Metallothionein
KW - Molecular dynamics simulation
KW - Mutants
KW - Neuronal cell culture
KW - Neuronal growth inhibitory factor (GIF)
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U2 - 10.1016/j.bbrc.2008.05.136
DO - 10.1016/j.bbrc.2008.05.136
M3 - Article
C2 - 18533104
AN - SCOPUS:45449083790
SN - 0006-291X
VL - 372
SP - 779
EP - 784
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 4
ER -