Incorporation of a glycine within the conserved TCPCP motif of human neuronal growth inhibitory factor significantly reduces its bioactivity.
It has been reported that the (6)CPCP(9) 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 (6)CPCP(9) 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 (6)CPCP(9) 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 (6)CPCP(9) 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 (6)CPCP(9) 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.
Chemical Biology Laboratory, Department of Chemistry, Fudan University, 220 Han-Dan Road, Shanghai 200433, China.
Zhi-Chun
ZC
Ding
Dong
D
Chen
Feng-Yun
FY
Ni
Qi
Q
Zheng
Bin
B
Cai
Wen-Hua
WH
Yao
Yang
Y
Wang
Guo-Ming
GM
Zhou
Zhong-Xian
ZX
Huang
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D017124:Conserved Sequence; D005998:Glycine / Q000737:chemistry / Q000235:genetics; D006801:Humans; D000090023:Metallothionein 3; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats
D000090023:Metallothionein 3; D009419:Nerve Tissue Proteins; D005998:Glycine