Important roles of the conserved linker-KKS in human neuronal growth inhibitory factor.
Metallothinein-3 (MT3), also named neuronal growth inhibitory factor (GIF), is attractive by its distinct neuronal growth inhibitory activity, which is not shared by other MT isoforms. The polypeptide chain of GIF is folded into two individual domains, which are connected by a highly conserved linker, KKS. In order to figure out the significance of the conserved segment, we constructed several mutants of human GIF (hGIF), including the K31/32A mutant, the K31/32E mutant and the KKS-SP mutant by site-directed mutagenesis. pH titration and DTNB reaction exhibited that all the three mutations made the beta-domain lower in stability and looser. More significantly, change of KKS to SP also altered the general backbone conformation and metal-thiolate cluster geometry. Notably, bioassay results showed that the bioactivity of the K31/32A mutant and the K31/32E mutant decreased obviously, while the KKS-SP mutant lost inhibitory activity completely. Based on these results, we proposed that the KKS linker was a crucial factor in modulating the stability and the solvent accessibility of the Cd(3)S(9) cluster in the beta-domain through domain-domain interactions, thus was indispensable to the biological activity of hGIF.
Ding
Zhi-Chun
ZC
Chemical Biology Laboratory, Department of Chemistry, Fudan University, Shanghai, China. 051022004@fudan.edu.cn
Teng
Xin-Chen
XC
Zheng
Qi
Q
Ni
Feng-Yun
FY
Cai
Bin
B
Wang
Yang
Y
Zhou
Guo-Ming
GM
Sun
Hong-Zhe
HZ
Tan
Xiang-Shi
XS
Huang
Zhong-Xian
ZX
D000595:Amino Acid Sequence; D000818:Animals; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D000090023:Metallothionein 3; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid
D000090023:Metallothionein 3; D009419:Nerve Tissue Proteins