title: Important roles of the conserved linker-KKS in human neuronal growth inhibitory factor.
abstract: 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.
authors.affiliation: Chemical Biology Laboratory, Department of Chemistry, Fudan University, Shanghai, China. 051022004@fudan.edu.cn
authors.forename: Zhi-Chun
authors.initials: ZC
authors.lastname: Ding
authors.forename: Xin-Chen
authors.initials: XC
authors.lastname: Teng
authors.forename: Qi
authors.initials: Q
authors.lastname: Zheng
authors.forename: Feng-Yun
authors.initials: FY
authors.lastname: Ni
authors.forename: Bin
authors.initials: B
authors.lastname: Cai
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.forename: Guo-Ming
authors.initials: GM
authors.lastname: Zhou
authors.forename: Hong-Zhe
authors.initials: HZ
authors.lastname: Sun
authors.forename: Xiang-Shi
authors.initials: XS
authors.lastname: Tan
authors.forename: Zhong-Xian
authors.initials: ZX
authors.lastname: Huang
mesh_terms: 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
chemical_list: D000090023:Metallothionein 3; D009419:Nerve Tissue Proteins