title: The structural and biological significance of the EAAEAE insert in the alpha-domain of human neuronal growth inhibitory factor.
abstract: Human neuronal growth inhibitory factor (hGIF) is able to inhibit the outgrowth of neurons. As compared with the amino acid sequences of metallothionein 1/2, hGIF contains two insertions: a Thr at position 5 and an acidic hexapeptide EAAEAE(55-60) close to the C-terminus. Moreover, all mammalian growth inhibitory factor sequences contain a conserved CPCP(6-9) motif. Previous studies have demonstrated that the TCPCP(5-9) motif is pivotal to its bioactivity, but no specific role has been assigned to the unique EAAEAE(55-60) insert. To investigate the potential structural and biological significance of the EAAEAE(55-60) insert, several mutants were constructed and investigated in detail. Notably, deletion of the acidic insert (the Delta55-60 mutant) reduced the inhibitory activity, whereas the bioactivities of other mutants did not change much. Then, spectroscopic characterization and molecular dynamics simulation were performed to investigate the potential causes of the reduced bioactivity of the Delta55-60 mutant. It was found that the domain-domain interaction mechanism of hGIF was different from that of metallothionein 2. It was also shown that the acidic insert could regulate the interdomain interactions in hGIF, leading to the structural change in the beta-domain, which resulted in the alteration of the solvent accessibility and metal release ability, thus playing an important role in the biological activity of hGIF. Our studies provided useful information on the domain-domain interaction at the molecular level for the first time, and shed new light on the mechanism of the bioactivity of hGIF.
authors.affiliation: Chemical Biology Laboratory, Department of Chemistry, Fudan University, Shanghai, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Cai
authors.forename: Zhi-Chun
authors.initials: ZC
authors.lastname: Ding
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhang
authors.forename: Feng-Yun
authors.initials: FY
authors.lastname: Ni
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Qi
authors.initials: Q
authors.lastname: Zheng
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.forename: Guo-Ming
authors.initials: GM
authors.lastname: Zhou
authors.forename: Ke-Qiang
authors.initials: KQ
authors.lastname: Wang
authors.forename: Hong-Zhe
authors.initials: HZ
authors.lastname: Sun
authors.forename: Hou-Ming
authors.initials: HM
authors.lastname: Wu
authors.forename: Zhong-Xian
authors.initials: ZX
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D017124:Conserved Sequence / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D000090023:Metallothionein 3; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D051381:Rats; D017208:Rats, Wistar; D015032:Zinc / Q000378:metabolism
chemical_list: D000090023:Metallothionein 3; D009419:Nerve Tissue Proteins; D015032:Zinc