The structural and biological significance of the EAAEAE insert in the alpha-domain of human neuronal growth inhibitory factor.
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.
Cai
Bin
B
Chemical Biology Laboratory, Department of Chemistry, Fudan University, Shanghai, China.
Ding
Zhi-Chun
ZC
Zhang
Qi
Q
Ni
Feng-Yun
FY
Wang
Hui
H
Zheng
Qi
Q
Wang
Yang
Y
Zhou
Guo-Ming
GM
Wang
Ke-Qiang
KQ
Sun
Hong-Zhe
HZ
Wu
Hou-Ming
HM
Huang
Zhong-Xian
ZX
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
D000090023:Metallothionein 3; D009419:Nerve Tissue Proteins; D015032:Zinc