The structural and biological significance of the EAAEAE insert in the α‐domain of human neuronal growth inhibitory factor

Abstract

DatabaseThe structures of the a-domain in Cd 7 -hGIF and the solo Cd 4 -a-domain have been deposited in the Protein Data Bank under the numbers 2FJ4 and 2FJ5, respectively *These authors contributed equally to this work (Received 16 October 2008, revised 19 March 2009, accepted 24 April 2009) doi:10.1111/j.1742-4658.2009 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 D55-60 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 D55-60 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 b-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.Abbreviations AD, Alzheimer's disease; GIF, neuronal growth inhibitory factor; hGIF, human neuronal growth inhibitory factor; hMT1g, human MT1 isoform g; MT, metallothionein; SNOC, S-nitrosocysteine; ZINCON, 2-(2-hydroxy-5-sulfoformazyl) benzoic acid.

Neuronal growth‐inhibitory factor (metallothionein‐3): structure–function relationships

“…Based on the backbone 1 H chemical shifts between the α‐domain of the holo‐hGIF and the single α‐domain, some residues (including Ser36, Pro39, Ala40, Glu41 and Ala46) were identified to be involved in the interdomain interaction [16]. However, no further conclusions could be made on the interdomain interaction by NMR unless structure of the β‐domain of hGIF could be identified.…”

Isolation of Metallothionein Genes and in silico Structural Characterization of Their Proteins Using Molecular Modeling from Yak (Bos grunniens)

“…Because the N-alkyl bond is fixed in the five-member ring, thus restricting the torsion angle, proline plays a key role in the secondary structures of polypeptides. T 5 CPCP 9 would form a particular conformation with two parallel Pro residues (Cai et al 2009), which could provide an interacting surface for protein-protein interactions (Williamson 1994). The insertion of T 5 places the first four residues (M 1 DPE 4 ) near the N-terminus farther from residues 23-26 in BgMT-III, which permits the structure of the b-domain to be looser.…”

“…The acidic insertion E 55 GAEAE 60 in the a-domain of BgMT-III could regulate particular interdomain interactions and lead to the conformational change in the b-domain, including the solvent accessibility of the metal-thiolate cluster and the metal-releasing ability of the protein (Fig. 8) (Cai et al 2009). …”

“…The target structures of BgMTs were simulated using the DeepView/Swiss-PdbViewer (version 4.0.1; Guex and Peitsch 1997). The metal-binding a-and b-domains of BgMTs were aligned according to previous reports (Cai et al 2009;Berman et al 2000;Karotki and Vašák 2009). Model data are available in the PMDB database under accession nos.…”

“…MT-I and MT-II are the most studied and most widely distributed isoforms (Coyle et al 2002); they can be induced easily by heavy metal ions, hormones, cytokines, inflammation, acute stress, and many chemicals. MT-III (also known as neuronal growth inhibitory factor, or GIF) was discovered initially as an inhibitory factor down-regulated in brains with Alzheimer's disease (Uchida et al 1991;Yu et al 2001;Chung et al 2002;Cai et al 2009). It is regulated very differently from MT-I and MT-II.…”