Effect of α-domain substitution on the structure, property and function of human neuronal growth inhibitory factor

Abstract

Human metallothionein-3 (hMT3), also named human neuronal growth inhibitory factor (hGIF), is attractive due to its distinct neuronal growth inhibitory activity, which is not shown by other human MT isoforms. It has been reported that the neuronal growth inhibitory activity arises from the N-terminal beta-domain rather than its C-terminal alpha-domain. However, previous bioassay results have shown that the single beta-domain is less effective at inhibiting the neuron growth than that in intact hMT3 on a molar basis, which suggests that the alpha-domain is indispensable to the neuronal growth inhibitory activity of hMT3. In order to confirm this assumption, we constructed two domain-hybrid mutants, the beta(MT3)-beta(MT3) mutant and the beta(MT3)-alpha(MT1) mutant, and investigated their structural and metal binding properties by UV-vis spectroscopy, CD spectroscopy, pH titration, DTNB reaction, EDTA reaction, etc. The results showed that stability of the Cd(3)S(9) cluster of the beta(MT3)-beta(MT3) mutant decreased significantly while the Cd(3)S(9) cluster of the beta(MT3)-alpha(MT1) mutant had a similar stability and solvent accessibility to that of hMT3. Interestingly, the bioassay results showed that the neuronal growth inhibitory activity of the beta(MT3)-beta(MT3) mutant decreased significantly, while the beta(MT3)-alpha(MT1) mutant showed similar inhibitory activity to hMT3. Based on these results, we conclude that the alpha-domain is indispensable and plays an important role in modulating the stability of the metal cluster in the beta-domain by domain-domain interactions, thus influencing the bioactivity of hMT3.

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

“…However, it has been well documented that the two domains of MT do not work independently, and domain–domain interactions do exist and affect the properties and reactivity of each domain [20–22]. Furthermore, our results, and those of other bioassays, showed that the inhibitory activity of the single β‐domain is less effective than that of intact hGIF on a molar basis [6,23]. Hence, it is suggested that the α‐domain might play some important roles in the neuronal growth‐inhibitory activity of hGIF.…”

Study on structure–property–reactivity–function relationship of human neuronal growth inhibitory factor (hGIF)

“…Actually, it was found that the inhibitory activity of the single b-domain is less effective than that of intact hGIF on a molar basis, implying that the a-domain might play some important roles in the neuronal growth inhibitory activity of hGIF [32]. thus altering zinc homeostasis in brain and influencing the bioactivity [42]. This conclusion is supported by our further studies.…”

“…the DT5, T5S, T5A mutant proteins; (2) mutants focused in the conserved 6 Based on these results, we favor to conclude that the neuronal growth inhibitory bioactivity of hGIF is regulated by multiple factors, including: (1) the defined conformation of residues 1-13, which is pivotal to the bioactivity of hGIF, no such conformation no GIF bioactivity appears; (2) domain-domain interactions, which may play important roles in modulating the stability of the metalthiolate cluster and the conformation of the b-domain; (3) the solvent accessibility of the metal-thiolate cluster, which is closely associated with the structure of the protein and mutual accessibility of metal-thiolate clusters with biologically sensitive small molecules such as NO. (4) The metal-releasing ability, determined by the stability and solvent accessibility of the metal-thiolate cluster in the b-domain of hGIF, is exquisitely modulated by the domaindomain interaction and the protein's conformation [31,37,42]. Our studies provide more and more evidences showing that the bioactivity of hGIF is mainly related to the essential metal release and its characteristic conformation.…”

“…the Cd 2+ -reconstituted forms for spectroscopic and metal-binding studies, while the Zn 2+ -reconstituted forms for bioassay experiments [39,40,42,47,51]. However, the native GIF contains both copper and zinc ions in its metal clusters [25,26,55,56].…”

The Δ33‐35 Mutant α‐Domain Containing β‐Domain‐Like M3S9 Cluster Exhibits the Function of α‐Domain with M4S11 Cluster in Human Growth Inhibitory Factor

“…However, the α -domain can affect the function of β -domain though domain-domain interactions do exist in MTs [21, 37–39], and it has been proved in our previous work [21, 37]. Taking all the experimental results together, for hGIF and its mutants, the stability and solvent accessibility of the metal-thiolate cluster in N-terminal β -domain are in good line with the bioactivity of the whole protein; so they may be very important to the neuronal growth inhibitory activity of hMT3.…”