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 β-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 Cd3S9 cluster in the β-domain through domain–domain interactions, thus was indispensable to the biological activity of hGIF.
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Abbreviations
- MT:
-
Metallothionein
- GIF:
-
Growth inhibitory factor
- AD:
-
Alzheimer’s disease
- PD:
-
Parkinson’s disease
- CNS:
-
Central nervous system
- cDNA:
-
Complimentary DNA
- IPTG:
-
Isopropyl β-D-thiogalactoside
- DTNB:
-
5,5′-Dithiobis-(1-nitrobenzoic acid)
- PCR:
-
Polymerase chain reaction
- DTT:
-
Dithithreitol
- ESI-MS:
-
Electrospray ionization mass spectrometry
- PSI:
-
Pounds per square inch
- UV-vis:
-
Ultraviolet-visible
- CD:
-
Circular dichroism
- LMCT:
-
Ligand-to-metal charge transfer
- NMR:
-
Nuclear magnetic resonance
- MTT:
-
3-(4,5)-dimethylthiahiazo (-2-y1)-2,5-di-phenytetrazolium bromide
- DMSO:
-
Dimethyl sulfoxide
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Acknowledgments
The authors are grateful to the financial support of the National Natural Science Foundation of China (Grant 29281005). We appreciate Professor J. H. R. Kägi from Department of Biochemistry of the University of Zürich for helpful discussion and valuable advice.
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Ding, ZC., Teng, XC., Zheng, Q. et al. Important roles of the conserved linker-KKS in human neuronal growth inhibitory factor. Biometals 22, 817–826 (2009). https://doi.org/10.1007/s10534-009-9228-1
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DOI: https://doi.org/10.1007/s10534-009-9228-1