C-Terminal Domain of the Human Zinc Transporter hZnT8 Is Structurally Indistinguishable from Its Disease Risk Variant (R325W)
Abstract
:1. Introduction
2. Results
2.1. The Maltose-Binding Protein (MBP) Tag Improves the Solubility of the CTDs
2.2. The CTDs Exist as Tetramers in Solution
2.3. The CTDs Bind Zn+2 with High Affinity
2.4. The CTDs Share Similar Structural Features in the Presence and Absence of Zn+2
3. Discussion
4. Materials and Methods
4.1. Cloning, Expression, and Purification of the CTDs
4.2. Gel Filtration
4.3. Analytical Ultracentrifugation
4.4. Mass Spectrometry
4.5. Negative-Stain EM
4.6. Metal Binding Analysis
4.7. CD Spectroscopy
4.8. FT-IR Spectroscopy
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
CD | Circular dichroism |
CDF | Cation diffusion facilitator |
CTD | C-terminal domain |
EM | Electron microscopy |
FT-IR | Fourier transform infrared |
GWAS | Genome-wide association study |
hZnT8 | Human zinc transporter 8 |
MBP | Maltose-binding protein |
MW | Molecular weight |
SEC | Size-exclusion chromatography |
SNP | Single nucleotide polymorphism |
T1D | Type 1 diabetes |
T2D | Type 2 diabetes |
TEV site | Tobacco etch virus protease recognition site |
TMD | Transmembrane domain |
WT | Wild-type |
ZIP | Zrt- and Irt-like proteins |
ZnT | Zinc transporter |
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Protein | α-Helix (%) | β-Sheet (%) | β-Turn (%) | Random Coil (%) | Technique |
---|---|---|---|---|---|
WT CTD | 41 | 15 | 13 | 31 | CD spectroscopy 1 |
46 | 10 | 17 | 27 | CD spectroscopy 2 | |
R325W CTD | 46 | 12 | 13 | 29 | CD spectroscopy 1 |
53 | 10 | 17 | 20 | CD spectroscopy 2 | |
WT CTD | 29.6 | 26.1 | 27 | 17.3 | FT-IR spectroscopy |
R325W CTD | 27 | 24 | 25 | 24 | FT-IR spectroscopy |
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Ullah, R.; Shehzad, A.; Shah, M.A.; March, M.D.; Ismat, F.; Iqbal, M.; Onesti, S.; Rahman, M.; McPherson, M.J. C-Terminal Domain of the Human Zinc Transporter hZnT8 Is Structurally Indistinguishable from Its Disease Risk Variant (R325W). Int. J. Mol. Sci. 2020, 21, 926. https://doi.org/10.3390/ijms21030926
Ullah R, Shehzad A, Shah MA, March MD, Ismat F, Iqbal M, Onesti S, Rahman M, McPherson MJ. C-Terminal Domain of the Human Zinc Transporter hZnT8 Is Structurally Indistinguishable from Its Disease Risk Variant (R325W). International Journal of Molecular Sciences. 2020; 21(3):926. https://doi.org/10.3390/ijms21030926
Chicago/Turabian StyleUllah, Raheem, Aamir Shehzad, Majid Ali Shah, Matteo De March, Fouzia Ismat, Mazhar Iqbal, Silvia Onesti, Moazur Rahman, and Michael J. McPherson. 2020. "C-Terminal Domain of the Human Zinc Transporter hZnT8 Is Structurally Indistinguishable from Its Disease Risk Variant (R325W)" International Journal of Molecular Sciences 21, no. 3: 926. https://doi.org/10.3390/ijms21030926