The correct compartmentalisation of cellular zinc is critical to its homeostasis in Eukarya. The vesicular subfamily of mammalian ZnTs mediates the transport of zinc from the cytosol into secretory vesicles in endocrine tissues and the brain. ZnT8 supplies the millimolar zinc concentrations of insulin granules in pancreatic β-cells, affecting insulin processing, crystallisation and secretion. In common with its bacterial homologue YiiP, ZnT8 is formed of a transmembrane and a cytosolic C-terminal domain (CTD). The mechanism of zinc transport in the bacterial proteins is well-described. While the transmembrane domain and the overall protein topology of the mammalian ZnTs is largely conserved from bacteria, there are significant differences in primary sequence in the metal-binding CTD. This domain has been shown to mediate protein-protein interactions in other mammalian ZnTs. A common mutation causing W325R in the CTD of ZnT8 increases the risk of developing type 2 and gestational diabetes, and affects autoantibody specificity in type 1 diabetes. This thesis details the purification and biophysical characterisation of the two common variants of the ZnT8 CTD. Both domains exhibit soluble recombinant protein expression in
Escherichia coli. The domains form the ferredoxin-like fold elucidated for the bacterial homologues. Zinc binds to the conserved interface site and to the novel cysteine-rich tail, suggesting that there is a different zinc:protein stoichiometry than is found in the bacterial homologues. The cysteine motifs are conserved in the vesicular ZnTs and may represent a specific metal uptake strategy by this subfamily. The W325R mutation affects dimer formation and stability of the ZnT8 CTD. Dimer formation is not reliant on disulphide formation or divalent metal coordination. The data presented herein begin to provide a molecular basis for the different diabetes susceptibility caused by the full-length ZnT8 proteins.
Structural and functional features of the C-terminal domain of ZnT8
Parsons, D. S. (Author). 1 Jun 2019
Student thesis: Doctoral Thesis › Doctor of Philosophy