C-Terminal Domain of the Human Zinc Transporter hZnT8 Is Structurally Indistinguishable from Its Disease Risk Variant (R325W).

Raheem Ullah,Mazhar Iqbal,Michael J Mcpherson,Aamir Shehzad,Silvia Onesti,Fouzia Ismat,Matteo De March,Majid Ali Shah,Moazur Rahman

doi:10.3390/ijms21030926

Abstract

The human zinc transporter 8 (hZnT8) plays important roles in the storage of insulin in the secretory vesicles of pancreatic β cells. hZnT8 consists of a transmembrane domain, with its N- and C-termini protruding into the cytoplasm. Interestingly, the exchange of arginine to tryptophan at position 325 in the C-terminal domain (CTD) increases the risk of developing type 2 diabetes mellitus (T2D). In the present study, the CTDs of hZnT8 (the wild-type (WT) and its disease risk variant (R325W)) were expressed, purified, and characterized in their native forms by biophysical techniques. The data reveal that the CTDs form tetramers which are stabilized by zinc binding, and exhibit negligible differences in their secondary structure content and zinc-binding affinities in solution. These findings provide the basis for conducting further structural studies aimed at unravelling the molecular mechanism underlying the increased susceptibility to develop T2D, which is modulated by the disease risk variant.

Highlights

Zinc (Zn+2) plays important roles in cellular growth and differentiation [1,2,3,4]
Cellular Zn+2 homeostasis is maintained by two families of Zn+2 transporters—the cation diffusion facilitator (CDF) family, known as the SLC30 family, that controls the Zn+2 concentration in the extracellular matrix or intracellular vesicles, and the Zrt- and Irt-like proteins (ZIP) family, known as the SLC39A family, that is implicated in controlling the intracellular Zn+2 uptake [6,7,8]
As the His tag has affinity for divalent metal ions, and may affect the Zn+2 binding analysis, the N-terminal tags were removed from the C-terminal domain (CTD) after the nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography (Figure S2, Supplementary Materials)

Summary

Introduction

Zinc (Zn+2) plays important roles in cellular growth and differentiation [1,2,3,4]. Cellular Zn+2 homeostasis is maintained by two families of Zn+2 transporters—the cation diffusion facilitator (CDF) family, known as the SLC30 family, that controls the Zn+2 concentration in the extracellular matrix or intracellular vesicles, and the Zrt- and Irt-like proteins (ZIP) family, known as the SLC39A family, that is implicated in controlling the intracellular Zn+2 uptake [6,7,8]. ZnT and ZIP-family members play key roles in various biological functions, and an understanding of their importance in health and disease continues to increase as more information on disease-related mutations in the corresponding protein-encoding genes comes to light [9]. Variations in the amino acid sequence of ZnT and ZIP-family members

Methods

Results

Discussion

Conclusion