The C‐terminal cytosolic domain of the human zinc transporter ZnT8 and its diabetes risk variant

Douglas S Parsons,Christer Hogstrand,Wolfgang Maret

doi:10.1111/febs.14402

Abstract

A significant aspect of the control of cellular zinc in eukarya is its subcellular re‐distribution. One of the four human vesicular zinc transporters, ZnT8, supplies the millimolar zinc concentrations of insulin granules in pancreatic β‐cells, affecting insulin processing, crystallisation and secretion. ZnT8 has a transmembrane and a C‐terminal cytosolic domain; the latter has important functions and purportedly mediates protein–protein interactions, senses cytosolic zinc and/or channels zinc to the transport site in the transmembrane domain (TMD). A common variant W325R in the C‐terminal domain (CTD) increases the risk to develop type 2 diabetes and affects autoantibody specificity in type 1 diabetes. To investigate the differences between the two protein variants, we purified and biophysically characterised both variants of the ZnT8 CTD [R325 variant of ZnT8 CTD (aa267–369) (ZnT8cR) and W325 variant of ZnT8 CTD (aa267–369) (ZnT8cW)]. The domains fold independently of the TMD. Remarkably, the ZnT8cW variant (diabetes protection in the full‐length protein) is less thermostable than the ZnT8cR variant (diabetes risk in the full‐length protein). The ZnT8cW monomers associate with higher affinity. Both CTD variants bind zinc with a stoichiometry that differs from bacterial homologues, emphasising the limitation of the latter as models for the structure and function of the human proteins. The relatively small but reproducible differences between the two ZnT8 CTD variants begin to provide a molecular basis for the different diabetes susceptibility caused by the full‐length ZnT8 proteins.

Highlights

Perturbations of cellular zinc metabolism through mutations in dozens of proteins are implicated in a growing number of human diseases [1]
Based on a comparison of the primary sequences of several human and two bacterial cation diffusion facilitator (CDF) C-terminal domain (CTD), for which 3D structures are known, the two ZnT8 CTD variants (ZnT8cR and W325 variant of ZnT8 CTD (ZnT8cW)) are predicted to adopt an abbab fold (Fig. 1A) observed in at least four bacterial CTDs of homologous zinc transporters. This fold is characteristic of the ‘heavy metal-associated domain’, called the ferredoxin fold babbab in different metalloproteins interacting with iron, copper or zinc [25]
A 3D model of the R325 variant of ZnT8 CTD (ZnT8cR) homodimer based on the structure of T. thermophilus CzrB was constructed (Fig. 1B)

Summary

Introduction

Perturbations of cellular zinc metabolism through mutations in dozens of proteins are implicated in a growing number of human diseases [1]. ZnT8 (SLC30A8), is highly expressed in the membrane of the dense insulin secretory granules of pancreatic bcells [4] It supplies zinc for the storage of insulin as a crystalline hexamer and for other aspects of the Abbreviations Ch. Int., charge interlock; CTD, C-terminal domain; ICP-MS, inductively coupled plasma mass spectrometry; MST, microscale thermophoresis; nDSF, nano differential scanning fluorimetry; T1D, type 1 diabetes; T2D, type 2 diabetes; TMD, transmembrane domain; ZnT8cR, R325 variant of ZnT8 CTD (aa267-369); ZnT8cW, W325 variant of ZnT8 CTD (aa267–369); ZnT, zinc transporter

Methods

Results

Conclusion