Abstract
The human zinc transporter ZnT8 provides the granules of pancreatic β-cells with zinc (II) ions for assembly of insulin hexamers for storage. Until recently, the structure and function of human ZnTs have been modelled on the basis of the 3D structures of bacterial zinc exporters, which form homodimers with each monomer having six transmembrane α-helices harbouring the zinc transport site and a cytosolic domain with an α,β structure and additional zinc-binding sites. However, there are important differences in function as the bacterial proteins export an excess of zinc ions from the bacterial cytoplasm, whereas ZnT8 exports zinc ions into subcellular vesicles when there is no apparent excess of cytosolic zinc ions. Indeed, recent structural investigations of human ZnT8 show differences in metal binding in the cytosolic domain when compared to the bacterial proteins. Two common variants, one with tryptophan (W) and the other with arginine (R) at position 325, have generated considerable interest as the R-variant is associated with a higher risk of developing type 2 diabetes. Since the mutation is at the apex of the cytosolic domain facing towards the cytosol, it is not clear how it can affect zinc transport through the transmembrane domain. We expressed the cytosolic domain of both variants of human ZnT8 and have begun structural and functional studies. We found that (i) the metal binding of the human protein is different from that of the bacterial proteins, (ii) the human protein has a C-terminal extension with three cysteine residues that bind a zinc(II) ion, and (iii) there are small differences in stability between the two variants. In this investigation, we employed nickel(II) ions as a probe for the spectroscopically silent Zn(II) ions and utilised colorimetric and fluorimetric indicators for Ni(II) ions to investigate metal binding. We established Ni(II) coordination to the C-terminal cysteines and found differences in metal affinity and coordination in the two ZnT8 variants. These structural differences are thought to be critical for the functional differences regarding the diabetes risk. Further insight into the assembly of the metal centres in the cytosolic domain was gained from potentiometric investigations of zinc binding to synthetic peptides corresponding to N-terminal and C-terminal sequences of ZnT8 bearing the metal-coordinating ligands. Our work suggests the involvement of the C-terminal cysteines, which are part of the cytosolic domain, in a metal chelation and/or acquisition mechanism and, as now supported by the high-resolution structural work, provides the first example of metal-thiolate coordination chemistry in zinc transporters.
Highlights
A subgroup of four out of ten human zinc transporters (ZnT2, 3, 4 and 8) of the cation diffusion facilitator (CDF) protein family exports Zn2+ ions from the cytosol into intracellular vesicles [1,2]
We demonstrate that the cytosolic C-terminal domain (CTD) of ZnT8 binds nickel ions with a stoichiometry similar to zinc ion binding
Titration of the protein with nickel ions in the presence and absence of cysteine modification and spectroscopic features indicate binding to sulfur in coordination environments that differ significantly in the two variants, revealing for the first time structural differences between the two variants and sulfur coordination in zinc transporter biochemistry, possibly involving redox biology in human ZnT zinc transporters
Summary
A subgroup of four out of ten human zinc transporters (ZnT2, 3, 4 and 8) of the cation diffusion facilitator (CDF) protein family exports Zn2+ ions from the cytosol into intracellular vesicles [1,2]. ZnT8 is present in α-cells of the endocrine pancreas, but its precise role in glucagon secretion has not yet been elucidated [4] Concepts of how these vesicular zinc transporters work were primarily based on the 3D structures of prokaryotic CDF proteins. Given the abundance of low molecular weight ligands in the cell, there are no Zn2+ ions with water ligands only in the cytosol, but several nM of zinc bound to small molecules with an overall approximate affinity of 100 fM [17]. Independent of these fundamental issues, there is no excess of zinc freely available for passive transmembrane transport. Additional issues for interpreting gradients across the membrane of granules are that given an average granule volume of only 7.2 aL, the estimated Zn2+ concentration of 120 nM would indicate less than one Zn2+ ion per granule, and that crystallization of insulin will further reduce the concentration in solution as it removes zinc from equilibria in solution [18]
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