Probing the Structure and Function of the Cytosolic Domain of the Human Zinc Transporter ZnT8 with Nickel(II) Ions.

The human zinc transporter ZnT8 provides the granules of pancreatic ß-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,ß 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.

Insights into the Dynamics of the Human Zinc Transporter ZnT8 by MD Simulations.

ZnT8 is a human zinc(II) transporter expressed at the membrane of secretory granules where it contributes to insulin storage importing zinc ions from the cytosol. In the human population, the two most common ZnT8 variants carry an arginine (R325) or a tryptophan (W325) in position 325. The former variant has the most efficient kinetics in zinc transport and has been correlated to a higher risk of developing insulin resistance. On the contrary, the W325 variant is less active and protects against type-2-diabetes. Here, we used molecular dynamics (MD) simulations to investigate the main differences between the R325 and W325 variants in the interaction with zinc(II) ions. Our simulations suggested that the position of the metal ion within the transport site was not the same for the two variants, underlying a different rearrangement of the transmembrane (TM) helices in the channel. The W325 variant featured a peculiar zinc environment not detected in the experimental structures. With respect to conformational dynamics, we observed that the R325 variant was significantly more flexible than W325, with the main role played by the transmembrane domain (TMD) and the C-terminal domain (CTD). This dynamics affected the packing of the TM helices and thus the channel accessibility from the cytosol. The dimer interface that keeps the two TM channels in contact became looser in both variants upon zinc binding to the transport site, suggesting that this may be an important step toward the switch from the inward- to the outward-facing state of the protein.

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

The human zinc transporter 8 (hZnT8) plays important roles in the storage of insulin in the secretory vesicles of pancreatic ß 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.

Insights into the structure-function relationship of both wild and mutant zinc transporter ZnT8 in human: a computational structural biology approach.

Polymorphism rs13266634 in SLC30A8 causes abnormal synthesis, maturation and secretion of insulin, resulting in decrease in efficiency of glucose metabolism and diabetes. SLC30A8 encodes Zinc transporter 8 protein (ZnT8). Due to lack of NMR/crystal structures of complete ZnT8 transporter, insights into the structure, function and its interaction with different drugs is still not known. Therefore, in-silico methods were adopted in the present studies for predicting three-dimensional structure of ZnT8 transporter via comparative modelling approach and studying the impact of mutation (p.ARG325TRP) on architecture and function via simulation studies. Wild-type protein comprises 15 α-helix and 3 ß-strands, while mutant consists of 12 α-helix and 2 ß-strands, respectively. Interaction studies of mutant ZnT8 transporter with phytochemicals/drugs screened the best phytochemicals, which can retain the wild-type property. Molecular docking studies reveal that mutant proteins have better binding energy with ligands of LY-2608204, Roseoside, and Luzonoid B. Further molecular dynamic simulation analysis exhibited a strong binding of these ligands with mutant protein and displaying similar behaviour as that of wild type. ALA79, ILE80, and ARG215 are the common interacting amino acids with ligand in all three complexes. As the ligands passed ADMET tests, these may be utilized as anti-diabetic drugs in near future. Although earlier studies have reported anti-diabetic property of LY-2608204 and Roseoside, for the first time, this study reporting Luzonoid B may have anti-diabetic property besides elucidating the structure and functions of ZnT8 transporter.

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

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 ß-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.

Detection of SNPs of T2DM susceptibility genes by a ligase detection reaction-fluorescent nanosphere technique.

OBJECTIVE: To establish a high throughput, low cost, and simple nanotechnology-based method for the detection of single nucleotide polymorphism (SNP) loci in type 2 diabetes mellitus (T2DM). METHODS: Multiplex ligase detection reaction (LDR) amplification was performed using fluorescently labeled magnetic nanosphere-bound upstream LDR probes and downstream probes labeled with a unique fluorescent group for each SNP locus. The amplified LDR products were separated by magnetic nanospheres and then scanned by fluorescence spectroscopy. Four SNP loci associated with T2DM were detected, including the rs13866634 locus in SLC30A8, rs10811661in CDKN2A/2B, rs1111875 in the HHEX gene, and rs7903146 in the TCF7L2 gene. The SNP genotype was also determined by DNA sequencing as a control. RESULTS: The SNP genotypes of the four gene loci determined by the nanosphere-based multiplex LDR method were consistent with the DNA sequencing results. The accuracy rate was 100%. CONCLUSION: A method based on multiplex PCR and LDR was established for simultaneous detection of four SNP loci of T2DM susceptibility genes.

Differential cytolocation and functional assays of the two major human SLC30A8 (ZnT8) isoforms.

The non-synonymous single nucleotide polymorphism (SNP) rs13266634 in human zinc transporter 8, ZnT8 (SLC30A8), leads to a R325 variant, which is associated with an increased risk of developing Type 2 Diabetes (T2D). Although the molecular details remain unknown, the mutation is thought to alter the kinetics of zinc transport into insulin granules in pancreatic ß-cells. Nevertheless, analysis of ZnT8 sequences from several animals shows that the amino acid at position 325 is poorly conserved. Apart from this particular SNP, human ZnT8 also has two isoforms (splice variants) that differ in length regarding a 49 amino acid N-terminal extension. When expressed in human embryonic kidney (HEK293) cells, the long isoform was present in the plasma membrane in addition to internal membranes, whereas the short isoform was localized mostly to internal membranes. Our observation that human ZnT8 variants and isoforms expressed in Xenopus laevis oocytes are all localized at the cell surface allowed us to develop a zinc transport assay using the radioactive isotope 65Zn. We found no detectable differences in zinc transport between W and R variants and no statistically significant differences between long and short isoforms of the W325 variant. Our findings of differential cytolocation of ZnT8 isoforms could be relevant for ß-cell zinc metabolism in health and disease.

Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance.

Cation diffusion facilitators (CDF) are highly conserved, metal ion efflux transporters that maintain divalent transition metal cation homeostasis. Most CDF proteins contain two domains, the cation transporting transmembrane domain and the regulatory cytoplasmic C-terminal domain (CTD). MamM is a magnetosome-associated CDF protein essential for the biomineralization of magnetic iron-oxide particles in magnetotactic bacteria. To investigate the structure-function relationship of CDF cytoplasmic domains, we characterized a MamM M250P mutation that is synonymous with the disease-related mutation L349P of the human CDF protein ZnT-10. Our results show that the M250P exchange in MamM causes severe structural changes in its CTD resulting in abnormal reduced function. Our in vivo, in vitro and in silico studies indicate that the CTD fold is critical for CDF proteins' proper function and support the previously suggested role of the CDF cytoplasmic domain as a CDF regulatory element. Based on our results, we also suggest a mechanism for the effects of the ZnT-10 L349P mutation in human.