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Three-dimensional structure of beta-cell-specific zinc transporter, ZnT-8, predicted from the type 2 diabetes-associated gene variant SLC30A8 R325W.

ABSTRACT: BACKGROUND:We examined the effects of the R325W mutation on the three-dimensional (3D) structure of the beta-cell-specific Zn2+ (zinc) transporter ZnT-8. METHODS:A model of the C-terminal domain of the human ZnT-8 protein was generated by homology modeling based on the known crystal structure of the Escherichia coli (E. coli) zinc transporter YiiP at 3.8 A resolution. RESULTS:The homodimer ZnT-8 protein structure exists as a Y-shaped architecture with Arg325 located at the ultimate bottom of this motif at approximately 13.5 A from the transmembrane domain juncture. The C-terminal domain sequences of the human ZnT-8 protein and the E. coli zinc transporter YiiP share 12.3% identical and 39.5% homologous residues resulting in an overall homology of 51.8%. Validation statistics of the homology model showed a reasonable quality of the model. The C-terminal domain exhibited an alphabetabetaalphabeta fold with Arg325 as the penultimate N-terminal residue of the alpha2-helix. The side chains of both Arg325 and Trp325 point away from the interface with the other monomer, whereas the epsilon-NH3+ group of Arg325 is predicted to form an ionic interaction with the beta-COO- group of Asp326 as well as Asp295. An amino acid alignment of the beta2-alpha2 C-terminal loop domain revealed a variety of neutral amino acids at position 325 of different ZnT-8 proteins. CONCLUSIONS:Our validated homology models predict that both Arg325 and Trp325, amino acids with a helix-forming behavior, and penultimate N-terminal residues in the alpha2-helix of the C-terminal domain, are shielded by the planar surface of the three cytoplasmic beta-strands and hence unable to affect the sensing capacity of the C-terminal domain. Moreover, the amino acid residue at position 325 is too far removed from the docking and transporter parts of ZnT-8 to affect their local protein conformations. These data indicate that the inherited R325W abnormality in SLC30A8 may be tolerated and results in adequate zinc transfer to the correct sites in the pancreatic islet cells and are consistent with the observation that the SLC30A8 gene variant R325W has a low predicted value for future type 2 diabetes at population-based level.

SUBMITTER: Weijers RN

PROVIDER: S-EPMC2890542 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Three-dimensional structure of beta-cell-specific zinc transporter, ZnT-8, predicted from the type 2 diabetes-associated gene variant SLC30A8 R325W.

Weijers Rob Nm RN

Diabetology & metabolic syndrome 20100605 1

<h4>Background</h4>We examined the effects of the R325W mutation on the three-dimensional (3D) structure of the beta-cell-specific Zn2+ (zinc) transporter ZnT-8.<h4>Methods</h4>A model of the C-terminal domain of the human ZnT-8 protein was generated by homology modeling based on the known crystal structure of the Escherichia coli (E. coli) zinc transporter YiiP at 3.8 A resolution.<h4>Results</h4>The homodimer ZnT-8 protein structure exists as a Y-shaped architecture with Arg325 located at the ...[more]

PMID: 20525392

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Project description:The human zinc transporter ZnT8 (SLC30A8) is expressed primarily in pancreatic β-cells and plays a key function in maintaining the concentration of blood glucose through its role in insulin storage, maturation and secretion. ZnT8 is an autoantigen for Type 1 diabetes (T1D) and is associated with Type 2 diabetes (T2D) through its risk allele that encodes a major non-synonymous single nucleotide polymorphism (SNP) at Arg325. Loss of function mutations improve insulin secretion and are protective against diabetes. Despite its role in diabetes and concomitant potential as a drug target, little is known about the structure or mechanism of ZnT8. To this end, we expressed ZnT8 in Pichia pastoris yeast and Sf9 insect cells. Guided by a rational screen of 96 detergents, we developed a method to solubilize and purify recombinant ZnT8. An in vivo transport assay in Pichia and a liposome-based uptake assay for insect-cell derived ZnT8 showed that the protein is functionally active in both systems. No significant difference in activity was observed between full-length ZnT8 (ZnT8A) and the amino-terminally truncated ZnT8B isoform. A fluorescence-based in vitro transport assay using proteoliposomes indicated that human ZnT8 functions as a Zn2+/H+ antiporter. We also purified E. coli-expressed amino- and carboxy-terminal cytoplasmic domains of ZnT8A. Circular dichroism spectrometry suggested that the amino-terminal domain contains predominantly α-helical structure, and indicated that the carboxy-terminal domain has a mixed α/β structure. Negative-stain electron microscopy and single-particle image analysis yielded a density map of ZnT8B at 20 Å resolution, which revealed that ZnT8 forms a dimer in detergent micelles. Two prominent lobes are ascribed to the transmembrane domains, and the molecular envelope recapitulates that of the bacterial zinc transporter YiiP. These results provide a foundation for higher resolution structural studies and screening experiments to identify compounds that modulate ZnT8 activity.

Dataset Information

Three-dimensional structure of beta-cell-specific zinc transporter, ZnT-8, predicted from the type 2 diabetes-associated gene variant SLC30A8 R325W.

Publications

Three-dimensional structure of beta-cell-specific zinc transporter, ZnT-8, predicted from the type 2 diabetes-associated gene variant SLC30A8 R325W.

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