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.

Department of Analytical Chemistry Faculty of Pharmacy in Hradec Králové Charles University Heyrovského 1203 500 05 Hradec Králové Czech Republic

Department of Pharmacology and Toxicology Faculty of Pharmacy in Hradec Králové Charles University Heyrovského 1203 500 05 Hradec Králové Czech Republic

Department of Radiology Boston University School of Medicine 670 Albany Street Boston MA 02118 USA

Departments of Biochemistry and Nutritional Sciences School of Life Course Sciences Faculty of Life Sciences and Medicine King's College London Franklin Wilkins Bldg 150 Stamford St London SE1 9NH UK

Institute of Biochemistry and Biophysics Polish Academy of Sciences Pawińskiego 5a 02 106 Warsaw Poland

Zobrazit více v PubMed

Hogstrand C., Fu D. Zinc. In: Maret W., Wedd A.G., editors. Binding, Transport and Storage of Metal Ions in Biological Cells. Royal Society of Chemistry; Cambridge, UK: 2014. pp. 666–694.

Kambe T., Tsuji T., Hashimoto A., Itsumura N. The Physiological, Biochemical, and Molecular Roles of Zinc Transporters in Zinc Homeostasis and Metabolism. Physiol. Rev. 2015;95:749–784. doi: 10.1152/physrev.00035.2014. PubMed DOI

Davidson H.W., Wenzlau J.M., O’Brien R.M. Zinc transporter 8 (ZnT8) and beta cell function. Trends Endocrinol. Metab. 2014;25:415–424. doi: 10.1016/j.tem.2014.03.008. PubMed DOI PMC

Solomou A., Philippe E., Chabosseau P., Migrenne-Li S., Gaitan J., Lang J., Magnan C., Rutter G.A. Over-expression of Slc30a8/ZnT8 selectively in the mouse alpha cell impairs glucagon release and responses to hypoglycemia. Nutr. Metab. 2016;13:46. doi: 10.1186/s12986-016-0104-z. PubMed DOI PMC

Lu M., Fu D. Structure of the zinc transporter YiiP. Science. 2007;317:1746–1748. doi: 10.1126/science.1143748. PubMed DOI

Lu M., Chai J., Fu D. Structural basis for autoregulation of the zinc transporter YiiP. Nat. Struct. Mol. Biol. 2009;16:1063–1067. doi: 10.1038/nsmb.1662. PubMed DOI PMC

Coudray N., Valvo S., Hu M., Lasala R., Kim C., Vink M., Zhou M., Provasi D., Filizola M., Tao J., et al. Inward-facing conformation of the zinc transporter YiiP revealed by cryoelectron microscopy. Proc. Natl. Acad. Sci. USA. 2013;110:2140–2145. doi: 10.1073/pnas.1215455110. PubMed DOI PMC

Cherezov V., Höfer N., Szebenyi D.M.E., Kolaj O., Wall J.G., Gillilan R., Srinivasan V., Jaroniec C.P., Caffrey M. Insights into the mode of action of a putative zinc transporter CzrB in Thermus thermophilus. Structure. 2008;16:1378–1388. doi: 10.1016/j.str.2008.05.014. PubMed DOI PMC

Higuchi T., Hattori M., Tanaka Y., Ishitani R., Nureki O. Crystal structure of the cytosolic domain of the cation diffusion facilitator family protein. Proteins. 2009;76:768–771. doi: 10.1002/prot.22444. PubMed DOI

Uebe R., Keren-Khadmy N., Zeytuni N., Katzmann E., Navon Y., Davidov G., Bitton R., Plitzko J.M., Schüler D., Zarivach R. The dual role of MamB in magnetosome membrane assembly and magnetite biomineralization. Mol. Microbiol. 2018;107:542–557. doi: 10.1111/mmi.13899. PubMed DOI

Zeytuni N., Offer T., Davidov G., Zarivach R. Crystallization and preliminary crystallographic analysis of the C-terminal domain of MamM, a magnetosome-associated protein from Magnetospirillum gryphiswaldense MSR-1. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2012;68:927–930. doi: 10.1107/S1744309112025638. PubMed DOI PMC

Udagedara S.R., La Porta D.M., Spehar C., Purohit G., Hein M.J.A., Fatmous M.E., Casas Garcia G.P., Ganio K., McDevitt C.A., Maher M.J. Structural and functional characterizations of the C-terminal domains of CzcD proteins. J. Inorg. Biochem. 2020;208:111087. doi: 10.1016/j.jinorgbio.2020.111087. PubMed DOI

Krężel A., Maret W. Zinc-buffering capacity of a eukaryotic cell at physiological pZn. J. Biol. Inorg. Chem. 2006;11:1049–1062. doi: 10.1007/s00775-006-0150-5. PubMed DOI

Vinkenborg J.L., Nicolson T.J., Bellomo E.A., Koay M.S., Rutter G.A., Merkx M. Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis. Nat. Methods. 2009;6:737–740. doi: 10.1038/nmeth.1368. PubMed DOI PMC

Maret W. Analyzing free zinc(II) ion concentrations in cell biology with fluorescent chelating molecules. Metallomics. 2015;7:202–211. doi: 10.1039/C4MT00230J. PubMed DOI

Chabosseau P., Woodier J., Cheung R., Rutter G.A. Sensors for measuring subcellular zinc pools. Metallomics. 2018;10:229–239. doi: 10.1039/C7MT00336F. PubMed DOI

Marszałek I., Goch W., Bal W. Ternary Zn(II) Complexes of FluoZin-3 and the Low Molecular Weight Component of the Exchangeable Cellular Zinc Pool. Inorg. Chem. 2018;57:9826–9838. doi: 10.1021/acs.inorgchem.8b00489. PubMed DOI

Goch W., Bal W. Stochastic or Not? Method to Predict and Quantify the Stochastic Effects on the Association Reaction Equilibria in Nanoscopic Systems. J. Phys. Chem. A. 2020;124:1421–1428. doi: 10.1021/acs.jpca.9b09441. PubMed DOI

Daniels M.J., Jagielnicki M., Yeager M. Structure/Function analysis of human ZnT8 (SLC30A8): A diabetes risk factor and zinc transporter. Curr. Res. Struct. Biol. 2020;2:144–155. doi: 10.1016/j.crstbi.2020.06.001. PubMed DOI PMC

Xue J., Xie T., Zeng W., Jiang Y., Bai X.C. Cryo-EM structures of human ZnT8 in both outward- and inward-facing conformations. eLife. 2020;9:e58823. doi: 10.7554/eLife.58823. PubMed DOI PMC

Parsons D.S., Hogstrand C., Maret W. The C-terminal cytosolic domain of the human zinc transporter ZnT8 and its diabetes risk variant. FEBS J. 2018;285:1237–1250. doi: 10.1111/febs.14402. PubMed DOI PMC

Boesgaard T.W., Žilinskaitė J., Vänttinen M., Laakso M., Jansson P.A., Hammarstedt A., Smith U., Stefan N., Fritsche A., Häring H., et al. The common SLC30A8 Arg325Trp variant is associated with reduced first-phase insulin release in 846 non-diabetic offspring of type 2 diabetes patients—The EUGENE2 study. Diabetologia. 2008;51:816–820. doi: 10.1007/s00125-008-0955-6. PubMed DOI

Merriman C., Huang Q., Rutter G.A., Fu D. Lipid-tuned Zinc Transport Activity of Human ZnT8 Protein Correlates with Risk for Type-2 Diabetes. J. Biol. Chem. 2016;291:26950–26957. doi: 10.1074/jbc.M116.764605. PubMed DOI PMC

Kim I., Kang E.S., Yim Y.S., Ko S.J., Jeong S.H., Rim J.H., Kim Y.S., Ahn C.W., Cha B.S., Lee H.C., et al. A low-risk ZnT-8 allele (W325) for post-transplantation diabetes mellitus is protective against cyclosporin A-induced impairment of insulin secretion. Pharm. J. 2011;11:191–198. doi: 10.1038/tpj.2010.22. PubMed DOI

Carvalho S., Molina-Lopez J., Parsons D., Corpe C., Maret W., Hogstrand C. Differential cytolocation and functional assays of the two major human SLC30A8 (ZnT8) isoforms. J. Trace Elem. Med. Biol. 2017;44:116–124. doi: 10.1016/j.jtemb.2017.06.001. PubMed DOI

Sala D., Giachetti A., Rosato A. Insights into the Dynamics of the Human Zinc Transporter ZnT8 by MD Simulations. J. Chem. Inf. Model. 2021;61:901–912. doi: 10.1021/acs.jcim.0c01139. PubMed DOI PMC

Wenzlau J.M., Liu Y., Yu L., Moua O., Fowler K.T., Rangasamy S., Walters J., Eisenbarth G.S., Davidson H.W., Hutton J.C. A common nonsynonymous single nucleotide polymorphism in the SLC30A8 gene determines ZnT8 autoantibody specificity in type 1 diabetes. Diabetes. 2008;57:2693–2697. doi: 10.2337/db08-0522. PubMed DOI PMC

Zhao J., Bertoglio B.A., Devinney M.J., Jr., Dineley K.E., Kay A.R. The interaction of biological and noxious transition metals with the zinc probes FluoZin-3 and Newport Green. Anal. Biochem. 2009;384:34–41. doi: 10.1016/j.ab.2008.09.019. PubMed DOI PMC

Marszałek I., Krężel A., Goch W., Zhukov I., Paczkowska I., Bal W. Revised stability constant, spectroscopic properties and binding mode of Zn(II) to FluoZin-3, the most common zinc probe in life sciences. J. Inorg. Biochem. 2016;161:107–114. doi: 10.1016/j.jinorgbio.2016.05.009. PubMed DOI

Kocyła A., Pomorski A., Krężel A. Molar absorption coefficients and stability constants of Zincon metal complexes for determination of metal ions and bioinorganic applications. J. Inorg. Biochem. 2017;176:53–65. doi: 10.1016/j.jinorgbio.2017.08.006. PubMed DOI

Sydor A.M., Lebrette H., Ariyakumaran R., Cavazza C., Zamble D.B. Relationship between Ni(II) and Zn(II) coordination and nucleotide binding by the Helicobacter pylori [NiFe]-hydrogenase and urease maturation factor HypB. J. Biol. Chem. 2014;289:3828–3841. doi: 10.1074/jbc.M113.502781. PubMed DOI PMC

Dietrich H., Maret W., Kozlowski H., Zeppezauer M. Active site-specifically reconstituted nickel(II) horse liver alcohol dehydrogenase: Optical spectra of binary and ternary complexes with coenzymes, coenzyme analogues, substrates, and inhibitors. J. Inorg. Biochem. 1981;14:297–311. doi: 10.1016/S0162-0134(00)80287-1. PubMed DOI

Krężel A., Wójcik J., Maciejczyk M., Bal W. May GSH and L-His contribute to intracellular binding of zinc? Thermodynamic and solution structural study of a ternary complex. Chem. Commun. 2003:704–705. doi: 10.1039/b300632h. PubMed DOI

Robertson R.P., Harmon J., Tran P.O., Poitout V. Beta-cell glucose toxicity, lipotoxicity, and chronic oxidative stress in type 2 diabetes. Diabetes. 2004;53(Suppl. 1):S119–S124. doi: 10.2337/diabetes.53.2007.S119. PubMed DOI

Árus D., Dancs Á., Nagy N.V., Gajda T. A comparative study on the possible zinc binding sites of the human ZnT3 zinc transporter protein. Dalton Trans. 2013;42:12031–12040. doi: 10.1039/c3dt50754h. PubMed DOI

Maret W., Vallee B.L. Cobalt as probe and label of proteins. Methods Enzymol. 1993;226:52–71. PubMed

Krężel A., Szczepanik W., Sokołowska M., Jeżowska-Bojczuk M., Bal W. Correlations between complexation modes and redox activities of Ni(II)-GSH complexes. Chem. Res. Toxicol. 2003;16:855–864. doi: 10.1021/tx034012k. PubMed DOI

Nieba L., Nieba-Axmann S.E., Persson A., Hamalainen M., Edebratt F., Hansson A., Lidholm J., Magnusson K., Karlsson A.F., Pluckthun A. BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip. Anal. Biochem. 1997;252:217–228. doi: 10.1006/abio.1997.2326. PubMed DOI

Ullah R., Shehzad A., Shah M.A., De March M., Ismat F., Iqbal M., Onesti S., Rahman M., McPherson M.J. C-Terminal Domain of the Human Zinc Transporter hZnT8 Is Structurally Indistinguishable from Its Disease Risk Variant (R325W) Int. J. Mol. Sci. 2020;21:926. doi: 10.3390/ijms21030926. PubMed DOI PMC

Cubillas C., Vinuesa P., Tabche M.L., Garcia-de los Santos A. Phylogenomic analysis of Cation Diffusion Facilitator proteins uncovers Ni2+/Co2+ transporters. Metallomics. 2013;5:1634–1643. doi: 10.1039/c3mt00204g. PubMed DOI

Wong W.P., Allen N.B., Meyers M.S., Link E.O., Zhang X., MacRenaris K.W., El Muayed M. Exploring the Association Between Demographics, SLC30A8 Genotype, and Human Islet Content of Zinc, Cadmium, Copper, Iron, Manganese and Nickel. Sci. Rep. 2017;7:473. doi: 10.1038/s41598-017-00394-3. PubMed DOI PMC

Hoch E., Lin W., Chai J., Hershfinkel M., Fu D., Sekler I. Histidine pairing at the metal transport site of mammalian ZnT transporters controls Zn2+ over Cd2+ selectivity. Proc. Natl. Acad. Sci. USA. 2012;109:7202–7207. doi: 10.1073/pnas.1200362109. PubMed DOI PMC

El Muayed M., Raja M.R., Zhang X., MacRenaris K.W., Bhatt S., Chen X., Urbanek M., O’Halloran T.V., Lowe W.L., Jr. Accumulation of cadmium in insulin-producing beta cells. Islets. 2012;4:405–416. doi: 10.4161/isl.23101. PubMed DOI PMC

Job P. Recherches sur la formation des complexes minéraux en solution, et sur leur stabilité. Ann. Chim. 1928;9:113–203.

Filipsky T., Riha M., Hrdina R., Vavrova K., Mladěnka P. Mathematical calculations of iron complex stoichiometry by direct UV-Vis spectrophotometry. Bioorganic Chem. 2013;49:1–8. doi: 10.1016/j.bioorg.2013.06.002. PubMed DOI

Sabel C.E., Neureuther J.M., Siemann S. A spectrophotometric method for the determination of zinc, copper, and cobalt ions in metalloproteins using Zincon. Anal. Biochem. 2010;397:218–226. doi: 10.1016/j.ab.2009.10.037. PubMed DOI

Fields G.B. Introduction to peptide synthesis. Curr. Protoc. Protein Sci. 2001;26:18.1.1–18.1.9. doi: 10.1002/0471140864.ps1801s26. PubMed DOI PMC

Gans P., Sabatini A., Vacca A. Investigation of equilibria in solution. Determination of equilibrium constants with the HYPERQUAD suite of programs. Talanta. 1996;43:1739–1753. doi: 10.1016/0039-9140(96)01958-3. PubMed DOI