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. 2021 Mar 14;22(6):2940.
doi: 10.3390/ijms22062940.

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

Maria Carmen Catapano  1   2 , Douglas S Parsons  1   3 , Radosław Kotuniak  4 , Přemysl Mladěnka  5 , Wojciech Bal  4 , Wolfgang Maret  1
Affiliations

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

Maria Carmen Catapano et al. Int J Mol Sci. .

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.

Keywords: C-terminal domain; ZnT8; diabetes type 2; nickel; zinc; zinc transporter.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
ZnT8c protein stability. Both ZnT8cR (circles) and ZnT8cW (triangles) were stored at −20 °C in 50 mM TRIS/HCl, pH 8, 500 mM NaCl, 300 mM imidazole, 2 mM DTT (dithiothreitol), 100 mM sucrose with 50% (v/v) glycerol. After each of three freeze–thaw cycles the concentration of soluble protein was determined spectrophotometrically.
Figure 2
Figure 2
ZnT8c UV absorbance changes with addition of zinc. Representative (n = 3) UV absorbance spectra of 35 µM of ZnT8cR (A) and ZnT8cW (B) in the absence of Zn2+ (blue), and with one (red) and two (green) molar equivalents of Zn2+, demonstrating that addition of Zn2+ ablates a broad peak at approximately 320 nm from both apo-ZnT8c variants. Spectra are buffer- and dilution-corrected.
Figure 3
Figure 3
Assessment of the Zincon-Ni(II) and FluoZin-3-Ni(II) complex stoichiometry at pH 7.0. (A) Complementary Approach. The molar concentration of Ni(II) ions was kept constant at 25 μM and the total molar concentration of Zincon was continuously varied from 6 to 100 μM. The line with the grey diamonds corresponds to the absorbance of the complex formed after an excess of nickel was added. The other lines represent the theoretical absorbances expected if the stoichiometries were 1:1 (squares), 2:1 (triangles) or 3:1 (crosses). The comparison between the measured absorbance (black crosses) with the theoretical lines establishes a stoichiometry of 1:1. (B) Job’s method. The ratios refer to chelating agent/nickel. The total molar concentration of FluoZin-3 and Ni(II) was 100 μM. The lines converge on a molar ratio of 1:1. The assessment was performed with three independent stock solutions.
Figure 4
Figure 4
Nickel affinity of the two ZnT8 C-terminal domain (CTD) variants before and after alkylation. ZnT8cW. Nickel binding in competition with FluoZin-3 (A) and Zincon (B). Measuring fluorescence at 515 nm and absorbance at 653 nm, 70 μM NiSO4 saturates 70 μM FluoZin-3/Zincon in 50 mM HEPES (4-(2-hydroxymethyl)-1-piperazineethanesulfonic acid), 300 mM NaCl, 100 mM sucrose, pH 8 in agreement with the stoichiometry of the complex (red circles). In competition with 5 µM ZnT8cW, no signals at 515 nm or 653 nm are detected until 10 μM NiSO4 is added, revealing two high-affinity nickel binding sites in ZnT8cW which outcompete FluoZin-3/Zincon (black squares). When ZnT8cW is incubated with iodoacetamide for 1 h prior to the FluoZin-3/Zincon competition assay, only 5 μM NiSO4 is required to elicit the initial signals at 515 and 653 nm and the stoichiometry decreases to 1 (blue triangles). ZnT8cR. Nickel binding in competition with FluoZin-3 (C) and Zincon (D). NiSO4 titration of FluoZin-3/Zincon alone in HEPES buffer (red circles), in competition with ZnT8cR (black squares), and in competition with ZnT8cR modified with iodoacetamide (blue triangles), demonstrates that ZnT8cR also contains two high affinity nickel binding sites and that one binding site is blocked by alkylation.
Figure 4
Figure 4
Nickel affinity of the two ZnT8 C-terminal domain (CTD) variants before and after alkylation. ZnT8cW. Nickel binding in competition with FluoZin-3 (A) and Zincon (B). Measuring fluorescence at 515 nm and absorbance at 653 nm, 70 μM NiSO4 saturates 70 μM FluoZin-3/Zincon in 50 mM HEPES (4-(2-hydroxymethyl)-1-piperazineethanesulfonic acid), 300 mM NaCl, 100 mM sucrose, pH 8 in agreement with the stoichiometry of the complex (red circles). In competition with 5 µM ZnT8cW, no signals at 515 nm or 653 nm are detected until 10 μM NiSO4 is added, revealing two high-affinity nickel binding sites in ZnT8cW which outcompete FluoZin-3/Zincon (black squares). When ZnT8cW is incubated with iodoacetamide for 1 h prior to the FluoZin-3/Zincon competition assay, only 5 μM NiSO4 is required to elicit the initial signals at 515 and 653 nm and the stoichiometry decreases to 1 (blue triangles). ZnT8cR. Nickel binding in competition with FluoZin-3 (C) and Zincon (D). NiSO4 titration of FluoZin-3/Zincon alone in HEPES buffer (red circles), in competition with ZnT8cR (black squares), and in competition with ZnT8cR modified with iodoacetamide (blue triangles), demonstrates that ZnT8cR also contains two high affinity nickel binding sites and that one binding site is blocked by alkylation.
Figure 5
Figure 5
Near-UV circular dichroism (CD) of the two ZnT8c variants. Representative (n = 3) near-UV CD spectra of 0.4 mg/mL ZnT8cR (blue) and ZnT8cW (green) in 50 mM Tris, 300 mM NaCl, 100 mM sucrose, 100 µM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), pH 8. Subtracting the ZnT8cR spectrum from that of ZnT8cW provides the expected CD of W325 in the protein alone (red).
Figure 6
Figure 6
CD spectra of ZnT8c with Ni2+ excess. ZnT8cR + 0.1 mM NiSO4 (black line) and ZnT8cW + 0.1 mM of NiSO4 (dotted line) in 50 mM Tris, 300 mM NaCl, 100 mM sucrose, 100 µM TCEP, pH 8, 10 mm pathlength. The concentrations of the ZnT8c proteins were 35 µM. Corrected for buffer baseline.
Figure 7
Figure 7
Speciation diagrams for the ZnT8 11-residue C-terminal peptide. (A) Protonation of 180 µM peptide (denoted L) during titration with NaOH according to data in Table 6. At pH 7.4 the dominant species is LH4 (red). (B) Modeling of 180 µM peptide with 200 µM Zn2+ using the stability constants in Table 8 reveals that at pH 7.4 there is a mix of 58% ZnLH (red) and 42% ZnLH2 (green) relative to the peptide concentration. In both experiments the ionic strength is 0.1 M and the temperature is 25 °C.
Figure 8
Figure 8
The two Zn2+-binding sites in the cytosolic C-terminal domain (CTD) of human ZnT8. The presentation is based on a recent Cryo-EM structure of the entire protein [20]. One metal ion is bound by two N-donors from histidines (301 & 318) and one O-donor from glutamate (352). An S-donor from cysteine (53) from the N-terminus (not present in the CTD) complements a tetrahedral binding geometry. The second site is assembled from donors of the C- and N-termini only: Two S-donors from cysteines (361 & 364) from the C-terminus and two N-donors from histidines (52 & 54) from the N-terminus. The N-terminal tripartite motif with adjacent metal-coordinating amino acid side chains (HCH) “seals off” the metal sites [20]. Both termini have to swing into the site to effect coordination of the second metal ion.
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