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. 2017 Jan 17;15(1):e2000094. doi: 10.1371/journal.pbio.2000094

Fig 3. Zinc directly bound the HIZR-1 ligand-binding domain.

Fig 3

(A) Glutathione S-transferase (GST) alone and the ligand-binding domain of HIZR-1 (residues 101–412) fused to GST were expressed in bacteria and partially purified by affinity chromatography. Increasing concentrations of either GST::HIZR-1(101–412 WT) or GST alone were incubated with a fixed concentration of radioactive zinc-65, and the amount of zinc-65 bound to protein was quantified by filter binding and scintillation counting. Values are the average +/- S.D. in counts per minute (CPM). At least two technical replicates were performed for each unique protein concentration. GST::HIZR-1(101–412 WT) displayed saturable binding, and a nonlinear regression was used to calculate a dissociation constant of 1.7 +/- 0.3 μM. X-ray crystallography studies indicate that GST binds one zinc molecule per protein [32]; our data are consistent with saturable, low-level zinc binding by GST alone. (B) GST alone and the ligand-binding domain of DAF-12 isoform A (residues 440–753) fused to GST were expressed in bacteria, partially purified by affinity chromatography, and analyzed for zinc binding using the method described above. GST again displayed saturable, low-level zinc binding. GST::DAF-12(440–753 WT) LBD displayed binding similar to GST alone, indicating that the LBD of DAF-12 does not bind an appreciable amount of zinc. (C) Partially purified GST::HIZR-1(101–412 WT) was incubated with radioactive zinc-65 and no additional metal (none) or 500 μM nonradioactive zinc, copper, nickel, or manganese. Bars indicate the amount of zinc-65 bound to protein +/- S.D. (n = 3) quantified by filter binding and scintillation counting. The values were normalized by setting the value of the sample with no additional metal equal to 1.0, defined as maximal binding. Low values indicate the nonradioactive metal competes effectively with radioactive zinc-65. For GST::HIZR-1(101–412 WT), compared to nonradioactive zinc, nickel and manganese displayed significantly lower effectiveness as competitors (*, p < 0.05). The value for copper was not significantly different than the value for zinc (p = 0.13), indicating copper effectively competes with zinc for binding.