Fig. 1: AflR-DBD combines a structured zinc cluster motif with disordered regions.

a Domain architecture of AflR (1–444) showing the zinc cluster motif (29–56) and truncation constructs designed for functional mapping. b SDS-PAGE analysis of purified TRX-tagged AflR truncations. The experiments have been repeated independently three times, and one representative result is shown. c Size exclusion chromatography (SEC) profiles of TRX-tagged AflR truncations with molecular weight standards. d Analytical gel filtration chromatography profiles of TRX-tagged AflR truncation interactions with the ver1 promoter DNA. Complex peaks (black arrow), free DNA peak (green arrow), and free protein peak (gray arrow) are indicated. Absorbance at 280 nm (mAU) indicates components eluting at different retention volumes. e Binding affinity determination using FCS titration of Cy3-labeled ver1 promoter with AflR truncations. Error bars represent the Mean ± SD from three independent experiments. f Far-UV CD spectra of AflR 8–98 and 15–79. Error bars represent the Mean ± SEM from three independent measurements. g ¹H-¹⁵N HSQC spectrum demonstrating partial disorder in AflR 15–79. Black labels indicate zinc cluster residues, blue labels show N-terminal residues, cyan labels show C-terminal residues. h Secondary structure propensity calculated from Cα and Cβ chemical shifts. i MD simulation-derived conformational ensemble showing the six most populated states with their relative populations. Helical regions are shown in blue, dynamic terminal regions are shown in red, zinc ions are shown in gold. j T₁/T₂ ratios of ¹⁵N-labeled AflR-DBD. T₁ and T₂ relaxation rates were estimated by fitting the peak intensities to single exponential decays. Error bars represent the uncertainty in relaxation rates derived from the fitting procedure⁴⁵. k ¹H-¹⁵N heteronuclear NOE of ¹⁵N-labeled AflR-DBD. NOE values were determined as the ratio of peak intensities in spectra recorded with and without proton saturation. Error bars were calculated based on the standard deviation of noise in the saturated and unsaturated spectra using error propagation. For (c–f), colors consistently represent AflR truncations: 8–98 (blue), 15–88 (green), 15–79 (yellow), 15–69 (pink), and 26–79 (purple).