Figure 1.

Structure of Nur and metal sites. (A) Ribbon diagram of Nur with the dimeric core veiled by transparent surface. Nickel ions and metal-coordinating residues are represented by spheres and sticks, respectively. M and Ni indicate M- and Ni-sites, respectively. A black circle indicates the plausible DNA-binding site. For clarity, secondary structure elements only for DB-domain are labeled. (B) Ribbon diagram of PaFur. M and Fe indicate M- and Fe-sites, respectively. The S1–S2 sheets are in green. Zinc ions and residues of M-site are shown by spheres and sticks, respectively. A black circle indicates the plausible DNA-binding site. (C) A structure-based sequence alignment of Nur with PaFur. Red and blue letters in the Nur (PaFur) sequence indicate residues of M- and Ni (Fe)-site, respectively. The hinge region is dark shaded. Two Cys-X-X-Cys motifs are boxed. (D) Stereo view of the final 2F_o – F_c electron density maps contoured at 1σ, showing M- and Ni-sites. Ni and Ni-coordinating residues are shown in spheres and sticks, respectively. (E) DNA-binding activity of Nur variants with substitution mutations of M-site residues. E101A mutant was examined in parallel for comparison. Electrophoretic mobility shift assay was performed for binding between the sodF promoter DNA fragment (–60 to +30 nt from transcription start site) and cell extracts containing either wild-type or mutant Nur proteins with H33A, H86A, H88A and H90A mutations. For each Nur variant, three separate reactions with increasing amounts of cell extracts were examined, along with the assessment of Nur protein levels by western blot analysis (lower panel). FP indicates a sample with free probe only. (F) DNA-binding activity of Nur variants with substitution mutations of Ni-site residues. Nur variants with H70A, H72A and H126A mutations were examined as described above. H75A mutant was examined in parallel for comparison. (G) Cα-tracings of DB-domains from Nur (green) and PaFur (violet) that are superposed using only one DB-domain.