FIGURE 4.

Structure of FrmR^E64H and inferred Zn(II)/Co(II)-sensing site. A, ribbon representation of the 2.19 Å resolution crystal structure of FrmR^E64H tetramer (Protein Data Bank code 5LCY; see Table 2 for a summary of the crystallographic data). Each monomer is colored differently, and secondary structural units are labeled on the cyan monomer. B, electrostatic surface potential of FrmR^E64H tetramer using Chimera (103). The color scale is from −10 (negative potential; red) to +10 (positive potential; blue) kcal/mol·e. C, anisotropy change upon titration of a limiting concentration (10 nm) of frmRAPro (solid symbols) or frmRAPro* (half-site defined in Fig. 2D; open symbols) with FrmR (circles) or FrmR^E64H (triangles) in the presence of 5 mm EDTA. The lines are fits of the data to a model describing a 2:1 protein tetramer (nondissociable)/DNA stoichiometry (binding with equal affinity) (50, 86). D, expansion of the dimeric interface with backbone helices from two different monomers shaded green and cyan (the same colors as used in A). The inferred Zn(II)/Co(II)-binding site comprises Cys³⁵ from α2′, and His⁶⁰ and His⁶⁴ from α2 (belonging to the XYZ motif required for metal binding in DUF156 members CsoR, RcnR, and InrS (39, 46, 68), with His³ from α1 (position W (46, 61)) and Asp⁶³ presenting candidate fourth ligands. E, analysis of fractions (0.5 ml) for protein by Bradford assay (open circles) and metal by inductively coupled plasma MS (filled circles) following size exclusion chromatography of FrmR, FrmR^E64H, FrmR^C35A (50 μm, monomer), or FrmR^H60L (in this case, [monomer] = 32.5 μm), preincubated with 150 μm ZnCl₂.