PMC

Two ribbon representation views of CzrA in which the backbone amide resonances derived from an ¹H-¹⁵N TROSY spectrum (see ) of ternary CzrA•Zn₂•DNA complex mimic those found in the apo-CzrA-DNA state (shaded yellow) or in Zn₂ CzrA (shaded green) or are found in a magnetically unique environment in each of the three states (shaded magenta). Backbone resonance assignments of the CzrA•Zn₂•DNA complex were obtained by inspection and included ≈50% coverage of the molecule (unassigned resonances shaded gray). Residues subjected to mutagenesis are indicated with Cα atoms represented as spheres.

The structures of Zn(II)-bound wild-type and V66A/L68V CzrAs are globally identical. (a) Global Cα wireframe superposition of wild-type (green) and V66A/L68V (red) CzrAs, with the positions of the zinc atoms shown in slate or darker slate. (b) Detailed superposition of the first and selected second coordination shell region of wild-type (green) and V66A/L68V (magenta) CzrAs. The interprotomer H97’-H67 (highlighted by the red arrow) and L68-Leu63 hydrogen bonds are indicated by the dashed lines. (c) and (d) Spacefilling representations of the van der Waals packing region in the vicinity of these hydrogen bonds in Zn₂ wild-type CzrA (panel c) and V66A/L68V CzrA (panel d). Side chains of residues 66 and 68 and Cα atoms are shaded orange and pale yellow, respectively.

Analysis by isothermal titration calorimetry of Zn(II) binding to various apo-CzrAs and the apo-CzrA-DNA complex. (a) Representative titrations of Zn(II) binding to wild-type (WT), V66A, L68V and V66A/L68V CzrA dimers. (b) Thermodynamic parameters obtained for Zn(II) binding to CzrAs obtained from experiments like those shown in panel A. t, total, obtained by summing parameters obtained for the first (Zn₁) and second (Zn₂) Zn(II) binding steps. (c) Representative titrations of Zn(II) into CzrO complexes formed by wild-type (WT) and V66A/L68V CzrAs. These titrations correspond to the “top” and “bottom” of the heterotropic coupling equilibrium that defines this system. (d) Graphical illustration of the coupling energetics of wild-type vs. V66A/L68V CzrAs derived from the first (Zn₁) and second (Zn₂) binding steps. t, total. Conditions: 38 µM dimer, 3 mM NTA as a zinc competitor, or 38 µM dimer-DNA complex, with 1 mM NTA as competitor in 10 mM Hepes, 0.4 M NaCl, pH 7.0, 25.0 °C.

Methylhistidine-substituted (H97MeH) CzrA and cavity mutant CzrAs show poor allosteric inhibition of DNA binding upon Zn(II) binding. (a) Ribbon representation of the CzrA dimer illustrating the relative disposition of the allosteric Zn(II) sites (Zn(II) ion, yellow spheres) and the DNA-binding helix-turn-helix (H-T-H) domain shaded in light blue. Residues of interest in this study are shown in stick representation. (b) Proposed allosteric coupling pathway that links each of the two ligand binding sites. (c) Normalized fluorescence anisotropy-based DNA binding isotherms of H96C (closed symbols) and H97MeH (open symbols) CzrAs acquired in the absence (circles) and presence (triangles) of 10 µM Zn(II). Curves represent the best fit using a 1:1 dimer:DNA binding model with the binding parameters compiled in . (d) Representative normalized fluorescence anisotropy-based DNA binding curves for Zn(II)-saturated wild-type (closed circles), L68V (open circles), L68A (closed squares), V66A (open squares) and V66A/L68V (triangles) CzrAs. Parameter values are compiled in .. Conditions: 10 mM Hepes, 0.23 M NaCl, 2.0 µM ZnSO₄, pH 7.0, 25.0 °C.

Structural representation of a statistical coupling analysis of ArsR family repressor reveals a coupled network connected to an allosteric “hot-spot”. (a) Network of coupled residues shown in spacefill on both protomers (CPK coloring shown in left protomer with protons shaded yellow and carbons slate; gray on right protomer). Key DNA binding residues (S57, H58) shaded CPK with protons white and carbon atoms shaded orange; V66 and L68 define an allosteric “hot-spot” with all atoms shaded magenta. CzrA is shown in its open or “flat” allosterically inhibited Zn(II)-bound low DNA binding affinity state. (b)-(d) Surface representations of (b) CzrA (1R1V); (c) CadC (1U2W) and (d) CmtR (2JSC) highlighting a sector of coevolving residues (shaded yellow), the allosteric metal site chelates (shaded blue), major energetic determinants of the DNA binding domain (shaded orange; corresponding to residues 54–58 in CzrA) and the V66/L68 in CzrA (shaded magenta). View from the DNA binding interface is shown on the left, with a view from the top of the molecule shown on the right. (e) Schematic rendering of the conformational transition from a high to a low DNA binding conformation with the sector schematized as a lever, and the V66/L68 “pivot” point indicated by the triangle with the α5 metal sites shown as circles.

Statistical coupling analysis of ArsR family repressors. (a) Residue-specific conservation plotted as relative entropy of residue positions 15–35, 41–87, 89–90 and 92–98 which correspond to CzrA residue positions. Gaps correspond to gaps in the multiple sequence alignment. Selected residues with the highest conservation are highlighted with CzrA residue type and number. (b) Schematic illustration of residues in physical contact arranged as two parts of the same network of 17 coupled residues (upper left; lower right, shaded gray), with selected pairwise statistical coupling energies shown in pink (see Methods). Three key residues that are strongly pairwise coupled to one another are shown boxed in red. The green box represents four residues in direct physical contact that connect β-wing and more peripheral regions of the DNA-binding site to the pivot point defined by V66/L68. (c) Normalized heat-map of all (17×17) significantly pairwise coupled interactions scaled from 0 (no coupling) to 1 (strong coupling). The diagonal is false-colored light blue and represents the mean value of the correlation map. Residues pairs 41/43 and 41/77 exhibit the strongest pairwise couplings. It is interesting to note that the CXC Cys pair in Pb(II)/Cd(II)-sensing CadCs (which correspond to residue positions 41/43 in CzrA) are ligands to the Cd/Pb ion.