(A) Global Cα wireframe superposition of wild-type (orange) and L34A (magenta, right) and V66L (green, left) CzrAs, with the positions of the zinc atoms shown as spheres. (B) Spacefilling representations of the van der Waals packing region in the vicinity of either the second coordination shell hydrogen bond23,26,39 (top) or the L34 surface (bottom) in Zn2 wild-type (orange), L34A (magenta), V66L (green), and V66A/L68V (light green) CzrAs. Summary of the allosteric coupling free energies measured by fluorescence anisotropy (ΔGc, Figure S1) and stepwise Zn(II) binding free energies for the binding of the first (ΔG1,Zn) and second (ΔG2,Zn) Zn ions to each CzrA homodimer.