Structural basis for the negative allostery between Ca2+- and Mg2+-binding in the intracellular Ca2+-receptor calbindin D-9k

The three-dimensional structures of the magnesium- and manganese-bound forms of calbindin D-9k were determined to 1.6 Angstrom and 1.9 Angstrom resolution, respectively, using X-ray crystallography. These two structures are nearly identical but deviate significantly from both the calcium bound form and the metal ion-free (apo) form. The largest structural differences are seen in the C-terminal EF-hand, and involve changes in both metal ion coordination and helix packing. The N-terminal calcium binding site is not occupied by any metal ion in the magnesium and manganese structures, and shows little structural deviation from the apo and calcium bound forms. H-1-NMR and UV spectroscopic studies at physiological ion concentrations show that the C-terminal site of the protein is significantly populated by magnesium at resting cell calcium levels, and that there is a negative allosteric interaction between magnesium and calcium binding. Calcium binding was found to occur with positive cooperativity at physiological magnesium concentration.