Structural basis for the negative allostery between Ca2+‐ and Mg2+ ‐binding in the intracellular Ca2+ ‐receptor calbindin D9k

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Abstract
The three‐dimensional structures of the magnesium‐ and manganese‐bound forms of calbindin D9% were determined to 1.6 Å and 1.9 Å 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. 1 H‐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.