Zn super(2+) binding to human calbindin D sub(28k) and the role of histidine residues

We have studied the binding of Zn super(2+) to the hexa EF-hand protein, calbindin D sub(28k)-a strong Ca super(2+)-binder involved in apoptosis regulation-which is highly expressed in brain tissue. By use of radioblots, isothermal titration calorimetry, and competition with a fluorescent Zn super(2+) chelator, we find that calbindin D sub(28k) binds Zn super(2+) to three rather strong sites with dissociation constants in the low micromolar range. Furthermore, we conclude based on spectroscopic investigations that the Zn super(2+)-bound state is structurally distinct from the Ca super(2+)-bound state and that the two forms are incompatible, yielding negative allosteric interaction between the zinc- and calcium-binding events. ANS titrations reveal a change in hydrophobicity upon binding Zn super(2+). The binding of Zn super(2+) is compatible with the ability of calbindin to activate myo-inositol monophosphatase, one of the known targets of calbindin. Through site-directed mutagenesis, we address the role of cysteine and histidine residues in the binding of Zn super(2+). Mutation of all five cysteines into serines has no effect on Zn super(2+)-binding affinity or stoichiometry. However, mutating histidine 80 into a glutamine reduces the binding affinity of the strongest Zn super(2+) site, indicating that this residue is involved in coordinating the Zn super(2+) ion in this site. Mutating histidines 5, 22, or 114 has significantly smaller effects on Zn super(2+)-binding affinity.