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Zn 2+ binding to human calbindin D 28k and the role of histidine residues
Author(s) -
Bauer Mikael C.,
Nilsson Hanna,
Thulin Eva,
Frohm Birgitta,
Malm Johan,
Linse Sara
Publication year - 2008
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.073381108
Subject(s) - histidine , chemistry , isothermal titration calorimetry , binding site , dissociation constant , allosteric regulation , calmodulin , titration , stereochemistry , biochemistry , receptor , amino acid , enzyme , inorganic chemistry
We have studied the binding of Zn 2+ to the hexa EF‐hand protein, calbindin D 28k —a strong Ca 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 2+ chelator, we find that calbindin D 28k binds Zn 2+ to three rather strong sites with dissociation constants in the low micromolar range. Furthermore, we conclude based on spectroscopic investigations that the Zn 2+ ‐bound state is structurally distinct from the Ca 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 2+ . The binding of Zn 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 2+ . Mutation of all five cysteines into serines has no effect on Zn 2+ ‐binding affinity or stoichiometry. However, mutating histidine 80 into a glutamine reduces the binding affinity of the strongest Zn 2+ site, indicating that this residue is involved in coordinating the Zn 2+ ion in this site. Mutating histidines 5, 22, or 114 has significantly smaller effects on Zn 2+ ‐binding affinity.