Zn2+ binding to human calbindin D(28k) and the role of histidine residues.
We have studied the binding of Zn2+ to the hexa EF-hand protein, calbindin D(28k)-a strong Ca2+-binder involved in apoptosis regulation-which is highly expressed in brain tissue. By use of radioblots, isothermal titration calorimetry, and competition with a fluorescent Zn2+ chelator, we find that calbindin D(28k) binds Zn2+ to three rather strong sites with dissociation constants in the low micromolar range. Furthermore, we conclude based on spectroscopic investigations that the Zn2+-bound state is structurally distinct from the Ca2+-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 Zn2+. The binding of Zn2+ 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 Zn2+. Mutation of all five cysteines into serines has no effect on Zn2+-binding affinity or stoichiometry. However, mutating histidine 80 into a glutamine reduces the binding affinity of the strongest Zn2+ site, indicating that this residue is involved in coordinating the Zn2+ ion in this site. Mutating histidines 5, 22, or 114 has significantly smaller effects on Zn2+-binding affinity.
Bauer
Mikael C
MC
Department of Biophysical Chemistry, Lund University, Chemical Center, SE-22100 Lund, Sweden. Mikael.Bauer@bpc.lu.se
Nilsson
Hanna
H
Thulin
Eva
E
Frohm
Birgitta
B
Malm
Johan
J
Linse
Sara
S
D015720:5'-Nucleotidase / Q000378:metabolism; D000085:Acetates / Q000737:chemistry; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D064026:Calbindins; D002118:Calcium / Q000378:metabolism; D002151:Calorimetry; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000235:genetics / Q000378:metabolism*; D014966:Xanthenes / Q000737:chemistry; D015032:Zinc / Q000378:metabolism*
D000085:Acetates; D064026:Calbindins; C484689:FluoZin-1; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; D014966:Xanthenes; D006639:Histidine; D015720:5'-Nucleotidase; D015032:Zinc; D003545:Cysteine; D002118:Calcium