HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Bauer, M. C. Articles by Linse, S. PubMed PubMed Citation Articles by Bauer, M. C. Articles by Linse, S. Social Bookmarking                   What's this?

Zn²⁺ binding to human calbindin D_28k and the role of histidine residues

¹ Department of Biophysical Chemistry, Lund University, Chemical Center, SE-22100 Lund, Sweden
² Laboratory Medicine, Section for Clinical Chemistry, Lund University, SE 20502 Malmö, Sweden

(RECEIVED December 3, 2007; FINAL REVISION January 24, 2008; ACCEPTED January 28, 2008)

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.

Keywords: EF-hand; zinc; histidine; mutagenesis

CiteULike

Connotea

Del.icio.us

Digg

Reddit

Technorati