Advanced

Zn2+ binding to human calbindin D(28k) and the role of histidine residues.

Bauer, Mikael LU ; Nilsson, Hanna LU ; Thulin, Eva LU ; Frohm, Birgitta LU ; Malm, Johan LU and Linse, Sara LU (2008) In Protein Science 17(4). p.760-767
Abstract
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+.... (More)
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. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
histidine, EF-hand, zinc, mutagenesis
in
Protein Science
volume
17
issue
4
pages
760 - 767
publisher
The Protein Society
external identifiers
  • pmid:18359862
  • wos:000254197800017
  • scopus:41649096584
ISSN
1469-896X
DOI
10.1110/ps.073381108
language
English
LU publication?
yes
id
c514981c-fe1f-4474-b0f4-fd05da3bedee (old id 1052240)
date added to LUP
2008-05-09 10:33:45
date last changed
2017-03-19 03:25:15
@article{c514981c-fe1f-4474-b0f4-fd05da3bedee,
  abstract     = {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.},
  author       = {Bauer, Mikael and Nilsson, Hanna and Thulin, Eva and Frohm, Birgitta and Malm, Johan and Linse, Sara},
  issn         = {1469-896X},
  keyword      = {histidine,EF-hand,zinc,mutagenesis},
  language     = {eng},
  number       = {4},
  pages        = {760--767},
  publisher    = {The Protein Society},
  series       = {Protein Science},
  title        = {Zn2+ binding to human calbindin D(28k) and the role of histidine residues.},
  url          = {http://dx.doi.org/10.1110/ps.073381108},
  volume       = {17},
  year         = {2008},
}