Zn2+ binding to human calbindin D(28k) and the role of histidine residues.
(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:
https://lup.lub.lu.se/record/1052240
- author
- Bauer, Mikael LU ; Nilsson, Hanna LU ; Thulin, Eva LU ; Frohm, Birgitta LU ; Malm, Johan LU and Linse, Sara LU
- organization
- publishing date
- 2008
- 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
- pmid:18359862
- 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
- 2016-04-01 11:44:03
- date last changed
- 2022-03-12 23:55:47
@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}},
keywords = {{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}},
doi = {{10.1110/ps.073381108}},
volume = {{17}},
year = {{2008}},
}