NMR studies of the E140Q mutant of the carboxy-terminal domain of calmodulin reveal global conformational exchange in the Ca2+-saturated state
Evenäs, Johan LU ; Thulin, Eva LU ; Malmendal, Anders LU ; Forsén, Sture LU and Carlström, Göran LU
(1997)
In Biochemistry
36(12).
p.3448-3457
- Abstract
In the present investigation, the Ca2+ activation of the C-terminal domain of bovine calmodulin and the effects of replacing the bidentate Ca2+-coordinating glutamic acid residue in the 12th and last position of loop IV with a glutamine are studied by NMR spectroscopy. The mutation E140Q results in sequential Ca2+ binding in this domain and has far-reaching effects on the structure of (Ca2+)2 TR2C, thereby providing further evidence for the critical role of this glutamic acid residue for the Ca2+- induced conformational change of regulatory EF-hand proteins. Analyses of the NOESY spectra of the mutant under Ca2+-saturated conditions, such that 97% of... (More)
In the present investigation, the Ca2+ activation of the C-terminal domain of bovine calmodulin and the effects of replacing the bidentate Ca2+-coordinating glutamic acid residue in the 12th and last position of loop IV with a glutamine are studied by NMR spectroscopy. The mutation E140Q results in sequential Ca2+ binding in this domain and has far-reaching effects on the structure of (Ca2+)2 TR2C, thereby providing further evidence for the critical role of this glutamic acid residue for the Ca2+- induced conformational change of regulatory EF-hand proteins. Analyses of the NOESY spectra of the mutant under Ca2+-saturated conditions, such that 97% of the protein is in the (Ca2+)2 form, revealed two sets of mutually exclusive NOEs. One set of NOEs is found to be consistent with the closed structure observed in the apo state of the C-terminal domain of the wild- type protein, while the other set supports the open structure observed in the Ca2+-saturated state. In addition, several residues in the hydrophobic core exhibit broadened resonances. We conclude that the (Ca2+)2 form of the mutant experiences a global conformational exchange between states similar to the closed and open conformations of the C-terminal domain of wild-type calmodulin. A population of 65 ± 15% of the open conformation and an exchange rate of (1-7) x 104 s-1 were estimated from the NMR data and the chemical shifts of the wild-type protein. From a Ca2- titration of the 15N-labeled mutant, the macroscopic binding constants (log(K1) = 4.9 ± 0.3 and log(K2) = 3.15 ± 0.10] and the inherent chemical shifts of the intermediate (Ca2+)1 form of the mutant were determined using NMR. Valuable information was also provided on the mechanism of the Ca2+ activation and the roles of the structural elements in the two Ca2+- binding events. Comparison with the wild-type protein indicates that the (Ca2+)1 conformation of the mutant is essentially closed but that some rearrangement of the empty loop IV toward the Ca2+-bound form has occurred.
(Less)
- author
- Evenäs, Johan
LU
; Thulin, Eva
LU
; Malmendal, Anders
LU
; Forsén, Sture
LU
and Carlström, Göran
LU
- organization
- publishing date
- 1997-03-25
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Biochemistry
- volume
- 36
- issue
- 12
- pages
- 10 pages
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- pmid:9131994
- scopus:0030951461
- ISSN
- 0006-2960
- DOI
- 10.1021/bi9628275
- language
- English
- LU publication?
- yes
- id
- b5a6baa3-840f-4928-b02a-7c309c4a7868
- date added to LUP
- 2019-01-16 13:25:53
- date last changed
- 2024-01-15 11:46:50
@article{b5a6baa3-840f-4928-b02a-7c309c4a7868,
abstract = {{<p>In the present investigation, the Ca<sup>2+</sup> activation of the C-terminal domain of bovine calmodulin and the effects of replacing the bidentate Ca<sup>2+</sup>-coordinating glutamic acid residue in the 12th and last position of loop IV with a glutamine are studied by NMR spectroscopy. The mutation E140Q results in sequential Ca<sup>2+</sup> binding in this domain and has far-reaching effects on the structure of (Ca<sup>2+</sup>)<sub>2</sub> TR<sub>2</sub>C, thereby providing further evidence for the critical role of this glutamic acid residue for the Ca<sup>2+</sup>- induced conformational change of regulatory EF-hand proteins. Analyses of the NOESY spectra of the mutant under Ca<sup>2+</sup>-saturated conditions, such that 97% of the protein is in the (Ca<sup>2+</sup>)<sub>2</sub> form, revealed two sets of mutually exclusive NOEs. One set of NOEs is found to be consistent with the closed structure observed in the apo state of the C-terminal domain of the wild- type protein, while the other set supports the open structure observed in the Ca<sup>2+</sup>-saturated state. In addition, several residues in the hydrophobic core exhibit broadened resonances. We conclude that the (Ca<sup>2+</sup>)<sub>2</sub> form of the mutant experiences a global conformational exchange between states similar to the closed and open conformations of the C-terminal domain of wild-type calmodulin. A population of 65 ± 15% of the open conformation and an exchange rate of (1-7) x 10<sup>4</sup> s<sup>-1</sup> were estimated from the NMR data and the chemical shifts of the wild-type protein. From a Ca<sup>2-</sup> titration of the <sup>15</sup>N-labeled mutant, the macroscopic binding constants (log(K<sub>1</sub>) = 4.9 ± 0.3 and log(K<sub>2</sub>) = 3.15 ± 0.10] and the inherent chemical shifts of the intermediate (Ca<sup>2+</sup>)<sub>1</sub> form of the mutant were determined using NMR. Valuable information was also provided on the mechanism of the Ca<sup>2+</sup> activation and the roles of the structural elements in the two Ca<sup>2+</sup>- binding events. Comparison with the wild-type protein indicates that the (Ca<sup>2+</sup>)<sub>1</sub> conformation of the mutant is essentially closed but that some rearrangement of the empty loop IV toward the Ca<sup>2+</sup>-bound form has occurred.</p>}},
author = {{Evenäs, Johan and Thulin, Eva and Malmendal, Anders and Forsén, Sture and Carlström, Göran}},
issn = {{0006-2960}},
language = {{eng}},
month = {{03}},
number = {{12}},
pages = {{3448--3457}},
publisher = {{The American Chemical Society (ACS)}},
series = {{Biochemistry}},
title = {{NMR studies of the E140Q mutant of the carboxy-terminal domain of calmodulin reveal global conformational exchange in the Ca<sup>2+</sup>-saturated state}},
url = {{http://dx.doi.org/10.1021/bi9628275}},
doi = {{10.1021/bi9628275}},
volume = {{36}},
year = {{1997}},
}