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The Solution Structures of Mutant Calbindin D9k's, As Determined by NMR, Show That the Calcium-Binding Site Can Adopt Different Folds

Johansson, Charlotta; Ullner, Magnus LU and Drakenberg, Torbjörn LU (1993) In Biochemistry 32(33). p.8429-8438
Abstract

The complete 1H NMR assignments have been obtained for five mutant proteins of calbindin D9k and the three-dimensional solution structures determined for two of the mutants. The structures have been determined using distance geometry and simulated annealing, with distance constraints from NMR. All mutants have modifications in the first calcium-binding site of calbindin (the N-terminal site designated the pseudo-EF-hand). The 3D structure of the mutant with the most extensive modifications in the pseudo-EF-hand shows that the site has turned inside-out and coordinates calcium as in the normal EF-hand (the C-terminal site). In a pseudo-EF-hand loop the calcium is coordinated by main-chain carbonyls, whereas calcium... (More)

The complete 1H NMR assignments have been obtained for five mutant proteins of calbindin D9k and the three-dimensional solution structures determined for two of the mutants. The structures have been determined using distance geometry and simulated annealing, with distance constraints from NMR. All mutants have modifications in the first calcium-binding site of calbindin (the N-terminal site designated the pseudo-EF-hand). The 3D structure of the mutant with the most extensive modifications in the pseudo-EF-hand shows that the site has turned inside-out and coordinates calcium as in the normal EF-hand (the C-terminal site). In a pseudo-EF-hand loop the calcium is coordinated by main-chain carbonyls, whereas calcium in the normal EF-hand is coordinated by side-chain carboxylates. The 3D structures and 1H NMR assignments show that in order to accomplish a change in the coordinating ligands of the pseudo-EF-hand the loop must be 12 residues long and have glycine in the sixth position. It does, however, seem possible to have alanine instead of aspartic acid in the first calcium coordinating position. The overall global fold of the proteins has not been affected by the mutations in the calcium-binding site, as compared to the wild-type calbindin D9k [Kördel, J., Skelton, N. J., Akke, M., & Chazin, W. J. (1993) J. Mol. Biol. (in press)]. The structures consist of two helix-calcium-binding loop-helix motifs, the so called EF-hands, and the loops are connected by a short antiparallel β-sheet. All helices are pairwise in an antiparallel orientation.

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author
organization
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type
Contribution to journal
publication status
published
subject
in
Biochemistry
volume
32
issue
33
pages
10 pages
publisher
The American Chemical Society
external identifiers
  • scopus:0027305754
ISSN
0006-2960
DOI
10.1021/bi00084a007
language
English
LU publication?
yes
id
c8f21d96-ce43-46af-b197-80e175df3e4c
date added to LUP
2018-03-29 10:43:15
date last changed
2018-06-24 05:21:30
@article{c8f21d96-ce43-46af-b197-80e175df3e4c,
  abstract     = {<p>The complete <sup>1</sup>H NMR assignments have been obtained for five mutant proteins of calbindin D<sub>9k</sub> and the three-dimensional solution structures determined for two of the mutants. The structures have been determined using distance geometry and simulated annealing, with distance constraints from NMR. All mutants have modifications in the first calcium-binding site of calbindin (the N-terminal site designated the pseudo-EF-hand). The 3D structure of the mutant with the most extensive modifications in the pseudo-EF-hand shows that the site has turned inside-out and coordinates calcium as in the normal EF-hand (the C-terminal site). In a pseudo-EF-hand loop the calcium is coordinated by main-chain carbonyls, whereas calcium in the normal EF-hand is coordinated by side-chain carboxylates. The 3D structures and <sup>1</sup>H NMR assignments show that in order to accomplish a change in the coordinating ligands of the pseudo-EF-hand the loop must be 12 residues long and have glycine in the sixth position. It does, however, seem possible to have alanine instead of aspartic acid in the first calcium coordinating position. The overall global fold of the proteins has not been affected by the mutations in the calcium-binding site, as compared to the wild-type calbindin D<sub>9k</sub> [Kördel, J., Skelton, N. J., Akke, M., &amp; Chazin, W. J. (1993) J. Mol. Biol. (in press)]. The structures consist of two helix-calcium-binding loop-helix motifs, the so called EF-hands, and the loops are connected by a short antiparallel β-sheet. All helices are pairwise in an antiparallel orientation.</p>},
  author       = {Johansson, Charlotta and Ullner, Magnus and Drakenberg, Torbjörn},
  issn         = {0006-2960},
  language     = {eng},
  number       = {33},
  pages        = {8429--8438},
  publisher    = {The American Chemical Society},
  series       = {Biochemistry},
  title        = {The Solution Structures of Mutant Calbindin D<sub>9k</sub>'s, As Determined by NMR, Show That the Calcium-Binding Site Can Adopt Different Folds},
  url          = {http://dx.doi.org/10.1021/bi00084a007},
  volume       = {32},
  year         = {1993},
}