Open AccessMutation of the pseudo‐EF‐hand of calbindin D 9k into a normal EF‐hand
Author(s) -
JOHANSSON Charlotta,
BRODIN Peter,
GRUNDSTRÖM Thomas,
FORSÉN Sture,
DRAKENBERG Torbjön
Publication year - 1991
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1991.tb16501.x
Subject(s) - ef hand , mutant , amino acid , calmodulin , chemistry , side chain , calcium , calcium binding protein , calbindin , stereochemistry , cysteine , binding site , biochemistry , enzyme , gene , organic chemistry , polymer
The two Ca 2+ ‐binding sites in calbindin D 9k , a protein belonging to the calmodulin superfamily of intracellular proteins, have sligtly different structure. The C‐terminal site (amino acids 54–65) is a normal EF‐hand as in the other proteins of the calmodulin superfamily, while the N‐terminal site (amino acids 14–27) contains two additional amino acids, one of which is a proline. We have constructed and studied five mutants of calbindin D 9k modified in the N‐terminal site. In normal EF‐hand structures the first amino acid to coordinate calcium is invariantly an Asp. For this reason Ala 15, is exchanged by an Asp in all mutants and the mutants also contain various other changes in this site. The mutants have been characterized by 43 Ca, 113 Cd and 1 H NMR and by the determination of the calcium binding constants using absorption chelators. In two of the mutants (one where Ala14 is deleted, Ala15 is replaced by Asp and Pro20 is replaced by Gly, the other where, in addition, Asn21 is deleted), we find that the structure has changed considerably compared to the wild‐type calbindin. The NMR results indicate that the calcium coordination has changed to mainly side‐chain carboxyls, from being octahedrally coordinated by mainly back‐bone carbonyls, and/or that the coordination number has decreased. The N‐terminal site has thus been turned into a normal EF‐hand, in which the calcium ion is coordinated by side‐chain carboxyls. Furthermore, the calcium binding constants of these two mutant proteins are almost as high as in the wild‐type calbindin D 9k . That is, the extensive alterations in the N‐terminal site have not disrupted the calcium binding ability of the proteins.