Characterization of the calcium‐binding sites of calcineurin B

FEBS Letters

Kennedy, Michael¹  Armitage, Ian M²  Kakalis, Lazaros T²  Rusnak, Frank¹  Sikkink, Robert¹ 

[1] Section of Hematology, Research, Mayo Foundation and Clinic, Rochester, MN 55905, USA;Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA

关键词: Calcineurin;    Calcium binding;    EF hand;    Cadmium-113 NMR;    CaN;    calcineurin;    CaNA;    calcineurin A;    CaNB;    calcineurin B;    rCaNB;    recombinant CaNB;    CaM;    calmodulin;    CsA;    cyclosporin A;    CyP;    cyclophilin;    FKBP;    FK506 binding protein;    NFAT;    nuclear factor of activated T cells;    Tris;    tris(hydroxymethyl)aminomethane;    PIPES;    piperazine-N;    N′-bis(2-ethanesulfonic acid);    HEPES;    N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid;    TnC;    troponin C;    DTT;    dithiothreitol;    NMR;    nuclear magnetic resonance;   

DOI  :  10.1016/0014-5793(95)00207-P

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Calcineurin (CaN) is a calcium- and calmodulin-dependent serine/threonine phosphatase whose inhibition by the immunosuppressant-immunophilin complexes (cyclosporin-cyclophilin and FK506-FKBP) is considered key to the mechanism of immunosuppression. CaN is a heterodimer, consisting of a 59 kDa catalytic subunit (A) and a 19 kDa calcium-binding regulatory subunit (B). The latter is postulated to harbor four calcium binding domains of the EF hand type. The titration of the CaN B apoprotein with the isomorphic Cd²⁺ was followed by ¹¹³Cd NMR and these data support one high-affinity metal binding site and three lower-affinity ones. Flow dialysis data with Ca²⁺ indicate one high affinity calcium binding site with K _d ∼ 2.4 × 10⁻⁸ M and three other sites with K _d ∼ 1.5 × 10⁻⁵ M. The chemical shifts of all four ¹¹³Cd resonances (−75, −93, −106 and −119 ppm) are in the same range as found in other ¹¹³Cd substituted calcium-binding proteins, and are indicative of all-oxygen coordination of pentagonal bipyramidal geometry.

【 授权许可】

Unknown