Characterization of the calcium‐binding sites of calcineurin B

Abstract

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 Kd ∼ 2.4 × 10−8 M and three other sites with Kd ∼ 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