Characterization of the calcium-binding sites of calcineurin B

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.