ESR study on calcineurin.

X-band electron spin resonance spectroscopy was used to investigate the binding of Mn2+ to the apo-forms of calcineurin and its A and B subunits. The results indicated the presence of 2 Mn2+ binding sites of different affinities (20 mumol/L and 60 mumol/L) in the calcineurin A subunit and 4 Mn2+ binding sites in the calcineurin subunit B, 2 high affinity and 2 low affinity binding sites with Kd's of 4 mumol/L and 90 mumol/L, respectively. Interestingly and quite surprisingly, Mn2+ binding to the holoenzyme was characterized by only 2 binding sites with Kd's of 7 mumol/L and 33 mumol/L. However, in the presence of calmodulin about 10 Mn2+ sites were detected, and the Mn2+ calmodulin-calcineurin complex exhibited enzymatic activity. These results, based on direct spectral measurements of the metal ligand, demostrate that Mn2+ binds to both free subunits of calcineurin in a manner distinct from binding to the holoenzyme. Also, the data suggest that conformational changes occur upon heterodimer formation and association of the holoenzyme with the regulatory protein calmodulin.