Effect of metal ions on the activity of the catalytic domain of calcineurin

Abstract

Calcineurin (CN) is a heterodimer, composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). There are four functional domains present in CNA, which are catalytic domain (CNa), CNB-binding domain (BBH), CaM-binding domain (CBH) and autoinhibitory domain (AI). It has been shown previously that the in vitro activity of calcineurin is relied primarily on the binding of metal ions. Mn²⁺ and Ni²⁺ are the most crucial cation-activators for this enzyme. In order to determine which domain(s) in CN is functionally regulated by metal ions, the rat CNA α subunit and its catalytic domain (CNa) were cloned and expressed in E. coli. The effects of Mn²⁺, Ni²⁺ and Mg²⁺ on the catalytic activity of these purified proteins were examined. Our results demonstrate that all the metal ions tested in this study activated either CNA or CNa. However, the activation degree of CNa by the metal ions was much higher than that of CNA. In term of different metal ions, the activating extents to CNA and CNa were different. To CNA, the activating order from high to low was Mg²⁺>>Ni²⁺>Mn²⁺, but Mn²⁺>Ni²⁺>>Mg²⁺ to CNa. No effect of CaM/Ca²⁺ and CNB/Ca²⁺ on the activity of CNa was observed in our experiments. Moreover, a weak interaction (or untight coordination binding) between metal ions and the enzyme molecule was also identified. These results suggest that the activation of these enzymes by the exogenous metal ions might be via both regulating fragment of CNA (including BBH, CBH and AI) and catalytic domain (CNa), and mainly via regulating fragment to CNA and mainly via catalytic domain to CNa. The activating extents of metal ions via catalytic domain were higher than that via regulating fragment. The results obtained in this study should be very useful for understanding the molecular mechanism underlying the interaction between calcineurin and metal ions, especially Mn²⁺, Ni²⁺ and Mg²⁺.