Abstract
Calcineurin (CaN) plays an important role in the T-cell activation, cardiac system development and nervous system function. Previous studies have suggested that the regulatory domain (RD) of CaN binds Calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the regulatory domain, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is at least partially disordered, and binding of CaM induces folding in the RD. Previous studies have shown that an α-helical structure forms in the N-terminal half of the RD, but organization may occur in the C-terminal half as well. Here, we are interested in the structural transition of the full length RD as it binds to CaM. Using nuclear magnetic resonance (NMR) spectroscopy, we have successfully assigned >85% of the 15N, 13Cα, 13Cβ and HN chemical shifts of the unbound, regulatory domain of CaN. While the protein is disordered, secondary chemical shifts indicate that some regions possess α-helical propensity, even in the unbound state. Our study of the CaM and CaN interaction supports the formation of a distal helix in the region between the AID and calmodulin-binding region. Heat capacity changes upon binding predict that 54 residues fold when CaM binds to CaN, consistent with the formation of this distal helix. Paramagnetic relaxation enhancement (PRE) studies of this interaction suggest a potential binding mode where the distal helix binds to CaM near residues I9-A15. Mutagenesis in the distal helix disrupts PREs, further supporting this hypothesis. Together, these data suggest that the interactions between CaM and the distal helix of CaN can be important in regulation of phosphatase activity.
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