NMR Investigation of Calmodulin Induced Folding in the Regulatory Domain of Calcineurin

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 97-residue regulatory domain (RD) of CaN binds Calmodulin (CaM) towards the N-terminal end. Calcium/Calmodulin 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 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, full-length regulatory domain of CaN. While the protein is unstructured, secondary chemical shifts indicate some regions with α-helical propensity, even in the free state. At present, we are studying how the spectrum changes as calcium-loaded CaM is added to the solution. In the long term, this work will identify how binding-induced folding changes the RD structure, which could ultimately lead to a better understanding of how CaM activates CaN.