Characterization of the Disordered Regulatory Domain from Calcineurin

Calcineurin (CaN) is a highly-conserved, ubiquitous Ser/Thr phosphotase that plays vital roles in memory development and retention, cardiac growth, and immune system activation. Alterations in the regulation of CaN can lead to disorders such as Down syndrome-related mental retardation and cardiac hypertrophy. CaN is also the target for the immunosuppressant drugs FK506 and cyclosporin A. The regulation of CaN function is not well understood at the molecular level. CaN is inactive until bound by calmodulin (CaM). CaM binds at a site towards the N-terminus of a 95 residue regulatory domain in CaN. This regulatory domain is believed to be disordered. The binding of CaM to CaN causes an autoinhibitory domain located C-terminal to the regulatory domain to be ejected from CaN's active site. We hypothesize that the CaN regulatory domain undergoes a folding transition upon CaM binding, and that this folding provides the driving force for pulling the autoinhibitory domain from the active site. We have made a fragment of CaN that consists of the regulatory domain, autoinhibitory domain and a short C-terminal domain. We will present data from CD spectroscopy, fluorescence, NMR and analytical ultracentrifugation experiments that indicate this fragment is largely disordered in the absence of CaM, and gains structure when CaM binds.