Date of Degree
PhD (Doctor of Philosophy)
Madeline A. Shea
Calcineurin (CaN), a heterodimeric Ca2+-calmodulin-dependent Ser/Thr phosphatase, regulates diverse pathways, from stress responses in yeast to T-cell activation and cardiac hypertrophy in humans. Calmodulin (CaM), an essential mediator of calcium–dependent signaling pathways, activates CaN in the presence of calcium by binding to an intrinsically disordered region of the enzyme and altering its conformation. My hydrodynamic studies have determined that CaM participates in a 1:1 complex with the CaM-binding domain of βCaN (CaNp, residues 400–423).
To explore the molecular mechanism of CaM association with CaN, I have used spectroscopic methods to determine the calcium-dependent and domain–specific interactions of CaM with CaNp. These studies revealed that the affinity of CaM1–148 for CaNp was weak in the absence of calcium, and very high (Kd in the nM to pM range) in the presence of calcium. I have demonstrated that CaNp binding to CaM increases the calcium–binding affinity of each domain of CaM1–148 to a similar degree, thereby retaining the property of sequential calcium binding to the domains, with preference for sites in the C–domain. This allows the N–domain to lag in response to an increase in cellular calcium and perhaps contribute to the regulation of CaN in a manner distinct from that of the C–domain.
NMR studies of calcium–saturated CaM1–148 demonstrated that the N–domain of CaM experienced a larger structural perturbation than the C–domain upon binding CaNp. Additional NMR studies revealed that CaNp adopts an anti–parallel orientation when bound to CaM, with the sole aromatic residue of CaNp contacting the N–domain of CaM. This contrasts with many CaM-target complexes in which the sole aromatic residue contacts the C–domain of CaM. Rigorous thermodynamic studies explored how mutations in the calcium-binding sites of mammalian CaM (mCaM) and mutations known to cause disruption of CaM–mediated ion channel regulation in Paramecia (PCaM) affected the allosteric interactions of the domains of CaM in the presence of CaNp. These studies demonstrated separable roles of the domains of CaM in recognition of CaNp. The consequences of a mutation depended upon its location within the complex. Collectively, research presented in this thesis provides insight into the mechanisms whereby the two domains of CaM contribute to recognition of CaN.
Copyright 2009 Susan Ellen O'Donnell