Calcium-Mediated Regulation of Calcineurin by a Dynamic Duo of EF-Hand Proteins

Abstract

Calcium-mediated regulation of early stages of heart development critically depends on calcineurin (CaN), a heterodimeric, calcium-activated phosphatase. CaN acts by dephosphorylating NF-AT, promoting its nuclear localization to launch transcription that controls cardiac muscle growth. Vulnerability of the embryonic heart to fluctuations in CaN activity propels our studies to understand calcium-dependent regulation of CaN. Calcium partially activates CaN by binding to CaNB, its intrinsic 4-EF-Hand subunit. Full activity occurs upon binding of calmodulin (CaM, another 4-EF-Hand protein) which recognizes a CaM-binding domain (CaMBD) near the CaNA auto-inhibitory domain. To probe sequential calcium-mediated activation of CaN, we found CaM binding to CaNA caused a thousand-fold increase in calcium-binding affinity of each domain of CaM, while preserving a 10-fold difference between the domains. To explore the molecular basis for calcium-triggering of CaN, we studied site-knockout mutants of CaNB and CaM, and mutagenized CaMBD, a 1-5-8-14 basic, amphipathic alpha-helix with picomolar affinity for calcium-saturated CaM. CaM binding to an auto-fluorescent biosensor (donor-CFP-CaMBD-acceptor-YFP) allows estimates of equilibrium constants. Site-knockout mutants of CaM had variable effects on the CaN-CaM interface. Mutagenized CaMBDs indicated energetic contributions of the 1-5-8-14 positions were dramatically different: Ile>Ala at position 5 lowered binding energy by 3 kcal/mol more than Ile>Ala at position 1 or 8. Conventional motif classifications for CaMBDs clearly do not indicate energetic contributions at interfaces. Further, Phe>Ala (substitution of the sole aromatic residue) only modestly affected WT CaM binding, indicated that Phe410 is not an “anchor residue”. We are pursuing connections between these protein-protein interfaces and enzymatic activity of CaN to understand how congenital defects may lead to faulty development of the cardiac muscle. NIH R01 GM57001, AHA 12GRNT12050395 to MAS, and AHA Predoctoral Fellowship to SEO.