Recognition of β-calcineurin by the domains of calmodulin: Thermodynamic and structural evidence for distinct roles

Calcineurin (CaN, PP2B, PPP3), a heterodimeric Ca2+‐calmodulin‐dependent Ser/Thr phosphatase, regulates swimming in Paramecia, stress responses in yeast, and T‐cell activation and cardiac hypertrophy in humans. Calcium binding to CaNB (the regulatory subunit) triggers conformational change in CaNA (the catalytic subunit). Two isoforms of CaNA (α, β) are both abundant in brain and heart and activated by calcium‐saturated calmodulin (CaM). The individual contribution of each domain of CaM to regulation of calcineurin is not known. Hydrodynamic analyses of (Ca2+)4‐CaM1–148 bound to βCaNp, a peptide representing its CaM‐binding domain, indicated a 1:1 stoichiometry. βCaNp binding to CaM increased the affinity of calcium for the N‐ and C‐domains equally, thus preserving intrinsic domain differences, and the preference of calcium for sites III and IV. The equilibrium constants for individual calcium‐saturated CaM domains dissociating from βCaNp were ∼1 μM. A limiting Kd ≤ 1 nM was measured directly for full‐length CaM, while thermodynamic linkage analysis indicated that it was approximately 1 pM. βCaNp binding to 15N‐(Ca2+)4‐CaM1–148 monitored by 15N/1HN HSQC NMR showed that association perturbed the N‐domain of CaM more than its C‐domain. NMR resonance assignments of CaM and βCaNp, and interpretation of intermolecular NOEs observed in the 13C‐edited and 12C‐14N‐filtered 3D NOESY spectrum indicated anti‐parallel binding. The sole aromatic residue (Phe) located near the βCaNp C‐terminus was in close contact with several residues of the N‐domain of CaM outside the hydrophobic cleft. These structural and thermodynamic properties would permit the domains of CaM to have distinct physiological roles in regulating activation of βCaN. Proteins 2011. © 2010 Wiley‐Liss, Inc.