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Recognition of ?-calcineurin by the domains of calmodulin: thermodynamic and structural evidence for distinct roles.

ABSTRACT: Calcineurin (CaN, PP2B, PPP3), a heterodimeric Ca(2+)-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 CaN(B) (the regulatory subunit) triggers conformational change in CaN(A) (the catalytic subunit). Two isoforms of CaN(A) (?, ?) 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 (Ca(2+))?-CaM(1-148) bound to ?CaNp, a peptide representing its CaM-binding domain, indicated a 1:1 stoichiometry. ?CaNp 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 ?CaNp were ?1 ?M. A limiting K(d) ? 1 nM was measured directly for full-length CaM, while thermodynamic linkage analysis indicated that it was approximately 1 pM. ?CaNp binding to ¹?N-(Ca(2+))?-CaM(1-148) monitored by ¹?N/¹HN HSQC NMR showed that association perturbed the N-domain of CaM more than its C-domain. NMR resonance assignments of CaM and ?CaNp, and interpretation of intermolecular NOEs observed in the ¹³C-edited and ¹²C-¹?N-filtered 3D NOESY spectrum indicated anti-parallel binding. The sole aromatic residue (Phe) located near the ?CaNp 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 ?CaN.

SUBMITTER: O'Donnell SE

PROVIDER: S-EPMC3057930 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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

O'Donnell Susan E SE Yu Liping L Fowler C Andrew CA Shea Madeline A MA

Proteins 20101206 3

Calcineurin (CaN, PP2B, PPP3), a heterodimeric Ca(2+)-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 CaN(B) (the regulatory subunit) triggers conformational change in CaN(A) (the catalytic subunit). Two isoforms of CaN(A) (α, β) are both abundant in brain and heart and activated by calcium-saturated calmodulin (CaM). The individual contribution of each domain of CaM ...[more]

PMID: 21287611

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Project description:Calcineurin (CaN) is a Ca2+/calmodulin-dependent Ser/Thr protein phosphatase, which plays essential roles in many cellular and developmental processes. CaN comprises two subunits, a catalytic subunit (CaN-A, 60 kDa) and a regulatory subunit (CaN-B, 19 kDa). CaN-A tightly binds to CaN-B in the presence of minimal levels of Ca2+, but the enzyme is inactive until activated by CaM. Upon binding to CaM, CaN then undergoes a conformational rearrangement, the auto inhibitory domain is displaced and thus allows for full activity. In order to elucidate the regulatory role of CaM in the activation processes of CaN, we used NMR spectroscopy to determine the structure of the complex of CaM and the target peptide of CaN (CaNp). The CaM/CaNp complex shows a compact ellipsoidal shape with 8 α-helices of CaM wrapping around the CaNp helix. The RMSD of backbone and heavy atoms of twenty lowest energy structures of CaM/CaNp complex are 0.66 and 1.14 Å, respectively. The structure of CaM/CaNp complex can be classified as a novel binding mode family 1-18 with major anchor residues Ile396 and Leu413 to allocate the largest space between two domains of CaM. The relative orientation of CaNp to CaM is similar to the CaMKK peptide in the 1-16 binding mode with N- and C-terminal hydrophobic anchors of target sequence engulfed in the hydrophobic pockets of the N- and C-domain of CaM, respectively. In the light of the structural model of CaM/CaNp complex reported here, we provide new insight in the activation processes of CaN by CaM. We propose that the hydrophobic interactions between the Ca2+-saturated C-domain and C-terminal half of the target sequence provide driving forces for the initial recognition. Subsequent folding in the target sequence and structural readjustments in CaM enhance the formation of the complex and affinity to calcium. The electrostatic repulsion between CaM/CaNp complex and AID may result in the displacement of AID from active site for full activity.

Dataset Information

Recognition of ?-calcineurin by the domains of calmodulin: thermodynamic and structural evidence for distinct roles.

Publications

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

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