Dataset Information

Methionine oxidation in the calmodulin-binding domain of calcineurin disrupts calmodulin binding and calcineurin activation.

ABSTRACT: Calcineurin is a Ca (2+)/calmodulin-activated Ser/Thr phosphatase important in cellular actions resulting in memory formation, cardiac hypertrophy, and T-cell activation. This enzyme is subject to oxidative inactivation by superoxide at low micromolar concentrations and by H 2O 2 at low millimolar concentrations. On the basis of the hypothesis that oxidation of Met residues in calmodulin-binding domains inhibits binding to calmodulin, purified calcineurin was used to study the susceptibility of Met residues to oxidation by H 2O 2. The rate for oxidation of Met 406 in the calmodulin-binding domain was determined to be 4.4 x 10 (-3) M (-1) s (-1), indicating a high susceptibility to oxidation. Functional repercussions of Met 406 oxidation were evaluated using native enzyme and a calcineurin mutant in which Met 406 was exchanged for Leu. Measurement of fluorescent calmodulin binding demonstrated that oxidation of Met 406 results in a 3.3-fold decrease in the affinity of calmodulin for calcineurin. Calcineurin activation exhibited a loss in cooperativity with respect to calmodulin following Met 406 oxidation as shown by a reduction in the Hill slope from 1.88 to 0.86. Maximum phosphatase activity was unaffected by Met oxidation. Changes in the calcineurin-calmodulin interaction were accompanied by a 40% loss in the ability of calmodulin to stimulate binding of immunophilin/immunosuppressant to calcineurin. All effects on calmodulin binding to the native enzyme by the treatment with H 2O 2 could be reversed by treating the enzyme with methionine sulfoxide reductase. These results indicate that the calmodulin-binding domain of calcineurin is susceptible to oxidation at Met 406 and that oxidation disrupts calmodulin binding and enzyme activation. Oxidation-dependent decreases in the affinity of calmodulin for calcineurin can potentially modulate calmodulin-dependent signaling and calmodulin distribution.

SUBMITTER: Carruthers NJ

PROVIDER: S-EPMC4167793 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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Methionine oxidation in the calmodulin-binding domain of calcineurin disrupts calmodulin binding and calcineurin activation.

Carruthers Nicholas J NJ Stemmer Paul M PM

Biochemistry 20080215 10

Calcineurin is a Ca (2+)/calmodulin-activated Ser/Thr phosphatase important in cellular actions resulting in memory formation, cardiac hypertrophy, and T-cell activation. This enzyme is subject to oxidative inactivation by superoxide at low micromolar concentrations and by H 2O 2 at low millimolar concentrations. On the basis of the hypothesis that oxidation of Met residues in calmodulin-binding domains inhibits binding to calmodulin, purified calcineurin was used to study the susceptibility of ...[more]

PMID: 18275158

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Project description:We have used electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and fluorescence spectroscopy to investigate the secondary and tertiary structural consequences that result from oxidative modification of methionine residues in wheat germ calmodulin (CaM), and prevent activation of the plasma membrane Ca-ATPase. Using ESI-MS, we have measured rates of modification and molecular mass distributions of oxidatively modified CaM species (CaMox) resulting from exposure to H2O2. From these rates, we find that oxidative modification of methionine to the corresponding methionine sulfoxide does not predispose CaM to further oxidative modification. These results indicate that methionine oxidation results in no large-scale alterations in the tertiary structure of CaMox, because the rates of oxidative modification of individual methionines are directly related to their solvent exposure. Likewise, CD measurements indicate that methionine oxidation results in little change in the apparent alpha-helical content at 28 degrees C, and only a small (0.3 +/- 0.1 kcal mol(-1)) decrease in thermal stability, suggesting the disruption of a limited number of specific noncovalent interactions. Fluorescence lifetime, anisotropy, and quenching measurements of N-(1-pyrenyl)-maleimide (PMal) covalently bound to Cys26 indicate local structural changes around PMal in the amino-terminal domain in response to oxidative modification of methionine residues in the carboxyl-terminal domain. Because the opposing globular domains remain spatially distant in both native and oxidatively modified CaM, the oxidative modification of methionines in the carboxyl-terminal domain are suggested to modify the conformation of the amino-terminal domain through alterations in the structural features involving the interdomain central helix. The structural basis for the linkage between oxidative modification and these global conformational changes is discussed in terms of possible alterations in specific noncovalent interactions that have previously been suggested to stabilize the central helix in CaM.

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

Methionine oxidation in the calmodulin-binding domain of calcineurin disrupts calmodulin binding and calcineurin activation.

Publications

Methionine oxidation in the calmodulin-binding domain of calcineurin disrupts calmodulin binding and calcineurin activation.

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