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

Biochemistry. 2008 Mar 11;47(10):3085-95. doi: 10.1021/bi702044x. Epub 2008 Feb 15.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Calcineurin / chemistry*
  • Calcineurin / metabolism*
  • Calmodulin / metabolism*
  • Circular Dichroism
  • Dithiothreitol / pharmacology
  • Enzyme Activation / drug effects
  • Fluorescence Polarization
  • Hydrogen Peroxide / pharmacology
  • Methionine / metabolism*
  • Models, Molecular
  • Oxidation-Reduction / drug effects
  • Protein Binding / drug effects
  • Protein Structure, Secondary
  • Protein Structure, Tertiary


  • Calmodulin
  • Methionine
  • Hydrogen Peroxide
  • Calcineurin
  • Dithiothreitol