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Oxidation-induced conformational changes in calcineurin determined by covalent labeling and tandem mass spectrometry.

Zhou X, Mester C, Stemmer PM, Reid GE - Biochemistry (2014)

Bottom Line: Here, the susceptibility of calcineurin methionine residues toward H2O2-induced oxidation were determined using a multienzyme digestion strategy coupled with capillary HPLC-electrospray ionization mass spectrometry and tandem mass spectrometry analysis.Importantly, methionine residues found to be highly susceptible toward oxidation, and the lysine residues exhibiting large increases in accessibility upon oxidation, were all located in calcineurin functional domains involved in Ca(2+)/CaM binding regulated calcineurin stimulation.These findings therefore provide initial support for the novel mechanistic hypothesis that oxidation-induced global and/or local conformational changes within calcineurin contribute to inactivation via (i) impairing the interaction between calcineurin A and calcineurin B, (ii) altering the low-affinity Ca(2+) binding site in calcineurin B, (iii) inhibiting calmodulin binding to calcineurin A, and/or (iv) by altering the affinity between the calcineurin A autoinhibitory domain and the catalytic center.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, and §Department of Biochemistry and Molecular Biology, Michigan State University , East Lansing, Michigan 48824, United States.

ABSTRACT
The Ca(2+)/calmodulin activated phosphatase, calcineurin, is inactivated by H2O2 or superoxide-induced oxidation, both in vivo and in vitro. However, the potential for global and/or local conformation changes occurring within calcineurin as a function of oxidative modification, that may play a role in the inactivation process, has not been examined. Here, the susceptibility of calcineurin methionine residues toward H2O2-induced oxidation were determined using a multienzyme digestion strategy coupled with capillary HPLC-electrospray ionization mass spectrometry and tandem mass spectrometry analysis. Then, regions within the protein complex that underwent significant conformational perturbation upon oxidative modification were identified by monitoring changes in the modification rates of accessible lysine residues between native and oxidized forms of calcineurin, using an amine-specific covalent labeling reagent, S,S'-dimethylthiobutanoylhydroxysuccinimide ester (DMBNHS), and tandem mass spectrometry. Importantly, methionine residues found to be highly susceptible toward oxidation, and the lysine residues exhibiting large increases in accessibility upon oxidation, were all located in calcineurin functional domains involved in Ca(2+)/CaM binding regulated calcineurin stimulation. These findings therefore provide initial support for the novel mechanistic hypothesis that oxidation-induced global and/or local conformational changes within calcineurin contribute to inactivation via (i) impairing the interaction between calcineurin A and calcineurin B, (ii) altering the low-affinity Ca(2+) binding site in calcineurin B, (iii) inhibiting calmodulin binding to calcineurin A, and/or (iv) by altering the affinity between the calcineurin A autoinhibitory domain and the catalytic center.

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Ratio of second-orderDMBNHS modification rate constants determinedfor observed lysine residues from oxidized CN (15 min reaction timepoint) over the DMBNHS modification rate constants determined forthe corresponding Lys residues from native CN. CN Lys residues locatedin the catalytic domain (purple solid circle), CaM binding domain(green solid box) and autoinhibitory region (orange solid pentagon)of CNA, the CNA binding region (blue solid up triangle) and Ca2+ binding domain (light blue solid down triangle) of CNB andnonfunctional regions (⧫). Data labels indicate the percentmodification observed in the native CN protein complex at a DMBNHSmolar excess of 100.
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fig7: Ratio of second-orderDMBNHS modification rate constants determinedfor observed lysine residues from oxidized CN (15 min reaction timepoint) over the DMBNHS modification rate constants determined forthe corresponding Lys residues from native CN. CN Lys residues locatedin the catalytic domain (purple solid circle), CaM binding domain(green solid box) and autoinhibitory region (orange solid pentagon)of CNA, the CNA binding region (blue solid up triangle) and Ca2+ binding domain (light blue solid down triangle) of CNB andnonfunctional regions (⧫). Data labels indicate the percentmodification observed in the native CN protein complex at a DMBNHSmolar excess of 100.

Mentions: The extent of CN conformational change uponoxidation was characterizedby dividing the DMBNHS modification rate constant of each observedlysine residue from the oxidized (15 min time point) CN protein complexby the DMBNHS modification rate constant of the same lysine residuefrom the native CN protein complex, yielding a fold change indicativeof each lysine residue’s DMBNHS modification rate constantas a function of oxidation (Figure 7). Datalabels in Figure 7 indicate the percent modificationfor each lysine residues that was observed in the native CN proteincomplex using a DMBNHS molar excess of 100. When the same lysine residuewas observed from different peptides, the DMBNHS modification rateconstants determined from all observed peptides were averaged andthen used to calculate the fold increase.


Oxidation-induced conformational changes in calcineurin determined by covalent labeling and tandem mass spectrometry.

Zhou X, Mester C, Stemmer PM, Reid GE - Biochemistry (2014)

Ratio of second-orderDMBNHS modification rate constants determinedfor observed lysine residues from oxidized CN (15 min reaction timepoint) over the DMBNHS modification rate constants determined forthe corresponding Lys residues from native CN. CN Lys residues locatedin the catalytic domain (purple solid circle), CaM binding domain(green solid box) and autoinhibitory region (orange solid pentagon)of CNA, the CNA binding region (blue solid up triangle) and Ca2+ binding domain (light blue solid down triangle) of CNB andnonfunctional regions (⧫). Data labels indicate the percentmodification observed in the native CN protein complex at a DMBNHSmolar excess of 100.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4222536&req=5

fig7: Ratio of second-orderDMBNHS modification rate constants determinedfor observed lysine residues from oxidized CN (15 min reaction timepoint) over the DMBNHS modification rate constants determined forthe corresponding Lys residues from native CN. CN Lys residues locatedin the catalytic domain (purple solid circle), CaM binding domain(green solid box) and autoinhibitory region (orange solid pentagon)of CNA, the CNA binding region (blue solid up triangle) and Ca2+ binding domain (light blue solid down triangle) of CNB andnonfunctional regions (⧫). Data labels indicate the percentmodification observed in the native CN protein complex at a DMBNHSmolar excess of 100.
Mentions: The extent of CN conformational change uponoxidation was characterizedby dividing the DMBNHS modification rate constant of each observedlysine residue from the oxidized (15 min time point) CN protein complexby the DMBNHS modification rate constant of the same lysine residuefrom the native CN protein complex, yielding a fold change indicativeof each lysine residue’s DMBNHS modification rate constantas a function of oxidation (Figure 7). Datalabels in Figure 7 indicate the percent modificationfor each lysine residues that was observed in the native CN proteincomplex using a DMBNHS molar excess of 100. When the same lysine residuewas observed from different peptides, the DMBNHS modification rateconstants determined from all observed peptides were averaged andthen used to calculate the fold increase.

Bottom Line: Here, the susceptibility of calcineurin methionine residues toward H2O2-induced oxidation were determined using a multienzyme digestion strategy coupled with capillary HPLC-electrospray ionization mass spectrometry and tandem mass spectrometry analysis.Importantly, methionine residues found to be highly susceptible toward oxidation, and the lysine residues exhibiting large increases in accessibility upon oxidation, were all located in calcineurin functional domains involved in Ca(2+)/CaM binding regulated calcineurin stimulation.These findings therefore provide initial support for the novel mechanistic hypothesis that oxidation-induced global and/or local conformational changes within calcineurin contribute to inactivation via (i) impairing the interaction between calcineurin A and calcineurin B, (ii) altering the low-affinity Ca(2+) binding site in calcineurin B, (iii) inhibiting calmodulin binding to calcineurin A, and/or (iv) by altering the affinity between the calcineurin A autoinhibitory domain and the catalytic center.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, and §Department of Biochemistry and Molecular Biology, Michigan State University , East Lansing, Michigan 48824, United States.

ABSTRACT
The Ca(2+)/calmodulin activated phosphatase, calcineurin, is inactivated by H2O2 or superoxide-induced oxidation, both in vivo and in vitro. However, the potential for global and/or local conformation changes occurring within calcineurin as a function of oxidative modification, that may play a role in the inactivation process, has not been examined. Here, the susceptibility of calcineurin methionine residues toward H2O2-induced oxidation were determined using a multienzyme digestion strategy coupled with capillary HPLC-electrospray ionization mass spectrometry and tandem mass spectrometry analysis. Then, regions within the protein complex that underwent significant conformational perturbation upon oxidative modification were identified by monitoring changes in the modification rates of accessible lysine residues between native and oxidized forms of calcineurin, using an amine-specific covalent labeling reagent, S,S'-dimethylthiobutanoylhydroxysuccinimide ester (DMBNHS), and tandem mass spectrometry. Importantly, methionine residues found to be highly susceptible toward oxidation, and the lysine residues exhibiting large increases in accessibility upon oxidation, were all located in calcineurin functional domains involved in Ca(2+)/CaM binding regulated calcineurin stimulation. These findings therefore provide initial support for the novel mechanistic hypothesis that oxidation-induced global and/or local conformational changes within calcineurin contribute to inactivation via (i) impairing the interaction between calcineurin A and calcineurin B, (ii) altering the low-affinity Ca(2+) binding site in calcineurin B, (iii) inhibiting calmodulin binding to calcineurin A, and/or (iv) by altering the affinity between the calcineurin A autoinhibitory domain and the catalytic center.

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