The C-terminus of ca
lmodu
lin (CaM) functions as a sensor of oxidative stress, with oxidationof methionine 144
and 145 inducing a nonproductive association of the oxidized CaM with the p
lasmamembrane Ca
2+-ATPase (PMCA)
and other target proteins to downregu
late ce
llu
lar metabo
lism. To betterunderst
and the structura
l underpinnings
and mechanism of this switch, we have engineered a CaM mutant(CaM-L7) that permits the site-specific oxidation of M144
and M145,
and we have used NMR spectroscopyto identify structura
l changes in CaM
and CaM-L7
and changes in the interactions between CaM-L7
andthe CaM-binding sequence of the PMCA (C28W) due to methionine oxidation. In CaM
and CaM-L7,methionine oxidation resu
lts in nomina
l secondary structura
l changes, but chemica
l shift changes
andline broadening in NMR spectra indicate significant tertiary structura
l changes. For CaM-L7 bound toC28W, main chain
and side chain chemica
l shift perturbations indicate that oxidation of M144
and M145
leads to
large tertiary structura
l changes in the C-termina
l hydrophobic pocket invo
lving residues thatcomprise the interface with C28W. Sma
ller changes in the N-termina
l domain a
lso invo
lving residuesthat interact with C28W are observed, as are changes in the centra
l linker region. At the C-termina
l he
lix,
1H
lpha.gif" BORDER=0>,
13C
lpha.gif" BORDER=0>,
and 13CO chemica
l shift changes indicate decreased he
lica
l character, with a comp
lete
loss ofhe
licity for M144
and M145. Using
13C-fi
ltered,
13C-edited NMR experiments, dramatic changes inintermo
lecu
lar contacts between residues in the C-termina
l domain of CaM-L7
and C28W accompanyoxidation of M144
and M145, with an essentia
lly comp
lete
loss of contacts between C28W
and M144
and M145. We propose that the inabi
lity of CaM to fu
lly activate the PMCA after methionine oxidationoriginates in a reduced he
lica
l propensity for M144
and M145,
and resu
lts primari
ly from a g
loba
lrearrangement of the tertiary structure of the C-termina
l g
lobu
lar domain that substantia
lly a
lters theinteraction of this domain with the PMCA.