Mediating Molecular Recognition by Methionine Oxidation: Conformational Switching by Oxidation of Methionine in the Carboxyl-Terminal Domain of Calmodulin
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- 作者：Asokan Anbanandam ; Ramona J. Bieber Urbauer ; Ryan K. Bartlett ; Heather S. Smallwood ; Thomas C. Squier ; and Jeffrey L. Urbauer
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：July 12, 2005
- 年：2005
- 卷：44
- 期：27
- 页码：9486 - 9496
- 全文大小：497K
- 年卷期：v.44,no.27(July 12, 2005)
- ISSN：1520-4995
文摘
The C-terminus of calmodulin (CaM) functions as a sensor of oxidative stress, with oxidationof methionine 144 and 145 inducing a nonproductive association of the oxidized CaM with the plasmamembrane Ca2+-ATPase (PMCA) and other target proteins to downregulate cellular metabolism. To betterunderstand the structural 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 structural 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 results in nominal secondary structural changes, but chemical shift changes andline broadening in NMR spectra indicate significant tertiary structural changes. For CaM-L7 bound toC28W, main chain and side chain chemical shift perturbations indicate that oxidation of M144 and M145leads to large tertiary structural changes in the C-terminal hydrophobic pocket involving residues thatcomprise the interface with C28W. Smaller changes in the N-terminal domain also involving residuesthat interact with C28W are observed, as are changes in the central linker region. At the C-terminal helix,1H
, 13C, and 13CO chemical shift changes indicate decreased helical character, with a complete loss ofhelicity for M144 and M145. Using 13C-filtered, 13C-edited NMR experiments, dramatic changes inintermolecular contacts between residues in the C-terminal domain of CaM-L7 and C28W accompanyoxidation of M144 and M145, with an essentially complete loss of contacts between C28W and M144and M145. We propose that the inability of CaM to fully activate the PMCA after methionine oxidationoriginates in a reduced helical propensity for M144 and M145, and results primarily from a globalrearrangement of the tertiary structure of the C-terminal globular domain that substantially alters theinteraction of this domain with the PMCA.
, 13C, and 13CO chemical shift changes indicate decreased helical character, with a complete loss ofhelicity for M144 and M145. Using 13C-filtered, 13C-edited NMR experiments, dramatic changes inintermolecular contacts between residues in the C-terminal domain of CaM-L7 and C28W accompanyoxidation of M144 and M145, with an essentially complete loss of contacts between C28W and M144and M145. We propose that the inability of CaM to fully activate the PMCA after methionine oxidationoriginates in a reduced helical propensity for M144 and M145, and results primarily from a globalrearrangement of the tertiary structure of the C-terminal globular domain that substantially alters theinteraction of this domain with the PMCA.