Mediating molecular recognition by methionine oxidation: Conformational switching by oxidation of methionine in the carboxyl-terminal domain of calmodulin
Mediating molecular recognition by methionine oxidation: Conformational switching by oxidation of methionine in the carboxyl-terminal domain of calmodulin
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DOI：
10.1021/bi0504963
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发表时间：
2005-07-12
期刊：
影响因子：
2.9
通讯作者：
Urbauer, JL
中科院分区：
文献类型：
--
作者：
Anbanandam, A;Urbauer, RJB;Urbauer, JL
钙调素（CaM）的C-末端作为氧化应激的传感器发挥作用，蛋氨酸144和145的氧化诱导氧化的CaM与质膜Ca 2 +-ATP酶（PMCA）和其他靶蛋白的非生产性缔合以下调细胞代谢。为了更好地理解这种开关的结构基础和机制，我们设计了一种CaM突变体（CaM-L7），它使M 144和M 145的位点特异性氧化，我们使用NMR光谱来鉴定CaM和CaM-L7的结构变化以及由于甲硫氨酸氧化引起的CaM-L7与PMCA（C28 W）的CaM结合序列之间的相互作用变化。在CaM和CaM-L7中，甲硫氨酸氧化导致标称二级结构变化，但NMR光谱中的化学位移变化和谱线增宽指示显著的三级结构变化。对于与C28 W结合的CaM-L7，主链和侧链化学位移扰动表明，M144和Mk 145的氧化导致C-末端疏水性口袋中涉及包含与C28 W的界面的残基的大的三级结构变化。观察到N-末端结构域中也涉及与C28 W相互作用的残基的较小变化，以及中央接头区的变化。在C-末端螺旋，H-1（α）、C-13（α）和（CO）-C-13化学位移变化表明螺旋特征降低，M144和M145的螺旋度完全丧失。使用C-13过滤的、C-13编辑的NMR实验，CaM-L7和C28 W的C-末端结构域中的残基之间的分子间接触的急剧变化伴随着M144和M145的氧化，C28 W与M144和M145之间的接触基本上完全丧失。我们建议，钙调素不能完全激活后，蛋氨酸氧化PMCA起源于减少螺旋倾向M144和M145，并主要从全球重排的三级结构的C-末端球状结构域，大大改变了这个域与PMCA的相互作用的结果。
The C-terminus of calmodulin (CaM) functions as a sensor of oxidative stress, with oxidation of methionine 144 and 145 inducing a nonproductive association of the oxidized CaM with the plasma membrane Ca2+-ATPase (PMCA) and other target proteins to downregulate cellular metabolism. To better understand the structural underpinnings and mechanism of this switch, we have engineered a CaM mutant (CaM-L7) that pen-nits the site-specific oxidation of M 144 and M 145, and we have used NMR spectroscopy to identify structural changes in CaM and CaM-L7 and changes in the interactions between CaM-L7 and the 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 and line broadening in NMR spectra indicate significant tertiary structural changes. For CaM-L7 bound to C28W, main chain and side chain chemical shift perturbations indicate that oxidation of M144 and Mk145 leads to large tertiary structural changes in the C-terminal hydrophobic pocet involving residues that comprise the interface with C28W. Smaller changes in the N-terminal domain also involving residues that interact with C28W are observed, as are changes in the central linker region. At the C-terminal helix, H-1(alpha), C-13(alpha), and (CO)-C-13 chemical shift changes indicate decreased helical character, with a complete loss of helicity for M144 and M145. Using C-13-filtered, C-13-edited NMR experiments, dramatic changes in intermolecular contacts between residues in the C-terminal domain of CaM-L7 and C28W accompany oxidation of M144 and M145, with an essentially complete loss of contacts between C28W and M144 and M145. We propose that the inability of CaM to fully activate the PMCA after methionine oxidation originates in a reduced helical propensity for M144 and M145, and results primarily from a global rearrangement of the tertiary structure of the C-terminal globular domain that substantially alters the interaction of this domain with the PMCA.