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首页> 外文期刊>Biochemistry >Mediating molecular recognition by methionine oxidation: Conformational switching by oxidation of methionine in the carboxyl-terminal domain of calmodulin
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Mediating molecular recognition by methionine oxidation: Conformational switching by oxidation of methionine in the carboxyl-terminal domain of calmodulin

机译：通过蛋氨酸氧化介导分子识别：通过钙调蛋白的羧基末端结构域的蛋氨酸氧化构象转换

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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.
机译：钙调蛋白（CaM）的C端可作为氧化应激的传感器，蛋氨酸144和145的氧化诱导氧化的CaM与质膜Ca2 + -ATPase（PMCA）和其他靶蛋白的非生产性结合，从而下调细胞代谢。为了更好地了解此开关的结构基础和机制，我们设计了一个CaM突变体（CaM-L7），该突变体可直接修饰M 144和M 145的位点特异性氧化，并且我们使用NMR光谱法来鉴定CaM和CaM-L7以及由于蛋氨酸氧化而引起的CaM-L7与PMCA（C28W）的CaM结合序列之间相互作用的变化。在CaM和CaM-L7中，蛋氨酸氧化会导致名义上的二级结构变化，但NMR谱中的化学位移变化和谱线展宽表明存在重要的三级结构变化。对于与C28W结合的CaM-L7，主链和侧链的化学位移扰动表明M144和Mk145的氧化会导致C端疏水性多肽中大的三级结构变化，涉及与C28W构成界面的残基。观察到N端结构域的较小变化，也涉及与C28W相互作用的残基，中央连接区的变化也是如此。在C末端螺旋上，H-1α，C-13α和（CO）-C-13的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，C-13编辑的NMR实验，CaM-L7和C28W的C末端结构域中的残基之间的分子间接触发生了剧烈变化，伴随着M144和M145的氧化，C28W之间的接触基本上完全丧失以及M144和M145。我们认为蛋氨酸氧化后CaM不能完全激活PMCA的原因是M144和M145的螺旋倾向降低，这主要是由于C端球状结构域三级结构的整体重排导致的，该重排实质上改变了该域与PMCA。

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