Use of cysteine-reactive cross-linkers to probe conformational flexibility of human DJ-1 demonstrates that Glu18 mutations are dimers.
Use of cysteine-reactive cross-linkers to probe conformational flexibility of human DJ-1 demonstrates that Glu18 mutations are dimers.
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DOI：
10.1111/jnc.12763
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发表时间：
2014-09
影响因子：
4.7
通讯作者：
Wilson MA
中科院分区：
文献类型：
--
作者：
Prahlad J;Hauser DN;Milkovic NM;Cookson MR;Wilson MA
帕金森病相关蛋白 DJ-1 中关键半胱氨酸残基 (Cys106) 的氧化可调节其防止氧化应激和线粒体损伤的能力。 Cys106 与邻近的质子化 Glu18 残基相互作用，稳定 DJ-1 的 Cys106-SO2−（亚磺酸）形式。为了研究这一重要的翻译后修饰，我们之前设计了几种改变 Cys106 氧化倾向的 Glu18 突变（E18N、E18D、E18Q）。然而，最近的结果表明这些 Glu18 突变会导致 DJ-1 二聚化丧失，这将严重损害该蛋白质的功能。本研究的目的是使用 X 射线晶体学、NMR 光谱、热稳定性分析、CD 光谱、沉降平衡超速离心和交联来最终确定这些突变体的寡聚状态。我们发现所有 Glu18 DJ-1 突变体都是二聚体。硫醇交联表明这些突变二聚体比野生型蛋白质更灵活，并且由于野生型蛋白质中难以接近的半胱氨酸残基的瞬时暴露，可以形成多个交联二聚体种类。 Glu18 DJ-1 突变体增强的灵活性为其在细胞中观察到的较低交联效率提供了简约的解释。此外，硫醇交联剂作为蛋白质构象灵活性的定性探针的价值可能被低估。
The oxidation of a key cysteine residue (Cys106) in the parkinsonism-associated protein DJ-1 regulates its ability to protect against oxidative stress and mitochondrial damage. Cys106 interacts with a neighboring protonated Glu18 residue, stabilizing the Cys106-SO2− (sulfinic acid) form of DJ-1. To study this important post-translational modification, we previously designed several Glu18 mutations (E18N, E18D, E18Q) that alter the oxidative propensity of Cys106. However, recent results suggest these Glu18 mutations cause loss of DJ-1 dimerization, which would severely compromise the protein’s function. The purpose of this study was to conclusively determine the oligomerization state of these mutants using X-ray crystallography, NMR spectroscopy, thermal stability analysis, CD spectroscopy, sedimentation equilibrium ultracentrifugation, and crosslinking. We found that all of the Glu18 DJ-1 mutants were dimeric. Thiol crosslinking indicates that these mutant dimers are more flexible than the wild-type protein and can form multiple crosslinked dimeric species due to the transient exposure of cysteine residues that are inaccessible in the wild-type protein. The enhanced flexibility of Glu18 DJ-1 mutants provides a parsimonious explanation for their lower observed crosslinking efficiency in cells. In addition, thiol crosslinkers may have an underappreciated value as qualitative probes of protein conformational flexibility.
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影响因子：
6.1
作者：
Hauser DN;Hastings TG
通讯作者：
Hastings TG
DOI：
10.1073/pnas.0911175107
发表时间：
2010-05-25
影响因子：
11.1
作者：
Hao, Ling-Yang;Giasson, Benoit I.;Bonini, Nancy M.
通讯作者：
Bonini, Nancy M.
影响因子：
4.8
作者：
Honbou, K;Suzuki, NN;Inagaki, F
通讯作者：
Inagaki, F
影响因子：
56.9
作者：
Bonifati, V;Rizzu, P;Heutink, P
通讯作者：
Heutink, P
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K