Crystal structures of the selenoprotein glutathione peroxidase 4 in its apo form and in complex with the covalently bound inhibitor ML162.
Crystal structures of the selenoprotein glutathione peroxidase 4 in its apo form and in complex with the covalently bound inhibitor ML162.
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DOI：
10.1107/s2059798320016125
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发表时间：
2021-02-01
期刊：
影响因子：
--
通讯作者：
Hillig RC
中科院分区：
文献类型：
--
作者：
Moosmayer D;Hilpmann A;Hoffmann J;Schnirch L;Zimmermann K;Badock V;Furst L;Eaton JK;Viswanathan VS;Schreiber SL;Gradl S;Hillig RC
本文测定了人含硒半胱氨酸蛋白谷胱甘肽过氧化物酶4(Gpx4)的晶体结构，其分辨率为1.0 。建立了一种基于质谱学的方法来监测活性部位硒半胱氨酸Sec46与共价抑制剂的加合物的形成。在1.54 的分辨率下，测定了含Sec 46的Gpx4与共价抑制剂ML162[(S)对映体]形成的络合物的晶体结构。将野生型人谷胱甘肽过氧化物酶4(Gpx4)与硒半胱氨酸插入序列结合蛋白2(SBP2)在人HEK细胞中共表达，以实现结构生物学制备规模的高效生产这种含硒半胱氨酸酶。该蛋白经纯化和结晶，并在1.0 á分辨率下测定了野生型Gpx4的晶体结构。与先前确定的Gpx4突变形式的晶体结构相比，Gpx4的整体折叠和活性部位是保守的。建立了一种基于质谱学的方法来监测活性部位硒半胱氨酸Sec46与共价抑制剂的反应。再加上表面突变体(Cys66Ser)的引入，使得Gpx4与共价抑制剂ML162[(S)-对映体]形成的络合物的晶体结构得以确定。这里描述的基于质谱学的方法打开了进一步在与共价抑制剂的络合物中进一步与这种潜在的抗癌药物靶标的共络合物晶体结构的途径。
The crystal structure of the human selenocysteine-containing protein glutathione peroxidase 4 (GPX4) was determined at 1.0 Å resolution. A mass-spectrometry-based approach was developed to monitor the formation of adducts of the active-site selenocysteine Sec46 with covalent inhibitors. The crystal structure of Sec46-containing GPX4 in complex with the covalent inhibitor ML162 [(S)-enantiomer] was determined at 1.54 Å resolution. Wild-type human glutathione peroxidase 4 (GPX4) was co-expressed with SBP2 (selenocysteine insertion sequence-binding protein 2) in human HEK cells to achieve efficient production of this selenocysteine-containing enzyme on a preparative scale for structural biology. The protein was purified and crystallized, and the crystal structure of the wild-type form of GPX4 was determined at 1.0 Å resolution. The overall fold and the active site are conserved compared with previously determined crystal structures of mutated forms of GPX4. A mass-spectrometry-based approach was developed to monitor the reaction of the active-site selenocysteine Sec46 with covalent inhibitors. This, together with the introduction of a surface mutant (Cys66Ser), enabled the crystal structure determination of GPX4 in complex with the covalent inhibitor ML162 [(S)-enantiomer]. The mass-spectrometry-based approach described here opens the path to further co-complex crystal structures of this potential cancer drug target in complex with covalent inhibitors.